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Article https://doi.org/10.1038/s41467-022-32799-8
Isoform-specific and ubiquitination depen-
dent recruitment of Tet1 to replicating het-
erochromatin modulates methylcytosine
oxidation
María Arroyo
1
,FlorianD.Hastert
1,2
, Andreas Zhadan
1
, Florian Schelter
3
,
Susanne Zimbelmann
1
, Cathia Rausch
1,4
, Anne K. Ludwig
1,5
, Thomas Carell
3
&
M. Cristina Cardoso
1
Oxidation of the epigenetic DNA mark 5-methylcytosine by Tet dioxygenases
is an established route to diversify the epigenetic information, modulate
gene expression and overall cellular (patho-)physiology. Here, we demon-
strate that Tet1 and its short isoform Tet1s exhibit distinct nuclear localiza-
tion during DNA replication resulting in aberrant cytosine modification
levels in human and mouse cells. We show that Tet1 is tethered away from
heterochromatin via its zinc finger domain, which is missing in Tet1s allowing
its targeting to these regions. We find that Tet1s interacts with and is ubi-
quitinated by CRL4(VprBP). The ubiquitinated Tet1s is then recognized by
Uhrf1 and recruited to late replicating heterochromatin. This leads to
spreading of 5-methylcytosine oxidation to heterochromatin regions, LINE 1
activation and chromatin decondensation. In summary, we elucidate a dual
regulation mechanism of Tet1, contributing to the understanding of how
epigenetic information can be diversified by spatio-temporal directed Tet1
catalytic activity.
Covalent modifications of the fifth cytosine carbon atom in mamma-
lian DNA play a crucial role in cellular homeostasis and faulty cytosine
modification patterns are linked to a multitude of diseases, namely
cancer1. Consequently, proteins responsible for these modifications or
capable of interacting with them were implicated in essential, phy-
siological and pathophysiological cellular processes2.Thebest-studied
eukaryotic DNA modification is 5-methylcytosine (5mC), which corre-
lates with transcriptional silencing. The levels of 5mC are maintained
during DNA replication in the S-phase of the cell cycle by DNA
methyltransferase 1 (Dnmt1), the founding member of the DNA
methyltransferase family (Dnmt)2,3. During DNA replication in
euchromatin and facultative heterochromatin, Dnmt1 associates with
the replication machinery via its polymerase clamp PCNA binding
domain (PBD)4. In late S-phase an important cofactor of Dnmt1 is the
E3-ligase Uhrf1 which plays a crucial role in cellular homeostasis and
maintenance of DNA methylation. Besides the E3-ligase activity medi-
ated by its RING-domain, Uhrf1 harbors an Ubl domain; the PHD and
TTD domains capable of binding different histone modifications; and
the SRA domain which recognizes modified cytosine5. Cooperative
binding of Uhrf1 to hemi-methylated CpG sites and trimethylated
Received: 6 August 2021
Accepted: 15 August 2022
Check for updates
1
Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.
2
Section AIDS and
newly emerging pathogens, Paul Ehrlich Institute, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany.
3
Department of Chemistry, LudwigMaximiliansUniversity,
Butenandstr. 5-13, 81377 Munich, Germany.
4
Present address:Luxembourg Centre for Systems Biomedicine, Universityof Luxembourg, 6, avenue du Swing,
L-4367 Belvaux, Luxembourg.
5
Present address: Department of Medicine, Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Im
Neuenheimer Feld 410, 69120 Heidelberg, Germany. e-mail: florian.hastert@pei.de;cardoso@bio.tu-darmstadt.de
Nature Communications | (2022) 13:5173 1
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H3K9, as well as H3K18/K23 ubiquitination, directs Dnmt1 to sites of
ongoing DNA replication in pericentric heterochromatin6,7,which
shows high levels of 5mC around centromeric regions.
Methylated cytosine (5mC) can successively be oxidized to
5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and
5-carboxylcytosine (5caC) by the members of the Ten-eleven translo-
cation (Tet) protein family2,8,9. All three Tet protein family members,
Tet1, Tet2 and Tet3, share the same catalytic activity and high
sequence similarities in their C-terminal catalytic domains10.Despite
this, major differences are observed in their respective expression
levels throughout different tissues and developmental stages, and
therefore their physiological roles. While Tet3 is predominantly
expressed during early embryogenesis and also in post-mitotic neu-
rons, Tet1 and Tet2 are found to be expressed more ubiquitously
across different tissues and developmental stages from embryonic
stem cells to somatic cells11.
A structural and functional feature that separates the three Tet
proteins from one another is their N-terminal zinc finger domain.
Tet2 lost its zinc finger during evolution through chromosomal
inversion and this function is now taken over by the genomically
adjacent Idax/CxxC4, which was found to negatively regulate Tet2
activity12. Tet1 and Tet3, in contrast, both kept their respective zinc
fingers, and the Tet3 CxxC domain was shown to bind caC, thereby
regulating neurodegeneration13. The Tet1 zinc finger domain, on the
other hand, was found to mostly bind non-modified DNA14 and was
implicated in preventing DNA methylation spreading in euchromatic
regions, but it is unclear how this is regulated15. While three different
Tet3 isoforms have been characterized to date13, a N-terminally
truncated Tet1 isoform (Tet1s), which lacks the zinc finger domain,
was discovered recently and attributed a role in reproduction control
and embryogenesis, but also in cancerogenesis16–18. In the latter, a
strong increase of Tet1s activity at non-CpG islands was observed
together with transcriptional activation, while genic regions were
mostly targeted by full-length Tet1 via its CxxC domain17. However,
how Tet1 and its short isoform are differentially regulated remains
elusive.
Besides the double-stranded beta helix (DSBH) domain, which
harbors the Fe(II) and 2-oxoglutarate cofactor binding sites, the
C-terminal catalytic core of all three Tet proteins also comprises
the cysteine-rich domain (CRD). Two studies identified mono-
ubiquitination by the CRL4(VprBP) E3-ligase complex of a conserved
lysine residue within the CRD of all three Tet proteins to be essential
for their catalytic activity, and a lysine to glutamate mutation was
shown to abrogate catalytic activity19,20.
As the two Tet1 isoforms were shown to target different genomic
regions, we hypothesized that they could have different subcellular
distributions, which in turn could be regulated during the cell cycle.
Hence, we aimed to elucidate the subcellular localization of the Tet
proteins and the mechanism regulating it. Furthermore, we investi-
gated the effectof the Tet1 isoforms on 5mC oxidation in euchromatic
and heterochromatic regions, and the resulting biological con-
sequences like repetitive DNA element activation and chromatin
decompaction. This study shows that the short isoform of Tet1 but not
the full-length Tet1 isoform localizes during S phase to sites of ongoing
DNA replication in heterochromatin in an Uhrf1- and CRL4(VprBP)
dependent manner, by ubiquitination of the conserved lysine residue
in the CRD of Tet1s. This results in a significant de novo 5hmC for-
mation, globally, and more so in heterochromatin, including LINE 1
interspersed DNA repeats leading to their activation. In addition, we
report Tet1 localization to be prevented by the N-terminal zinc finger
domain of full-length Tet1 by a passive mechanism that is based on
retention of Tet1 in euchromatin by non-sequence specific chromatin
binding. Taken together, we delineate a dual mechanism thatregulates
the subnuclear localization of Tet1 and its short isoform and conse-
quently their catalytic activity.
Results
The short isoform of Tet1 is recruited to heterochromatin dur-
ing ongoing DNA replication in S-phase and increases 5mC
oxidation
A hallmark of many cancers are aberrant DNA cytosine modification
levels and, in particular, global hypomethylation concomitantly with
local hypermethylation. Hypermethylationis often found in promoters
and coding regions of tumor suppressor genes, which are both usually
hypomethylated in normal tissues21. Interestingly, also the canonical
Tet1 promoter was found to become hypermethylated in many
cancers22, resulting in the use of an alternative promoter as well as an
alternative transcription start site. This consequently leads to the
expression of a N-terminally truncated, but catalytically active, short
isoform Tet1s (Fig. 1A)17.
Based on these findings, we first addressed differences in TET1
isoforms and cytosine modification levels between MCF7 human
breast adenocarcinoma cells and MCF10a non-transformed human
breast epithelial cells. While MCF7 cells expressed high levels of
both TET1 isoforms, MCF10a cells showed comparatively low levels.
However, both MCF cell lines expressed similar levels of TET2, and
very low levels of TET317 (Supplementary Fig. 1A). To address the
cytosine modification levels in these cell lines, cells were immu-
nostained with antibodies against 5mC and 5hmC, 5fC or 5caC
(Fig. 1B and Supplementary Fig. 1B). Levels of 5hmC, 5fC or 5caC
were measured and normalized against the respective normalized
5mC levels to compensate for the epigenetic heterogeneity in
cancer cells23. As expected, MCF7 cells showed significantly lower
levels of 5mC in heterochromatin, compared to MCF10a cells.
Normalized 5mC levels in pericentric heterochromatin regions of
MCF7 cells were reduced by almost 50% in comparison to MCF10a
cells, but the strong loss of 5mC is also underlined by the ratio of
5hmC to 5mC levels in pericentric heterochromatin. The 5hmC/5mC
ratio is significantly increased in MCF7 cells compared to MCF10a
cells, which is mostly due to the substantial 5mC decrease. In con-
trast, levels of 5hmC, not so much 5fC, but especially 5caC were
elevated in MCF7 cells (Fig. 1C), which is in line with previous
findings in breast cancer24. Supplementary Fig. 1C shows repre-
sentative image s of cytosine modifications immunostaining in these
cell lines. These results were reproduced using a different immu-
nostaining protocol for detecting DNA modifications (Supplemen-
tary Fig. 1D).
The levels of the different oxidative 5mC derivatives were
previously found to peak at the end of S-phase similar to 5mC25,26
and in addition, Tet1 has been implicated as a maintenance DNA
demethylase that prevents aberrant methylation spreading15 .These
findings and our just described observations prompted us to
investigate the subnuclear localization of Tet1 and its short iso-
form, particularly during ongoing DNA replication in S-phase. Due
to the different expression levels of Tet1 and Tet1s in the two cell
types and to avoid fixation artifacts, we made use of live-cell
microscopy of fluorescently tagged variants of Tet1 as well as PCNA
(proliferating cell nuclear antigen) as a marker for S-phase sub-
stages. PCNA is the DNA polymerase processivity clamp and shows
distinct subnuclear patterns during the replication of different
chromatin domains. While replication of euchromatic domains is
characterized by many small, homogeneously distributed sub-
nuclear puncta, larger PCNA clusters are observed during hetero-
chromatin replication27,28. We, therefore, compared cells with PCNA
patterns that could clearly be assigned to DNA replication of
euchromatin or heterochromatin domains, hence to early and late
S-phase, respectively. In doing so, we found that neither full-length
Tet1 nor Tet1s showed recruitment during euchromatin replication
in either cell type (Fig. 1D, E). The accumulation of TET1 or TET1s in
replicating heterochromatin was quantified by measuring the
respective TET1-X levels and dividing it by the levels in the
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 2
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nucleoplasm. Furthermore, TET1 did not show any noteworthy
accumulations in sites of ongoing DNA replication in hetero-
chromatin. Of note, TET1s showed a clear overlap with PCNA at late
S-phase sites as verified by line-profile colocalization analyses
(Fig. 1E), indicating a r ecruitment during the replication of 5mC- rich
heterochromatin.
Tet1s oxidation of heterochromatin increases LINE 1 DNA repeat
activation
Next, we tested the impact of TET1s recruitment to replicating hetero-
chromatin regions and the consequences of 5hmC increase in these
genomic loci. Euchromatin and heterochromatin are differentially
populated by interspersed repetitive DNA sequences29–31. Alu repetitive
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 3
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DNA elements are among the most abundant SINEs (short interspersed
nuclear elements). Alu are located mostly in gene-rich euchromatin,
whereas retrotransposon-related long elements (LINEs) are located
mostly in heterochromatic regions32–34. They are preferentially found at
AT-rich and gene poor regions, corresponding to G-bands and DAPI-
bright bands of metaphase chromosomes. Here, we focused on the LINE
1 (L1) element, and in particular, one of the products of its transcription
and translation, the ORF1 protein (ORF1p)35,36. We aimed to determine
whether TET1s-mediated 5hmC formation at heterochromatic regions
leads to reactivation of LINE 137. For this, we analyzed the levels of TET1s
and LINE 1 ORF1p by immunofluorescence using high-content micro-
scopy. Briefly, MCF10a and MCF7 cells were synchronized and fixed at
late S phase. Then, cells were immunostained for LINE 1 ORF1p and
compared with non-synchronized cells. While in MCF7 cells higher
expression of TET1/TET1s corresponded to a significantincreaseinLINE
1 ORF1p levels, they were very low for MCF10a cells (Fig. 1F). Accord-
ingly, LINE 1 ORF1p formation was increased in MCF7 cells and was even
higher in S phase synchronized cells. Nonetheless, MCF10a cells showed
lower ORF1p levels independently of the cell cycle stage. Representative
images from confocal microscopy are shown in Fig. 1GandSupple-
mentary Fig. 1E, where a cytoplasmic distribution for LINE 1 ORF1p is
observed. ORF1 proteins bind to their own RNA in the cytosol to form a
ribonucleoprotein particle (RNP), which facilitates the re-import of LINE
1 RNA to the nucleus36. For this reason, cytoplasmic levels were used in
our analysis. Additionally, representative images showing the nuclear
distribution of endogenous TET1/TET1s in MCF7 cells are shown in
Fig. 1G. TET1/TET1s accumulation and colocalization with PCNA was
visible during late S phase in dense DAPI regions, while MCF10a cells
showedverylowlevelsofendogenousTET1/TET1sproteins(Supple-
mentary Fig. 1E). In addition, using immunofluorescence staining and
high-content microscopy we verified that the levels of endogenous
TET1/TET1s did not change more than 0.24% during the cell cycle
(Supplementary Fig. 1F). This indicates that the biological effects of
TET1s on 5mC oxidation and LINE 1 activation are not a reflection of a
protein level change throughout the cell cycle but are a reflection of its
subnuclear recruitment to heterochromatin during late S phase.
Tet1 heterochromatin association maps to the catalytic domain
To investigate the differences in the subnuclear S-phase dynamics of
Tet proteins in more detail, we selected C2C12 mouse myoblasts as
model system, as their S-phase behavior and substage replication
patterns are clearly distinguishable and have been extensively
studied38,39. Also importantly, C2C12 cells, in their undifferentiated
state, show low levels of all three Tet proteins40 as well as Mbd
proteins41, which we have previously shown to counteract Tet catalytic
activity42–44.
To avoid secondary effects from prolonged Tet overexpression and
concomitant 5mC oxidation, cells were subjected to live-cell time lapse
microscopy 8 h post-transfection. Live-cell imaging was initially chosen
for protein accumulation analysis, as the observed localization of Tet1s
was partially lost upon fixation (Supplementary Fig. 2A). Ectopically
overexpressing Tet1 or Tet1s together with PCNA as a marker for
S-phaseprogression,wefoundTet1s,but not Tet1, to associate with sites
of ongoing DNA replication in pericentric heterochromatin during late
S-phase analogous to our results in human cells (Fig. 2A). Moreover, the
observed recruitment of Tet1s was exclusively found in this substage of
S-phase, hence, no accumulation during the replication of euchromatin
in early S-phase or in G2 were observed (Fig. 2A, Supplementary
Movies 1, 2). The accumulation of Tet1s was also measured using a
marker for heterochromatic regions, H3K9me345,togetherwithPCNA.
C2C12 cells were transfected with Tet1s or Tet1 and immunostained
against H3K9me3 and PCNA. Confocal microscopy Z-stacks were taken
of cells in early and late S phase and then analyzed for colocalization
between Tet1-X and H3K9me3 using Fiji (Coloc2 plugging). After the
analysis, Pearson coefficient values were obtained, with Tet1s showing
values around 0.5 during late S phase, values close to 0 (no correlation)
during early S phase, and Tet1 showing values around 0 or negative
(anti-correlation) in both early or late S phase (Fig. 2B). Additionally, the
accumulation of Tet1 or Tet1s in PCNA marked heterochromatin was
quantified as described for MCF cells (Fig. 2C). To verify the biological
significance of C2C12 cells as a model system, we measured Tet1 levels in
MCF7, MCF10a, C2C12 and C2C12 cells overexpressing different Tet1
constructs (Tet1-X). To this end, cells were immunostained against Tet1
and the respective sum nuclear Tet1 intensities normalized to the
average sum intensity in non-transfected C2C12 cells (Fig. 2DandSup-
plementary Fig. 2B). We validated that the anti-Tet1 antibody used reacts
with both mouse and human proteins equally (Supplementary Fig. 2E).
Transfected C2C12 cells were grouped according to their GFP or
mcherry levels and plotted as low, mid and high overexpressing groups
(Supplementary Fig. 2B, C). Non-transfected C2C12 cells showed the
lowest Tet1 levels, while transfected C2C12 cells from the high over-
expressing group showed the highest level. Tet1-X mid-overexpressing
cells (group selected for most of the analyses) showed levels similar to
MCF7 cells (Fig. 2D and Supplementary Fig. 2D), indicating that the
selected ectopic expression levels in C2C12 cells emulate the endo-
genous Tet1 level in these cells. Our results furthermore imply that
C2C12 cells are de facto phenotypically Tet1 negative, while low over-
expression levels correspond to the non-tumor human cell line MCF10a.
In previous reports, it was demonstrated that the C-terminal cat-
alytic domains of all three Tet family members are sufficient for their
catalytic activity9and that they share high sequence similarities10.
Hence, besides the full-length proteins, we also investigated the sub-
nuclear localization of the respective catalytic domains (TetX-CD)
during the replication of heterochromatin by live-cell microscopy. In
doing so, we found that neither Tet2 nor Tet3 nor their respective
catalytic domains showed any noteworthy accumulations in late
S-phase (Fig. 2F), whereas Tet1-CD showed a strong accumulation at
replicating pericentric heterochromatin in late S-phase (Fig. 2E). In
order to verify the observed Tet1-CD accumulation by other methods,
chromatin immunoprecipitation experiments (ChIP) followed by qPCR
Fig. 1 | Breast cancer cells show aberrant 5mC oxidation and increased LINE 1
ORF1p level. A Gene structure of the human TET1 locus. Non-coding exons 1 and 3
harbor different transcription start sites, targeted by a different promoter. Trans-
lation starts in exon 2 (TET1) and 4 (TET1s). TET1 protein functional domains are
indicated. Graphical representation of cytosine modifications and their main-
tenance during the cell cycle. BFiji-based in situ cytosine modifications analysis
procedure. MCF cells were immunostained and imaged using confocal microscopy.
Binary nuclear and heterochromatin masks were generated based on DNA signals
(DAPI). Mean fluorescence intensities in the respective areas were measured.
CBoxplots showing normalized mean intensity of cytosine modifications at het-
erochromatin regions. n(5mC) = 23 (MCF10a) - 17 (MCF7), n(5hmC) = 19 (MCF10a ) -
20 (MCF7), n(5fC) = 21 (MCF10a) - 17 (MCF7), n(5caC) = 17 (MCF10a) - 16 (MCF7)
cells. D,ELive-cell analysis of Tet1 and Tet1s subnuclear localization: confocal
images of MCF10a and MCF7 cells expressing EGFP-Tet1s/EGFP-Tet1 and mRFP-
PCNA 8 h post-transfection. Colocalization of Tet1-X with PCNA was examined by
line-profile analysis and relative protein accumulation in early and late S-phase. n
(TET1) = 14 (MCF10a) - 13 (MCF7), n(Tet1s) = 16 (MCF10a) - 17 (MCF7) cells.
FSynchronized MCF cells immunostained against LINE 1 ORF1p. Cytoplasmic
fluorescence mean intensity levels were plotted. Representativeconfocal images for
these immunostainings, including endogenous TET1/TET1s, are shown in G.Forall
boxplots, the box represents 50% of the data, starting in the first quartile (25%) and
ending in the third (75%). The line inside represents the median. The whiskers
represent the upper and lower quartile. Statistical significance was tested with a
paired two-samples Wilcoxon test (n.s. not significant, is given for p-values ≥0.05;
one star (*) for p-values < 0.05 and ≥0.005; two stars (**) is given for values < 0.005
and ≥0.0005; three stars (***) is given for values < 0.0005). N-numbers and p-values
are shown in Supplementary Data 1. Source data are provided as a Source Data file.
Scale bars = 5 µm.
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 4
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of major satellite repeats (MajSat) were performed. GFP-Tet1-CD
binding to these genomic regions was immunoprecipitated using the
GBP beads. GFP and GFP-MeCP2 (methyl-CpG binding protein 2)46
were used as negative and positive controls, respectively, and GFP-
input fraction was used for normalization in all samples. qPCR using
primers for MajSat showed amplification of these sequences for Tet1-
CD pulldown in cells synchronized in late S phase in a similar level to
MeCP2, while no amplification was found for GFP and Tet1. Interest-
ingly, MeCP2 binding fraction at late S phase showed slightly lower
levels of amplification for MajSat repeats, corresponding to a slight
displacement of this protein during replication (Fig. 2G)47.Similar
levels of amplification were found for the input fraction in all samples.
Article https://doi.org/10.1038/s41467-022-32799-8
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Based onthe increased levels of 5mC oxidation products incancer
cells that express Tet1s and on our previous findings that Tet1-CD
overexpression results in a significant increase of 5mC oxidation37,43,44,
we addressed the catalytic activity of Tet1s compared to full-length
Tet1. For this purpose, we overexpressed fluorescently tagged Tet1 or
Tet1s constructs or GFP alone in C2C12 cells and 24 h later immunos-
tained them against 5hmC. While only cells that overexpressed high
levels of full-length Tet1 showed a significant global increase of 5hmC
compared to the GFP control, already low expression levels of Tet1s
resulted in a significant increase compared to the GFP control or Tet1
(Fig. 3A). We furthermore aimed to investigate whether the over-
expression of Tet1s in C2C12 cells had similar effects as those observed
in MCF7 cells with higher levels of TET1 proteins. For this purpose, we
performed immunofluorescence against LINE 1 ORF1p in C2C12 cells
comparing Tet1s transfected versus non-transfected cells after syn-
chronization in S phase. Representative images of these immuno-
fluorescence signals are shown in Fig. 3C. Grouping the cells according
to their GFP-Tet1s levels as we described before (Supplementary
Fig. 2B), we found an increase in the LINE 1 ORFp levels after Tet1s
overexpression compared with non-transfected cells. This increase
was proportional to the level of Tet1s and overall higher in late S-phase
synchronized cells (Fig. 3B).
The zinc finger domain of Tet1 impedes DNA replication asso-
ciation and prevents aberrant 5mC oxidation
One major functional domain that sets apart Tet1 and Tet1s is the
N-terminal zinc finger (ZF) domain only present in the full-length
protein (Fig. 1A). The ZF-domain has before been implicated in tar-
geting Tet1 to non-methylated chromatin14. Besides the ZF-domain one
additional regulatory domain in the very N-terminus was recently
identified and termed BC (before zinc finger) domain16. We, therefore,
tested the subnuclear localization of different N-terminal deletion
mutants of Tet1 and also its ZF-domain alone during late S-phase.
Hence, C2C12 cells were transfected with mRFP-PCNA and constructs
encoding GFP-tagged Tet1Δ1-389 lacking the BC-domain, Tet1Δ566-
833 lacking the ZF-domain or Tet1-ZF, the zinc finger domain alone.
Cells in late S-phase were imaged live 8 h post-transfection and the
relative Tet1 accumulation at PCNA foci of late S-phase cells was
quantified as described above. While Tet1 constructs containing the
zinc finger domain showed a very homogeneous nuclear pattern and
no accumulation, Tet1Δ566-833 showed a significantly increased
accumulation (Fig. 4A). Based on this finding, we investigated the
subnuclear localization of a synthetic construct, we termed Tet1-ZF-
CD, composed of the Tet1 zinc finger domain fused to Tet1-CD. In
contrast to Tet1-CD, the minimal catalytically active part of Tet1
recruited to replicating heterochromatin (Fig. 2E), Tet1-ZF-CD did not
localize to sites of ongoing DNA replication in heterochromatin
(Fig. 4B). We continued to investigate differences in the catalytic
activity of Tet1-CD and Tet1-ZF-CD. For this purpose, C2C12 were
transfected with either of the two constructs and 24h later, 5hmC
levels were detected by immunostaining. Tet1-CD increased 5hmC
significantly more than Tet1-ZF-CD, independent of the respective
overexpression levels (Fig. 4B, bottom). Analyzing cells that over-
expressed either construct, we found the 5hmC staining pattern to be
markedly different. While the 5hmC signal in cells transfected with
Tet1-CD overlapped with DAPI-dense regions, hence, pericentric het-
erochromatin, Tet1-ZF-CD overexpressing cells showed only small
punctated signals outside of pericentric heterochromatin (Fig. 4C).
We, therefore, analyzed the 5hmC levels in heterochromatin, by
masking the cells based on their DAPI signal. Again, Tet1-CD trans-
fected cells showed a significant increase of 5hmC compared to Tet1-
ZF-CD overexpressing cells (Fig. 4C, bottom). Dotplots for Fig. 4B, C
are shown in Supplementary Fig. 3A, B. Based on these findings, we
investigated differences in mobility and DNA binding kinetics between
Tet1-CD and Tet1-ZF-CD since the zinc fingerdomain facilitates binding
and its deletion results in decreased chromatin loading16. To this end,
we performed fluorescence recovery after photobleaching (FRAP)
experiments in C2C12 transfected with Tet1-CD or Tet1-ZF-CD
(Fig. 4D). Eight hours after transfection, FRAP measurements were
performed by selecting cells with a homogeneous nuclear distribution
of these proteins. Representative images of FRAP experiments are
shown in Supplementary Fig. 3C. Compared with freely nuclear dis-
tributed Tet1-CD, Tet1-ZF-CD had notably slower recovery kinetics,
thus, a decreased mobility indicated by a significantly increased half-
time (Fig. 4E).
S-phase localization of Tet1s is independent of its catalytic
activity and substrate
To further test, whether the observed localization ofTet1-CD depends
on its catalytic activity, we addressed the localization of a catalytically
dead mutant (Tet1-CDmut), which is still able to bind its substrate but
cannot oxidize it due to point mutations in its cofactor binding sites
(H1652Y, D1654A)44. Albeit being catalytically impaired, Tet1-CDmut
localized to late-replicating heterochromatin like the catalytically
active Tet1-CD (Fig. 2E). Thus, catalytic activity is dispensable for this
localization.
To furthermore exclude substrate-dependent localization, we
tested whether the observed localization of Tet1 depends on 5mC
abundance and how the loss of 5mC can affect the observed accu-
mulation at replicating heterochromatin. For this purpose, we made
use of cells that are deficient for the maintenance methyltransferase
Dnmt1, which colocalizes with PCNA during DNA replication in peri-
centric heterochromatin48. Mouse embryonic fibroblasts deficient for
Dnmt1, were also deficient for p53 (MEF-PM), as primary fibroblasts
Fig. 2 | Tet1s localizes at heterochromatin during DNA replication. A Live-cell
microscopy images of C2C12 cells expressing mRFP-PCNA as DNA replication
marker andEGFP-Tet1-X. Cells showing an early S-phase pattern were imaged every
20 min. Time points in early, late S and G2 phases are shown. BImmunostaini ng
againstPCNA, H3K9me3 (as heterochromatin marker) and Tet1-X. C2C12 cells were
transfected, immunostained, and imaged in S-phase. Pearson coefficient values
(colocalization) were plotted for early versus late S-phase cells. CTet1-X accumu-
lation analysis in late S-phase cells. Mean fluorescence intensities of three nuclear
areas inside and outside of DNA replication sites were measured and averaged.
Mean fluorescence intensities of Tet1-X in PCNA foci were divided by the mean
fluorescence in nucleoplasmic regions. Boxplot depicts the results of quantifica-
tion. DImmunostaining against Tet1/Tet1s inMCF10a, MCF7 and C2C12 cells. Sum
nuclear levels of Tet1/Tet1s and mean nuclear levels of EGFP were measured by
wide-field high-contentmicroscopy. TransfectedC2C12 cells were grouped by their
mean EGFP fluorescence (low, mid, high) (See Supplementary Fig. 2B, C). Boxplot
shows Sum nuclear Tet1/Tet1s normalized by the average sum nuclear intensity in
non-transfected C2C12. ERepresentative images of transfected C2C12 cells (mRFP-
PCNA and EGFP-Tet1-CD/Tet1-CDmut). Boxplot shows Tet1-X relative accumula-
tion. FLate S-phase C2C12 cells transfected with mRFP-PCNA and EGFP-Tet2/Tet3
proteins or respective catalytic domains. Boxplots show relative accumulation of
Tet-X at replicating heterochromatin. GChromatin immunoprecipitation (ChIP)
followed by qPCR for MajSat sequences. C2C12 cells were transfected with Tet1-X
and MeCP2 (positive control) and synchronized in G1/late S-phase. Barplots show
the average value of amplification levels in input and chromatin binding fractions
normalized to the GFP-input (red line). The whiskers represent the standard
deviation with a 95% confidence interval. For allboxplots, the box represents 50%of
the data, starting in the first quartile (25%) and ending in the third (75%). The line
inside representsthe median. The whiskers represent theupper and lower quartile.
Statistical significancewas tested with a pairedtwo-samples Wilcoxon test (n.s., not
significant, is given for p-values ≥0.05; one star (*) for p-values < 0.05 and ≥0.005;
two stars (**) is given for values < 0.005 and ≥0.0005; three stars (***) is given for
values < 0.0005). N-numbers and p-values are shown in Supplementary Data 1.
Source data are provided as a Source Data file. Scale bar = 5 μm.
Article https://doi.org/10.1038/s41467-022-32799-8
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from Dnmt1 negative and p53 proficient embryos proved nonviable49.
Cells deficient for Dnmt1, are characterized by global
hypomethylation48,49, and retain only residual levels of 5mC in major
satellite repeats, which is accompanied by decondensed pericentric
heterochromatin50. This makes them a suitable model to study the
effects of 5mC depletion on Tet1s localization during ongoing DNA
replication. To this end, we co-transfected MEF-PM and MEF control
cells, with mcherry-Tet1s, miRFP-PCNA and EGFP-MaSat, a synthetic
polydactyl zinc finger protein51 binding to pericentric, major satellite
containing, heterochromatinand imaged thecells live approximately 8
h later, to assess the localization of Tet1s during late S-phase. The
control cells, and also MEF-PM, showed a clear accumulation of Tet1s
at PCNA labeled pericentricheterochromatin (Supplementary Fig. 4A),
indicating that the global loss of DNA methylation does not affect the
association of Tet1s with sites of ongoing DNA replication in
heterochromatin.
S-phase localization of Tet1s is dependent on Uhrf1 via its DNA
binding domain but not its E3-ligase activity
Next, we addressed whether the loss of the accessory protein Uhrf1,
which plays an important role in Dnmt1 recruitment for DNA methyla-
tion maintenance (Fig. 5A)48, could affect Tet accumulation. The multi-
domain protein Uhrf1 (Fig. 5B) is mostly implicated to serve as a facil-
itating factor for Dnmt1 mediated DNA methylation maintenance. This is
achieved by interpreting the combined information of the DNA
methylation status and different histone modifications in the vicinity of
hemi-methylated CpGs during ongoing DNA replication. This triggers
the E3 ubiquitin ligase activity of Uhrf1 towards lysines in the histone H3
tail, which recruits Dnmt15,7. The loss of Uhrf1 is accompanied by severe
global hypomethylation, similar to the loss of Dnmt1. As loss of Uhrf1 is
eventually lethal during embryonic development and differentiation,
the effects of Uhrf1 deficiency were tested in mouse embryonic stem
cells (ESC)48,52,specifically in E14 mouse embryonic stem cells lacking
Uhrf1 (Uhrf1−/−). Wild-type and Uhrf1-deficient cells were transfected with
mcherry-Tet1s, miRFP-PCNA and EGFP-MaSat and imaged live approxi-
mately 10 h post-transfection. While E14 wild-type cells showed a clear
colocalization of Tet1s, PCNA and MaSat, no accumulation of Tet1s was
observed in Uhrf1-deficient E14 cells, at PCNA marked heterochromatin,
i.e. during replication (Fig. 5C). To map interactions of Uhrf1 and Tet1-
CD, co-immunoprecipitation experiments were performed. To this end,
GFP-tagged wild-type Uhrf1 or five different constructs with single
domains of Uhrf1 were co-overexpressed together with mcherry-Tet1-
CD in HEK293-EBNA cells. Immunoprecipitation was performed with a
GFP-binding nanobody (GBP)53 and analyzed by western blotting with
antibodies against GFP and RFP. In doing so, we found wild-type Uhrf1
andalsotheSRAortheRINGdomainalonetobeabletopulldownTet1-
CD (Fig. 5D).
To test whether Tet1s localization at heterochromatin could be
rescued in Uhrf1-deficient E14 ESCs, the cells were transfected with
GFP, GFP-Uhrf1 or GFP-Uhrf2 together with mcherry-Tet1s and miRFP-
Fig. 3 | C2C12 cells overexpressing Tet1s show aberrant 5mC oxidation and
higher levels of LINE 1 ORF1p. A Cells transfected with EGFP-Tet1, EGFP-Tet1s or
EGFP alone, were immunostained against 5hmC. Fluorescence intensity levels of
overexpressed proteins and 5hmC were measured, sum nuclear 5hmC levels were
normalized to the sum nuclear DAPI intensity and grouped as described in Sup-
plementary Fig. 2B,D. BC2C12 cells non-transfectedand transfectedwith GFP-Tet1s
were synchronized and immunostained against LINE 1 ORF1 protein. Fluorescence
intensity levels of the protein were measured and mean cytoplasmic levels were
plotted. Representative confocal images for LINE 1 ORF1p and TET1/TET1s
immunostainings are shown in C. For all boxplots, the box represents 50% of the
data, starting in the first quartile (25%) and ending in the third (75%). The line insi de
represents the median. The whiskers represent the upper and lower quartile. Sta-
tistical significance was tested with a paired two-samples Wilcoxon test (n.s. not
significant, is given for p-values ≥0.05; one star (*) for p-values < 0.05 and ≥0.005;
two stars (**) is given for values < 0.005 and ≥0.0005; three stars (***) is given for
values < 0.0005). N-numbers and p-values are shown in Supplementary Data 1.
Source data are provided as a Source Data file. Scale bar = 5 μm.
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PCNA (Fig. 5E). Cells with a heterochromatic replication pattern were
imaged approximately 14h after transfection and the accumulation of
Tet1s in PCNA foci was scored. For this, three regions inside and out-
side of PCNA foci were chosen, the respective mcherry-Tet1s levels
were measured and the signal in heterochromatin was divided by the
nucleoplasmic signal. E14 Uhrf1−/−transfected with GFP as control
showed nearly no elevated Tet1s levels at replicating heterochromatin.
In contrast, expression of Uhrf1, but not Uhrf2, could rescue the
localization of Tet1s and resulted in a significant accumulation in het-
erochromatin (Fig. 5E-left and Supplementary Fig. 4B). In addition, we
tested if any of the single domain deletion mutants of Uhrf1 was able to
rescue Tet1s localization. Interestingly, from the two domains that
were able to pull down Tet1-CD (SRA and RING domain) the deletion of
the RING domain (ΔRING), did not affect the rescue of the Tet1s
accumulation at late-replicating heterochromatin, while deletion of
the SRA domain (ΔSRA) resulted in levels of accumulation similar to
E14 Uhrf1−/−transfected with GFP (Fig. 5E-middle). On the other hand,
E14 Uhrf1−/−cells transfected only with the SRA domain showed levels
of Tet1s accumulation similar to E14 wild type (Fig. 5E-right and Sup-
plementary Fig. 4C). Hence, the association of the SRA domain of Uhrf1
with heterochromatin is sufficient to target Tet1-CD to these regions.
To better characterize the Tet1s-Uhrf1 interaction, we performed
immunofluorescence against PCNA and Uhrf1 in C2C12 cells trans-
fected with EGFP-Tet1s. Interestingly, a clear association of Uhrf1 to
replicating DNA was found from early to late S-phase, and cells showed
a clear colocalization between PCNA, Tet1s and Uhrf1 at replicating
heterochromatin. Additionally, both PCNA and Uhrf1 patterns looked
similar: Uhrf1 signal matched with replication foci during S-phase,
while a homogeneous nuclear distribution was found in non-S-phase
cells (Fig. 5F). Next, we made use of a fluorescent three-hybrid assay
Fig. 4 | The zinc finger domain of Tet1 impedes S-phase dependent hetero-
chromatin association and prevents aberrant 5mC oxidation. A Domain orga-
nization of Tet1 with locations of amino acids corresponding to N-terminal deletion
mutants. Representative images of C2C12 cells in late S-phase expressing EGFP-
tagged Tet1/Tet1Δ1-389/Tet1Δ566−833/Tet1-ZF domain and mRFP-PCNA. Boxplot
shows quantification of relative accumulation. BC2C12 cells were transfected with
mcherry-Tet1CD/Tet1-CD-ZF and EGFP-PCNA. Representative images of cells in late
S-phase are shown. In situ 5hmC levels were analyzed 24 h after transfection by
wide-field high-content microscopy. Boxplots show sum nuclear 5hmC levels nor-
malized by averaged levels in non-transfected cells and sum nuclear DAPI. Cells
were grouped by their mean mcherry fluorescence intensities into low, mid, and
high expressing (see Supplementary Fig. 2B, C). CRepresentative images of C2C12
from 4B. Selected ROIs in pericentric heterochromatic regions were magnified. Fiji-
based in situ 5hmC quantification was performed and results are shown in boxplot
(5hmC levels in pericentric heterochromatin in dark-gray). DFRAP experiments
scheme in cells expressing mcherry-tagged Tet1-CD/Tet1-CD-ZF. The mcherry signal
was photobleached with a 561 nm laser and recovery of fluorescence was followed
by time lapse microscopy. EFluorescence recovery curves and T-half times were
calculated using easyFRAP. Line plots show normalized averaged fluorescence
recovery values, and error bands show the respective standard deviation. 95%
confidence intervals are indicated in the plot. Representative images are shown in
Supplementary Fig. 3C. For all boxplots, the box represents 50% of the data, starting
in the first quartile (25%) and ending in the third (75%). The line inside represents the
median. The whiskers represent the upper and lower quartile. Statistical significance
was tested with a paired two-samples Wilcoxon test (n.s. not significant, is given for
p-values ≥0.05; one star (*) for p-values < 0.05 and ≥0.005; two stars (**) is given for
values < 0.005 and ≥0.0005; three stars (***) is given for values < 0.0005).
N-numbers and p-values are shown on Supplementary Data 1. Source data are
provided as a Source Data file. Scale bar = 5 μm. White scale bar = 2.5 μm.
Article https://doi.org/10.1038/s41467-022-32799-8
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(F3H) to further analyze the timing of the interaction between Tet1-CD
and Uhrf1_SRA during S-phase. In this assay, the major satellite
recognizing zinc finger protein described before, fused to a GFP-
binding nanobody (GBP-MaSat)50 is co-transfected with a GFP-fusion
protein as bait and a differently tagged protein as prey. GFP or GFP-
fusion proteins are tethered to pericentric heterochromatin and a
colocalization with the prey protein is observed in case of protein-
protein interactions. If a cell-cycle independent protein-protein inter-
action is observed, this colocalization persists throughout the differ-
ent S-phase and non-S-phase stages, as it was reported, for example,
for Mbd143. Hence, we co-transfected miRFP-PCNA as a cell cycle
S-phase marker together with mcherry-Tet1-CD and GFP-Uhrf1_SRA
with or without GBP-MaSat to anchor the GFP at pericentric hetero-
chromatin. GFP-positive cells with S-phase patterns were imaged live
8 h post-transfection. While in cells with a GBP-MaSat mediated tar-
geting resulted in a colocalization of mcherry-Tet1-CD with GFP-
Uhrf1_SRA in 20% of scored early S-phase cells, we observed an
increase to 95% during late S-phase. Cells without GBP-MaSat showed
colocalization only during late S-phase (Fig. 5G). Taken together, this
in vivo interaction assay suggests an S-phase substage dependent Tet1-
CD/Uhrf1 interaction since a cell-cycle dependent protein-protein
interaction is observed with colocalization observed only during late
S-phase. Tethering Uhrf1 to pericentric heterochromatin could not
initiate a premature Tet1-CD recruitment before late S-phase, which
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furthermore hinted to a replication dependent recruitment mechan-
ism for Tet1s.
We next investigated the mechanism underlying the requirement
of the E3 ubiquitin-protein ligase Uhrf1 for Tet1s recruitment to repli-
cating heterochromatin. Since it was shown that overexpression of
Uhrf1 enhances ubiquitinationlevels of Dnmt1 in vivo54 and twostudies
identified monoubiquitination of a conserved lysine residue withinthe
CRD of all three Tet proteins by the CRL4(VprBP) E3-ligase complexto
modulate their catalytic activity and chromatin association19,20,we
tested Tet1 ubiquitination levels in the E14 wild type cells versus E14
Uhrf1−/−. For this purpose, we transfected E14 cells with GFP-HA-
ubiquitin or GFP alone as negative control. Immunoprecipitation was
performed with GFP-binding nanobody (GBP) and analyzed by western
blotting with antibodies against GFP (negative control) and Tet1/Tet1s.
We could pull downTet1 and also Tet1s from wild-type E14 and also E14
Uhrf1−/−cells (Fig. 5H). This shows that, while Tet1s recruitment to
replicating heterochromatin is abrogated in E14 Uhrf1−/−cells, ubiqui-
tination of Tet1s still takes place. Similar results were reproduced in
HEK293-EBNA cells co-transfected with GFP-HA-ubiquitin and
mcherry-Uhrf1 and compared with cells only transfected with GFP-HA-
ubiquitin. Overexpression of Uhrf1 was found to increase ubiquitina-
tion of Dnmt1 but did not increase ubiquitination of Tet1/Tet1s (Sup-
plementary Fig. 4D). As we found Tet1s recruitment to sites of late DNA
replication to be unaffected by DNA hypomethylation but affected by
loss of Uhrf1, which showed a clear replication pattern in immuno-
fluorescence (Fig. 5F), we deemed replication association to be more
important than heterochromatin binding.
Tet1s recruitment to heterochromatin is dependent on DNA
replication and its CRD but not on its PCNA binding domain
In order to test if replication association was more important than
heterochromatin binding, we made use of a system we developed
before, based on the decoupling of the replisome via reversible
inhibition of eukaryotic DNA polymerases by aphidicolin
treatment55,56. The polymerase inhibition via aphidicolin leads to the
disassembly of proteins involved in DNA replication elongation like
PCNA55,57. C2C12 cells were transfected with mcherry-Tet1-CD and
miRFP-PCNA. As control, Z-stacks of the cells were acquired before
adding aphidicolin or DMSO, and after the addition of aphidicolin
cells were imaged every 5 min over a period of 30 min (Supple-
mentary Fig. 4E). Already after 5 min of drug treatment, a clear
reduction of Tet1-CD accumulation and PCNA dissociation from
replicating heterochromatin was observed, and levels of Tet1-CD
relative accumulation at the different time points were measured as
was explained before. This observation confirmed that Tet1-CD
recruitment to heterochromatin during late S-phase occurs in a
replication dependent manner.
Many proteins that associate with sites of ongoing DNA replica-
tion, do so via the interaction with the clamp loader protein PCNA.
Dnmt1, for example, harbors a so-called PCNA binding domain (PBD), a
short peptide with a conserved sequence motif that facilitates this
interaction. The consensus sequence of classical PBDs is characterized
by an initial glutamine (Q), followed by two variable amino acids, a
hydrophobic amino acid, like leucine (L) or isoleucine (I), two variable
residues and finally two aromatic amino acids, like phenylalanine (F),
tryptophan (W),tyrosine (Y) or histidine (H). This consensus sequence
is often followed by basic residues like arginine (R) or lysine (K)58
(Supplementary Fig. 4F). Interestingly, PCNA was reported to be an
interactor of Tet1 and to regulate its dioxygenase activity throughout
the cell cycle and, thereby, protect cells from aberrant DNA
methylation59. To identify a putative PBD in Tet1, and potentially also in
Tet2 and Tet3, the respective amino acids sequences were screened for
the just described consensus sequence. While there were nohits in the
sequences of Tet2 or Tet3, a short peptide that harbors a similar
sequence was identified in the CRD of Tet1, we termed Tet1 putative
PBD (Tet1-pPBD). However, a GFP-tagged Tet1-pPBD showed no
accumulation at sites of ongoing DNA replication (Supplementary
Fig. 4F). As Tet1-CD is the minimal catalytically active part of Tet1 that
localizes to replicating heterochromatin, we sought to investigate the
localization of the Tet1-CD main domains, the CRD and the DSBH. For
this, we overexpressed GFP-tagged Tet1-CRD or Tet1-DSBH together
with RFP-PCNA in C2C12 cells and quantified their accumulation in
heterochromatic PCNA foci as described above. Here, we found a
slight enrichment of the CRD but not the DSBH (Supplementary
Fig. 5A), while neither domain alone was able to increase 5hmC levels
(Supplementary Fig. 5B). This led us to delete the CRD from Tet1s and
investigate the S-phase localization of the resulting deletion mutant.
The respective fusion protein showed a clear nuclear signal but no
S-phase accumulation (Supplementary Fig. 5C). Additionally, no
increased 5hmC formation was observed, when overexpressing Tet1s-
ΔCRD (Supplementary Fig. 5D). These findings match with structural
data of Tet2, showing that the CRD and DSBH together form a compact
catalytic core60 to facilitate the dioxygenase activity of Tet proteins.
A conserved lysine in Tet1 is crucial for its S-phase hetero-
chromatin localization and targeted catalytic activity
In contrast to the putative but non-functional PBD that isonly found in
Tet1, all three Tet proteins share a conserved lysine residue in their
CRD, which was shown to become monoubiquitinated in a
CRL4(VprBP) dependent manner. The lysine itself resides within a
short peptide that is conserved between all three Tet protein family
membersand also between human and mouse Tet (Fig. 6A). This short
amino acid stretch was shown to stabilize the DNA around the mod-
ified cytosine target, by interacting with the phosphate backbone60
Fig. 5 | Uhrf1 physically interacts with Tet1 and is required for its S-phase
localization. A DNA methylation maintenance throughout the cell cycle: Dnmt1 is
recruited to sites of ongoing DNA replication by Uhrf1 and PCNA, ensuring faithful
inheritance of the DNA methylome. Tet1s is recruited to heterochromatin during
late S-phase. BDomain organization of Uhrf1 and Uhrf2: ubiquitin-like (UBL)
domain, histone modifications binding tandem tudor domain (TTD) and plant
homeodomain (PHD),DNA interacting SET and RING associated (SRA) domain and
the really interesting new gene (RING) domain. Sequence homology is shown in
percentage. CRepresentative images of wild-type E14 mouse embryonic stem cells
or Uhrf1-deficient cells (E14 Uhrf1−/−) expressing mcherry-Tet1s, miRFP-PCNA and
EGFP-MaSat. DHEK293-EBNA cells were transfected with EGFP/EGFP-Uhrf1 and
mcherry-Tet1CD.Cell extracts were analyzed by immunoprecipitation using a GFP-
binding nanobody and western blotting. Cut-outs show the bound GFP-fractions
and the input (I) and bound (B) mcherry fractions.EBoxplots depict quantification
of Tet1s accumulation in E14 wildtype and E14−/−co-transfected with PCNA, Tet1s
and either Uhrf1 (left) or different Uhrf1 mutant constructs (mid/right boxplot).
Representative images shown in Supplementary Fig. 4B, C. FUhrf1 and PCNA
immunostaining in C2C12 cells transfected with EGFP-Tet1s. Representativeimages
for 3 independent experiments are shown. GF3H assay in C2C12 transfected with
miRFP-PCNA, mcherry-Tet1-CD, EGFP-Uhrf or EGFP, and GBP-MaSat. Percentage of
cells with Tet1-CD localized at pericentric heterochromatin. HUbiquitination of
Tet1/Tet1s assayed by immunoprecipitation: E14 wildtype and E14−/−were trans-
fected with EGFP or EGFP-HA-tagged ubiquitin, immobilized using GFP-binding
nanobody, and analyzed by western blotting (antibodies against GFP, Tet1/Tet1s
and Dnmt1). The cut-outs show the bound GFP-fractions and the input and bound
Tet1/Tet1s fractions. Two independent experiments were performed. For all box-
plots, the box represents 50% of the data, starting in the first quartile (25%) and
ending in the third (75%). The line inside represents the median. The whiskers
represent the upper and lower quartile. Statistical significance was tested with a
paired two-samples Wilcoxon test (n.s. not significant, is given for p-values ≥0.05;
one star (*) forp-values < 0.05 and ≥0.005; two stars(**) is given for values < 0.005
and ≥0.0005; threestars (***) is given for values< 0.0005). N-numbers andp-values
are shown in Supplementary Data 1. Source data are provided as a Source Data file.
Scale bar = 5 μm.
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(Fig. 6B). Mutations of the respective lysine have been found to result
in a decrease or even loss of catalytic activity in all three Tet proteins
and monoubiquitination of this lysine was found to be important
during oocyte development and for Tet2 chromatin binding, but its
biological consequences have mostly been addressed only for Tet2
and Tet319,20. We therefore mutated lysine (K) 852 in Tet1s to glutamate
(E) or arginine (R) to invert or keep the respective charge and abrogate
a putative ubiquitination. We continued to investigate the DNA repli-
cation association capability of the respective constructs and their
effect on 5hmC generation. Mutating Tet1s lysine 852 to a glutamate
(Tet1s-K852E) or arginine (Tet1s-K852R) destroyed the ability to loca-
lize to sites of ongoing DNA replication in heterochromatic regions
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(Fig. 6C). Interestingly and in contrast to our observations on the
localization, global 5hmC levels were even higher in cells that over-
expressed Tet1s-K852R compared to wild-type Tet1s. Cells transfected
with the glutamate mutant Tet1s-K852E, on the other hand, showed a
very minor 5hmC increase (Fig. 6D). This observation is in line with
data from structural studies on Tet2, where a glutamate at thisposition
disrupted the stabilizing effectof the peptide stretch onthe phosphate
backbone of the DNA60. To analyze subnuclear 5hmC deposition in
more detail, we acquired high magnification Z-stacks and masks were
created to threshold the heterochromatin and the surrounding
nucleoplasm to subsequently measure respective mean fluorescence
intensities (Fig. 6E). While Tet1s transfected cells showed a significant
increase of 5hmC mostly in heterochromatin (Fig. 6F and Supple-
mentary Fig. 5E), 5hmC levels were also significantly elevated in the
nucleoplasm of Tet1s-K852R transfected cells, explaining the stronger
global increase (Fig. 6F). Tet1s-K852E transfection, on the other hand,
resulted only in minor 5hmC depositions in heterochromatin or
nucleoplasm, compared to Tet1s or Tet1s-K852R.In addition, we found
that cells that overexpressed Tet1s showed less compacted pericentric
heterochromatin compared with non-transfected cells or cells trans-
fected with Tet1s-K852E or Tet1s-K852R (Supplementary Fig. 5E). To
quantify this observation, we divided the DAPI-dense pericentric het-
erochromatin area by the total nuclear area and found that Tet1s
transfected cells exhibit higher relative heterochromatin area com-
pared with Tet1s-K852E and Tet1s-K852R transfected cells. This is
accompanied by a decrease in the standard deviation for DAPI inten-
sity values in Tet1s transfected cells due to the decondensation of
highly compact heterochromaticregions (Supplementary Fig. 5F). This
prompted us to perform a detailed quantitative analysis of changes in
chromatin structure. We analyzed the 3D nuclear landscape and the
spatial nuclear DNA organization using confocal microscopy, which
allowed us to assess different chromatin compaction levels (1 to 7) in
individual cell nuclei61. Comparing Tet1 versus Tet1s transfected cells in
late S-phase, we found differences in the distribution of the sub-
compartments that were calculated based on the DAPI intensities. For
Tet1s, an increase in the fraction for lower compaction classes was
observed, together with a reduction in higher compacted classes, in
comparison to Tet1 transfected cell s. Overall, this indicates a r eduction
in the fraction of inactive compartments (compacted core of chro-
matincluster),whichisinlinewithpreviousresultsshowingdecon-
densation of heterochromatic regions (Supplementary Fig. 5F). In
summary, mutating the conserved lysine to arginine leads to increased
levels of 5hmC globally in contrast to Tet1s. Overexpression of the
latter results in increased levels of 5hmC in heterochromatin and
accompanied decondensation of these regions, producing significant
changes in heterochromatin structure.
To verify the lack ofaccumulation at replicating heterochromatin
for Tet1s-K852E and Tet1s-K852R mutants, we performed chromatin
immunoprecipitation followed by qPCR as we described before
(Fig. 2G). Accordingly, qPCR amplification of MajSat sequences was
found for Tet1-CD during late S, while no amplification occurred for
Tet1s-K852E and Tet1s-K852R independently of the cell cycle stage
(Fig. 6G). The differences in the recruitment to replicating hetero-
chromatin and ChIP results, prompted us toinvestigate whether Tet1s-
K852E and Tet1s-K852R DNA binding kinetics are affected. To this end,
we performed FRAP analysis to measure the mobility of Tet1s and Tet1s
lysine mutants. Due to the unique ability of Tet1s to localize at repli-
cating heterochromatin during late S-phase, and to the higher mobility
of Tet1-CD found in previous FRAP experiments (Fig. 4E), we decided
to perform these analyses making a distinction between not late S
(homogeneous nuclear distribution of Tet1s) and late S-phase (when
Tet1s is localized at replicating pericentric heterochromatin). To this
end, we co-transfected C2C12 cells with GFP-Tet1s, GFP-Tet1s-K852E or
Tet1s-K852R together with mRFP-PCNA. Eight hours after transfection,
FRAP measurements were performed by bleaching a region of the
nucleus during not late S, excluding those areas with PCNA replication
foci, and bleaching a heterochromatic region during late S-phase
where PCNA and also Tet1s was located (Fig. 6H). Compared to
homogeneously distributed Tet1s and its lysine mutants during not
late S, heterochromatin accumulated Tet1s in late S-phase showed
much slower recovery kinetics and, thus, decreased mobility. In con-
trast to this, Tet1s-K852E and Tet1s-K852R had similar kinetics inde-
pendently of their S-phase substage with fast recovery kinetics and a
high mobility (Fig. 6I).
The CRL4(VprBP) complex ubiquitinates Tet1s and is needed for
Tet1s S-phase association via Uhrf1 interaction
We next checked the levels of ubiquitination of Tet1s-K852E and Tet1s-
K852R compared with Tet1s, and prospective changes in these levels
by the overexpression of either VprBP or Uhrf1. VprBP (Vpr-Binding
Protein/DDB1 And CUL4 Associated Factor 1) is a serine/threonine
kinase62 that serves as an adapter protein for DDB1 (DNA damage-
binding protein 1) and Cul4A/B (Cullin-4A/B)63, binds Tet proteins and,
thereby, brings them together with DDB1 and CRL4 E3-ligase
complex19,20. For that purpose, we co-transfected HEK293-EBNA cells
with HA-ubiquitin and GFP-tagged Tet1s, Tet1s-K852E or Tet1s-K852R,
and mcherry-Uhrf1 or mcherry-VprBP. Immunoprecipitation was per-
formed with GFP-binding nanobody (GBP) and analyzed by western
blotting with antibodies against GFP and HA. As expected, we found
Tet1s to be able to pull down HA-ubiquitin, but this ubiquitin pulldown
was reduced for Tet1s-K852E and Tet1s-K852R, pointing to a reduction
in ubiquitination of the lysine mutants (Supplementary Fig. 6A). By
Fig. 6 | Lysine residue K852 in the CRD domain of Tet1 is crucial for Tet1s
S-phase localization and targeted catalytic activity. A Localization of the con-
served lysine residue K852 in Tet1s and sequence alignment between human and
mouse Tet1. BModel of mouse Tet1s generated by homology modeling on the
refined human Tet2 crystal structure (4NM6). The CRD is shown in red (conserved
lysine 852 in pink), and the DSBH in yellow. The bound DNA helix appears in blue
with 5mC flipped out of the double helix in green. CRepresentative images of
C2C12 cells expressing mRFP-PCNA and EGFP-Tet1s/Tet1s-K852R/Tet1s-K852E.
Boxplots showing heterochromatin accumulation of Tet1-X. DC2C12 cells from C
were immunostained against 5hmC 24 h after transfection. Nuclear 5hmC levels
were measured by high-content microscopy. Boxplot shows sum nuclear 5hmC
levels normalizedto the averaged 5hmClevels of background cells and against sum
nuclear DAPI intensity. Cells were grouped according to their mean EGFP fluores-
cence intensities (see Supplementary Fig. 2B, C). E,FFiji-based in situ 5hmC ana-
lysis: Binary nucleoplasm and heterochromatin masks were generated and mean
fluorescence intensities in the respective areaswere measured. Boxplots in Fshow
5hmC levels in pericentric heterochromatin (dark-gray) and the surrounding
nucleoplasm (light-gray).Representative images andconstitutive heterochromatin
relative areas are shown in Supplementary Fig. 5E, F. GChromatin
immunoprecipitation experiments followed by qPCR for MajSat sequences. C2C12
cells weretransfectedwith Tet1s-K852R/Tet1s-K852E andTet1-CD (positive control)
and synchronized in G1/late S phase. Barplots show the average value of amplifi-
cation levels in input and chromatin binding fractions normalized with GFP-input
(red line). The error bars represent the standard deviation with a 95% confidence
interval. HFRAP experiments in transfected C2C12 cells (EGFP-tagged Tet1s/Tet1s-
K852R/Tet1s-K852E). The EGFP signalwas photobleached with a 488 nm laser.IFor
analysis, fluorescence recovery curves and T-half times were calculated using
easyFRAP. Line plots show normalized averaged fluorescence recovery values, and
error bands show the respective standard deviation. 95% confidence intervals are
indicated in the plot. For all boxplots,the box represents50% of the data, starting in
the first quartile (25%) and ending in the third (75%). The line inside represents the
median. The whiskers represent the upper and lower quartile. Statistical sig-
nificance wastested with a paired two-samples Wilcoxon test (n.s. notsignificant, is
given for p-values ≥0.05; one star (*)for p-values < 0.05and ≥0.005; two stars(**) is
given forvalues < 0.005 and ≥0.0005;three stars (***)is given for values < 0.0005).
N-numbers and p-values are shown in Supplementary Data 1. Source data are pro-
vided as a Source Data file. Scale bar = 5 µm.
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved
these pulldown experiments, we validated that ubiquitination of Tet1s
took place and was negatively affected in Tet1s-K852E and Tet1s-K852R
mutants as was the recruitment to replicating heterochromatin.
Overexpression of Uhrf1 did not increase ubiquitination for Tet1s (see
also Supplementary Fig. 4D) nor of Tet1s-K852E or Tet1s-K852R.
Interestingly, overexpression of VprPB increased ubiquitination of
Tet1s but not Tet1s-K852E or Tet1s-K852R (Supplementary Fig. 6A). As
the pull-downs and ubiquitination assays were performed by ectopi-
cally expressing tagged ubiquitin, we tested the ubiquitination of Tet1-
CD with endogenous levels of ubiquitin. For this, HEK293-EBNA cells
were transfected with GFP-Tet1-CD and GFP-PCNA as a positive con-
trol, and treated with MG-13264, a potent cell-permeable proteasome
inhibitor, followed by co-immunoprecipitation and western blot. Ubi-
quitinated Tet1-CD and PCNA were detected using an anti-ubiquitin
antibody (Supplementary Fig. 6B) validating the results with tagged
ubiquitin.
Next, we analyzed the cell cycle dependent subnuclear localiza-
tion of VprBP as the protein involved in the monoubiquitination of this
lysine residue in Tet1s. For this, we performed immunofluorescence
against PCNA and VprBP in fixed C2C12 cells transfected with EGFP-
Tet1s with the PCNA patterns used to discriminate S-phase substages.
A clear colocalization with PCNA was found, especially during late
S-phase. In addition, wefound colocalization of Tet1s, VprBP andPCNA
at replicating heterochromatin during late S, while homogeneous
cellular distribution of VprBP was found outside S-phase (Fig. 7A). In
addition, we co-transfected GFP-Tet1s, mcherry-VprBP and miRFP-
PCNA in C2C12 myoblasts and analyzed their cell cycle localization by
live-cell time lapse microscopy. Cells in early S-phase were chosen and
followed over time while acquiring Z-stack images every 20 min.
Although VprBP showed a pancellular distribution, a slight
cytoplasmic-to-nuclear translocation was observed from early to late
S-phase (Supplementary Fig. 6C). These observations are in line with a
previous study that found the chromatin association of VprBP to
increase throughout S-phase63. Furthermore, line-profile analysis of a
late S-phase replication focus showed thatthe VprBP signal follows the
distribution of PCNA and Tet1s (Fig. 7A and Supplementary Fig. 6C).
These observations together with the previous immunoprecipitation
results (Supplementary Fig. 6A) suggests a model in which
CRL4(VprBP) mediated Tet1s ubiquitination is responsible for its
replication association in late S-phase.
To test whether Tet1s and its lysine mutants interact with
CRL4(VprBP), we performed co-immunoprecipitation of tagged VprBP
and Tet1-CD or Tet1-ZF-CD, where the latter clarifies a potential dis-
ruption of the interaction due to the zinc finger domain. First, we co-
transfected HEK293-EBNA cells with GFP-Tet1s or its lysine mutants
and mcherry-VprBP. Immunoprecipitation was performed with GFP-
binding nanobody (GBP) and analyzed by western blotting with anti-
bodies against GFP and RFP. Tet1s, Tet1s-K852E and Tet1s-K852R were
able to pulldown VprBP (Fig. 7B left). Secondly, we co-transfected GFP-
VprBP and mcherry-Tet1-CD or mcherry-Tet1-ZF-CD and we performed
immunoprecipitation as described above. In all cases, GFP-VprBP
(Fig. 7B right) was able to immunoprecipitate Tet1-CD and Tet1-ZF-CD.
GFP-VprBP was also able to immunoprecipitate endogenous Cul4, as
shown by western blotting with antibodies against Cul4 and Cul4B
(Fig. 7B bottom). To further verify Tet1-X interactions with VprBP/Cul4
and Uhrf1 at endogenous levels, we performed immunoprecipitation
in MCF7 and MCF10a cells. For this purpose, we immobilized Tet1
proteins using protein G agarose beads preincubated with Tet1 anti-
body and analyzed cell extracts by western blotting with antibodies
against Tet1/Tet1s, VprBP, Cul4, Cul4B and Uhrf1 (Fig. 7C). Endogenous
Tet1 proteins were able to precipitate Cul4A/B, VprBP and Uhrf1 in the
tumor cell line MCF7, which showed higher levels of Tet1s. These data
indicated that Tet1s and Tet1-CD interact with VprBP independently of
the mutation on the lysine or the insertion of the zinc finger domain.
Immunoprecipitated Tet1 proteins from MCF10a extracts were able to
pulldown Cul4 but not VprBP or Uhrf1 (Fig. 7C). The endogenous
immunoprecipitation confirmed the previously mentioned interac-
tions in HEK293-EBNA cells and also showed differences in the pull
down of VprBP and Uhrf1 between these cell lines.
TofurtherelucidatetheroleofCul4A/BinTet1srecruitment,we
made use of the NEDD8-activating enzyme (NAE)-inhibitor pevonedi-
stat to abrogate SUMOylation of Cul465, which in turn prevents ubi-
quitination of the conserved lysine residue in Tet1s by the
CRL4(VprBP) complex. In parallel, we tested the effect of the knock-
down of VprBP. We imaged cells live as described before 8 h post-
transfection. For C2C12 cells co-transfected with mcherry-Tet1-CD,
EGFP-PCNA as a marker for S-phase progression and miRFP-MaSat as a
marker for pericentric heterochromatin, we could validate Tet1-CD
association with sites of ongoing DNA replication during late S-phase.
The accumulation of Tet1-CD was, though, lost in cells that were
transfected with siRNA_VprBP or after 5 h of treatment with pevone-
distat (Fig. 7D). Moreover, the accumulation of Tet1-CD at PCNA and
MaSat marked heterochromatin was quantified as described above
and the knockdown of VprBP wa s verified by westernblotting (Fig.7D).
In summary, indirect inhibition of the CRL4(VprBP) complex abrogates
the recruitment of Tet1-CD to replicating heterochromatin. Impor-
tantly, immunofluorescence staining against 5hmC in Tet1-CD trans-
fected cells after 24 h of treatment with pevonedistat resulted in
significantly lower 5hmC levels in heterochromatin but not in
euchromatin relative to control treated cells (Fig. 7E). This indicates
that the increase on 5hmC levels after Tet1s overexpression was a
consequence of Tet1s cell cycle dependent recruitment to hetero-
chromatic regions.
Finally, we tested whether treatment with pevonedistat affects the
ubiquitination of Tet1-CD and whether it negatively affects the inter-
action with Uhrf1, which we showed earlier (Fig. 5)tobeanessential
player in mediating the Tet1s subnuclearlocalization. For this purpose,
we made use of the ubiquitination assay using extracts from HEK293-
EBNA cells co-transfected with GFP-Tet1-CD or GFP-Dnmt1, as control
for ubiquitination, and HA-ubiquitin. After 24h treatment with pevo-
nedistat or DMSO as control, we immunoprecipitated GFP-tagged
proteins using the GFP-binding nanobody (GBP) and analyzed the cell
extracts and precipitated material by western blotting with antibodies
against GFP and the HA-tag. HA-ubiquitin was detected in the pulldown
fraction of DMSO-treated cells, but not of cells treated with pevone-
distat (Fig. 7F). Last, we co-transfected HEK293-EBNA cells with GFP-
Uhrf1 and mcherry-Tet1CD and again treated these cells with pevone-
distator DMSO as control. After treatment with the NAE-inhibitor,GFP-
Uhrf1 was not able to pulldown Tet1-CD anymore, compared with
control cells (Fig. 7G). This indicates that the protein-protein interac-
tion between Uhrf1 and Tet1-CD depends on Cul4-mediated ubiquiti-
nation of Tet1-CD.
Tet1 and Tet1s differently affect 5mC and 5hmC levels of het-
erochromatic LINE 1 elements compared with euchromatic Alu
elements
To relate the effect of the different Tet1 isoforms and mutants on5mC
oxidation withits physiological consequences on transcriptionalnoise,
we generated different MCF7 cell lines using CRISPR/Cas9 genome
editing (Fig. 8A). Firstly, we performed the knockout of TET1, but not
of TET1s, to show the effects on cytosine modification levels of the
short isoform without the interference of TET1 (Supplementary
Fig. 7A–C). For the characterization of these cell lines, we performed
PCR amplification and sequencing of the exon 1 to confirm the geno-
mic deletion (Supplementary Fig. 7A). Furthermore, we performed
measurements of TET1 protein levels by western blot and immuno-
fluorescence followed by high-content microscopy analysis (Supple-
mentary Fig. 7B, C). For immunofluorescence we used different
fixation protocols to distinguish between TET1 and TET1s (the latter
not fixable with formaldehyde fixation, Supplementary Fig. 2A and
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Supplementary Fig. 7C right). While with standard formaldehyde
treatment only TET1 is fixable, the gradual increase of formaldehyde
concentration increases the fixability of TET1s (Supplementary Fig. 7C
left)42. This allowed us to detect the nuclear localization of both TET1
proteins in the MCF7 TET1 KO (Supplementary Fig. 7C center). Sec-
ondly, using the TET1 KO cell line, we generated the TET1s-K852R
mutant by point mutation (Supplementary Fig. 6B and Supplementary
Fig. 7D). We focused on the K to R mutant since the K to E lacks its
catalytic activity and, therefore, physiological significance. We con-
firmed the insertion of the mutation by amplification of exon 8 by PCR
followed by DNA sequencing (Supplementary Fig. 7D). Lastly, we cre-
ated a full knockout for both TET1 and TET1s, using the strategy and
Article https://doi.org/10.1038/s41467-022-32799-8
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the gRNA described in a previous study17. As before, western blot and
immunofluorescence analysis for TET1/TET1s levels in these cell lines
were performed to confirm the double knockout, in addition to PCR
amplification and DNA sequencing of exon 11 (Supplementary
Fig. 7E–G).
Using these cell lines, we compared cytosine modification levels
of MCF7 wild-type and MCF10a cells. To this end, we used ultra-high
performance liquid chromatography coupled to tandem mass spec-
trometry (UHPLC-MS/MS) to quantitatively assess the levels of 5mC,
5hmC, 5fC, and 5caC in genomic DNA. Two different cell clones were
analyzed as biological replicates. We compared these measurements
with immunofluorescence results in these newly generated cell lines
for 5mC and 5hmC (Fig. 8B, C) and found that they followed a similar
trend. Abundance of genomic 5mC, 5hmC, 5fC, and 5caC is plotted as
the fraction of total modified cytosines, and DNA modification levels
are expressed as percentage (%) (Supplementary Data 1, 2). Cytosine
modification levels for MCF10a and MCF7 cells matched previous
immunofluorescence results in Fig. 1C. As predicted, MCF7 TET1 KO
and MCF7 TET1 KO/TET1s-K852R showed lower or similar 5mC levels
compared with MCF7 wild-type, while 5hmC, 5fC and 5caC were all
higher. Global DNA methylation (5mC) was increased for MCF7 TET1/
TET1s KO cells to levels similar to MCF10a (with low levels of both TET1
proteins, Supplementary Fig. 1A). Along with 5mC increase, the levels
of 5hmC, 5fC, and 5caC decreased from MCF7 wild-type to MCF7 TET1/
TET1s KO (Fig. 8C).
Next, we analyzed the occurrence of 5mC and 5hmC at selected
genomicloci. We compared the 5´ UTR region of the LINE 1 DNA repeat
element, as heterochromatic loci as mentioned before (see, e.g.,
Fig. 1F)30,34, versus the Alu DNA element (SINEs) as a repetitive DNA
sequence located in euchromatin32,33. These interspersed repeat ele-
ments exist in half a million to over one million copies throughout the
human genome but correlate with different chromatin states. Hence,
they are well suited to report on the DNA modification state of
euchromatic versus heterochromatic loci throughout the human
genome. For this purpose, we performed DNA glucosylation followed
by MspI and HpaII restriction enzyme digestion and PCR-based 5hmC
and 5mC detection (GluMS-PCR). Genomic DNA was treated with T4-
BGT, which adds glucose to 5hmC yielding 5ghmC but not to 5mC.
Then, we performed endonuclease treatment with MspI and HpaII,
both recognizing “CCGG”but sensitive to different methylation states.
HpaII cleaves only unmodified sites, while MspI cleaves 5mC and
5hmC, but not 5ghmC. Finally, we used primers for PCR amplification
of these genomic loci. The primers flank the target site of the endo-
nucleases: if the CpG site contains 5hmC a band is detected after glu-
cosylation (and conversion to 5ghmC) and digestion, but not in the
non-glucosylated control reaction (scheme in Fig. 9A)37,43,66.Resultsof
end-point PCR comparing MCF10a, MCF7 wild-type, MCF7 TET1 KO,
MCF7 TET1 KO/TET1s-K852R, and TET1/TET1s KO cells (including bio-
logical replicates) are shown in Fig. 9B. Quantification of 5mC and
5hmC levels in heterochromatic and euchromaticgenomic regions was
performed by image analysis of density (aka intensity) of bands in
agarose gels using Fiji67 and normalizing to non-digested samples. For
LINE 1 element at heterochromatic regions, MCF10a cells showed the
highest level of 5mC and levels of 5hmC close to 0 (corresponding to
the absence of a band for MspI digestion), and the same was observed
for MCF7 TET1/TET1s KO (Fig. 9B). In contrast, MCF7 wild-type cells
showed lower levels of 5mC and higher levels of 5hmC, and these
differences are increased for MCF7 TET1 KO cells, in line with mass
spectrometry and immunofluorescence results (Fig. 8B, C). As expec-
ted, MCF7 TET1 KO/TET1s-K852R showed a reduction in 5hmC levels
(Fig. 9B) due to the lack of TET1s accumulation on heterochromatic
regions (Fig. 6C, G). Nevertheless, they still showed some levels of
5hmC in heterochromatic repeats, which is consistent with their high
catalytic activity producing a global increase of 5hmC, 5fC and 5caC
(Figs. 6D, F, 8C). We, next, analyzed 5mC and 5hmC levels in an Alu
element as an euchromatic region33,34. After HpaII treatment for
MCF10a and MCF7 wild-type cells, there was no PCR amplification
indicating that these DNA sequences are mostly unmodified. For TET1
KO, we found an increase in 5mCthat is consistent withthe role of TET1
avoiding 5mC spreading into euchromatic regions. Interestingly, 5mC
increase after TET1 KO is reduced and likely oxidized to 5hmC in the
TET1 KO/TET1s-K852R mutants, which showed a higher catalytic
activity and mobility (Fig. 6), as we demonstrated earlier. In line with
these results, TET1/TET1s KO cells showed 5mC increase as observed
for TET1 KO (Fig. 9B). As these cell lines express the different TET1
versions from the endogenous loci and do not change throughout the
cell cycle, an async hronous population of cells showed consistent da ta.
In addition to the analysis described above, we performed bisul-
fiteandTet-assistedbisulfite (TAB) sequencing experiments in these
loci in order to investigate the 5mC and 5hmC levels atbase resolution
level. Bisulfite sequencing itself cannot differentiate 5mC from 5hmC,
as both resist deamination during the treatment of DNA with sodium
bisulfite (Fig. 9C)68. However, we can distinguish between 5mC and
5hmC combining bisulfite sequencing with two additional steps: pro-
tection of 5hmC through glucosylation and Tet1-CD mediated oxida-
tion of 5mC to 5caC. After subsequent bisulfite conversion, the
protected β-glucosyl-5-hydroxymethylcytosine (5ghmC) is read as C in
the sequence, whereas 5caC and C are read as T, enabling single-base
resolution sequencing of 5hmC69.5fCcanalsobeoxidizedbyTet
proteins to 5caC70. Thus, only protected 5ghmC will read as C in TAB-
sequencing while in bisulfite sequencing both 5mC and 5hmC are read
as C (Fig. 9D). Firstly, we verified the activity of purified Tet1-CD used
Fig. 7 | The CRL4(VprBP) complex ubiquitinates Tet1s and this is needed for
Tet1s recruitment to late-replicating heterochromatin. A VprBP and PCNA
immunostaining in C2C12 cells expressing EGFP-Tet1s: representative images of 3
independent experiments andline-profile analysis are shown. BHEK293-EBNAcells
were transfected with EGFP or EGFP-tagged Tet1-X/VprBP, or mcherry fusions
(VprBP/Tet1-X). Cell extracts were analyzed by immunoprecipitation with immo-
bilized GFP-binding nanobody, followed by detection with antibodies against GFP,
RFP, Cul4 and Cul4B. The cut-outs show input/bound GFP and input/bound
mcherry fractions. CEndogenous co-immunoprecipitation: MCF cellextracts were
analyzed using immobilized Tet1/Tet1s followed by detection with antibodies
against Tet1/Tet1s, Cul4B, Cul4, VprBP and Uhrf1. MIN antigen (attP-peptide) was
used as negative control. The cut-outs show the input/bound Tet1/Tet1s fractions.
DC2C12 cells were transfected with mcherry-Tet1-CD, EGFP-PCNA and miRFP-
MaSat. Additionally, cells were transfected with siRNA_VprBP or treated with
pevonedistat to indirectly inhibit Cul4/DMSO for 5 h before live-cell imaging.
Representative images are shown. Boxplots depict Tet1-CD accumulation at het-
erochromatin. Western blotting with antibody against VprBP validates the knock-
down inC2C12 cells. EInsitu 5hmC analysisafter 24 h treatment withCul4 inhibitor.
Boxplots show the quantification of 5hmC levels in C2C12 cells in euchromatin
(light-gray) versus heterochromatin (dark-gray). FHEK293-EBNA cells were trans-
fected with EGFP-tagged Tet1-CD/Dnmt1 and HA-ubiquitin, treated with pevone-
distat/DMSO for 24 h, and analyzed by immunoprecipitation with immobilized
GFP-binding nanobody and detection with antibodies againstGFP and HA. The cut-
outs show the input/bound GFP-fractions and the input/bound HA-Ubi fractions.
GHEK293-EBNA cells were transfected with EGFP-Uhrf1 and mcherry-Tet1-CD and
treated with pevonedistat/DMSO for 24 h. Cell extracts were analyzed by immu-
noprecipitation with immobilized GFP-binding nanobody, and detection with
antibodies against GFP and RFP. The cut-outs show the input/bound GFP-fractions,
and the input/bound mcherry fractions. In B,C,G, two independent experiments
were performed. For boxplots, the box represents 50% of the data, starting in the
first quartile (25%) and ending in the third (75%). The line inside represents the
median. The whiskers represent the upper and lower quartile. Statistical sig-
nificance wastested with a paired two-samples Wilcoxon test (n.s. notsignificant, is
given for p-values ≥0.05; one star (*)for p-values < 0.05and ≥0.005; two stars(**) is
given forvalues < 0.005 and ≥0.0005;three stars (***)is given for values < 0.0005).
N-numbers and p-values are shown in Supplementary Data 1. Source data are
provided as a Source Data file. Scale bar = 5 µm.
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved
for these experiments by an oxidation reaction with genomic DNA
(gDNA) of MCF10a and MCF7 cells. Afterwards, we performed slot
blotting of the DNA incubating with antibodies against 5hmC and 5caC.
Compared with non-treated gDNA samples, where MCF7 gDNA
showed higher levels of 5hmC than MCF10a, after Tet1-CD oxidation
reaction, 5hmClevels were reduced while 5caC levels wereincreased in
both celllines. The same amount of DNA, confirmed by methyleneblue
staining, was loaded for all samples (Fig. 9E). Bisulfite sequencing
analysis of cytosines in the LINE 1 5´UTR promoter (Fig. 9FandSup-
plementary Fig. 8A) showed strong methylation and hydro-
xymethylation for all MCF10a and MCF7 cell lines with the exception of
the TET1 KO/TET1s-K852R mutants. On the other hand, for the Alu
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved
repeats, MCF10a and MCF7 cell lines showed low percentage of
methylation and hydroxymethylation, in line with previous results
(Fig. 9B). For TAB-sequencing experiments, which analyzed exclusively
5hmC levels in these loci, MCF7 wild-type cells and TET1 KO showed
higher levels of hydroxymethylated cytosines for the heterochromatic
region selected, compared with MCF10a and MCF7 TET1/TET1s KO.
Interestingly, MCF7 TET1 KO/TET1s-K852R cell line showed a reduction
of the percent of hydroxymethylation compared with MCF7 and TET1
KO cell lines (Fig. 9G and Supplementary Fig. 8B). However, only MCF7
TET1 KO/TET1s-K852R showed high levels of hydroxymethylated C for
the euchromatic region analyzed, the Alu repeat. In conclusion,
MCF10a and MCF7 TET1/TET1s KO cells, both with low to no level of
TET1 isoforms, showed higher levels of methylated C for the hetero-
chromatic loci. On the other hand, for the euchromatic loci only MCF7
TET1 KO and MCF7 TET1/TET1s KO showed a high percent of methy-
lation, in line with previous results (Fig. 9B).
Altogether, the combination of global genomic DNA and
sequence specific eu- versus heterochromatic loci analyses highlights
the very different functions of the two TET1 isoforms based on their
cell cycle dependent subnuclear targeting.
Discussion
In this study, we describe the recruitment of the Tet1s isoform to
heterochromatic regions during ongoing DNA replication, the con-
sequent spreading of 5hmC to heterochromatic regions of thegenome
with activation of LINE 1 elements and chromatin decondensation, and
we elucidate the dual mechanism underlying this spatio-temporally
directed Tet1 catalytic activity (Fig. 10).
The subnuclear association of Tet1s at heterochromatin was
observed in non-tumorigenic and breast cancer human cell lines,
and also in murine cells. This finding points to a conserved molecular
mechanism for Tet1s recruitment that is not affected by the
malignant transformation. However, cancer cells are in general
hypomethylated71. In agreement with this, we found that MCF7 breast
cancer cells show a significant reduction of 5mC and increased 5hmC
levels at heterochromatic regions, compared to non-tumorigenic
mammary gland cells, and we showed that MCF7 cells overexpress
Tet1s. Non-tumorigenic MCF10a mammary gland cells, on the other
hand, expressed only minor levels of Tet1 isoforms. Considering the
recruitment and targeted catalytic activity of Tet1s in hetero-
chromatin, the 5mC reduction in MCF7 cells could thus be accounted
for by the high Tet1s level in these cells. Nonetheless, some of the
changes on global DNA methylation levels could also be attributed to
DNA modification fluctuations between individuals, as the MCF7 and
MCF10a cells were obtained from two different donors72.Asabout5%
of all cytosines in the human genome are estimated to be methylated,
of which a large number is found in a heterochromatic CpG context73,
the decrease of about 50% in methylation levels we measured,
accounts for a considerable number of cytosines that are unlikely to be
explained simply by variation between individuals. Comparatively, the
minor changes on 5fC levels we measured could be attributed to this
modification being an intermediate product of the 5mC oxidation
cascade. Additionally, 5fC and 5caC can be excised by the DNA repair
enzyme thymine-DNA glycosylase (TDG), followed by replacement
with unmodified cytosine74. Overall, the observed 5mC reduction,
especially in heterochromatin, is supported by our findings in murine
cells. In this model, Tet1s overexpression at levels mimicking the ones
in MCF7 cells and its targeted catalytic activity to heterochromatin,
resulted in a significant 5mC oxidation, and increased 5hmC
production.
We expanded the investigation of the cell cycle-dependent
nuclear localization of the Tet protein family and found late S-phase
recruitment only for Tet1s, but not for Tet1, Tet2 or Tet3. A structural
feature that separates these proteins from one another is their zinc
finger domain, while they share a conserved C-terminal catalytic
domain60. Indeed, only constructs lacking the zinc finger domain, not
present in Tet1s, were able to show late S-phase accumulation and the
zinc finger added to Tet1-CD prevented it. This indicates that the zinc
finger is the domain responsible for tethering Tet1 away from het-
erochromatin. Previous studies have shown that the zinc finger of Tet1
mostly binds non-modified DNA14 and is implicated in preventing DNA
methylation spreading in euchromatic regions15. Additionally, the zinc
finger and the BC domains were found to concomitantly increase the
DNA binding ability of Tet1 and their deletion was shown to result in
decreased chromatin loading16. In agreement with that, we found that
the zinc finger domain decreases the mobility of the protein by
increasing its DNA binding ability, and this diminishes its hetero-
chromatin recruitment and 5hmC spreading into these genomic
regions. In the absence of the zinc finger domain in Tet1s isoform, a
separate mechanism independent of catalytic activity targets this iso-
form to heterochromatic regions during late S-phase.
A previous study found Tet1 to interact with PCNA, which was
shown to mediate Tet1 activity59. The major difference between the
putative PBD of Tet1 and the PBD consensus sequence, is a hydro-
philic lysine that replaces the central hydrophobic amino acid
residue in canonical PBDs75. On the one hand, early S-phase cells did
not show Tet1s accumulation at replication foci. On the other hand,
the putative PBD did not recruit Tet1s to late S-phase replicating
heterochromatin. Hence, Tet1s heterochromatin accumulation
during late S-phase is likely not dependent on its interaction with
PCNA. Nonetheless, aphidicolin treatment, which inhibits DNA
polymerase activity, displaced Tet1s from heterochromatin regions
concomitant with PCNA dissociation, showing that Tet1s recruit-
ment is replication dependent. It would be interesting to back
mutate the putative PBD and analyze the consequences for genomic
distribution of DNA modifications. A successful reactivation of the
putative PBD, and therefore a restored interaction with PCNA,
would hint to an initial function that may have been lost during
evolution or replaced by a different mode of recruitment. A PCNA
and Uhrf1-dependent mode of recruitment has evolved and been
preserved in Dnmt1, which is the enzyme responsible for the
maintenance of DNA methylation7,76.
Fig. 8 | Comparison of global levels of cytosine modifications in MCF10a cell
line versus MCF7 and TET1-X mutants. A Scheme illustrating the different MCF7
cell lines generated by CRISPR/Cas9 genome editing, showing positions of gRNA
targets in the TET1 locus. MCF7 TET1 KO (full isoform KO), MCF7 TET1 KO/TET1s-
K852R (full isoform KO and TET1s lysine mutant) and MCF7 TET1/TET1s KO (both
isoforms KO) were generated. Two different clones were selected and used as
biological replicates. BImmunofluorescence analysis of 5mC and 5hmC nuclear
levels in MCF10a, MCF7 wild-type and MCF7 TET1-X mutants by high-content
microscopy. Levels were compared with 5mC/5hmC shown in C.Meanintensity
values werenormalized to theaverage for MCF7 wild-type (discontinuousred line).
CBarplots showing levels of 5mC, 5hmC, 5fC, and 5caC in genomic DNA measured
by ultra-high performance liquid chromatography coupled to tandem mass spec-
trometry (UHPLC-MS/MS). The abundance of genomic cytosine modifications was
plotted as the fraction of to tal modified cytosines, and DNA modification levels are
expressed as percentage (%). Average levels in MCF7 wild-type cells are indicated
with a discontinuous red line for all cytosine modifications. The error bars repre-
sent thestandard deviation witha 95% confidence interval. Forall boxplots,the box
represents 50% ofthe data, startingin the first quartile (25%) and ending in the third
(75%). Theline inside represents the median. The whiskers represent theupper and
lower quartile. Statistical significance was tested with a paired two-samples Wil-
coxon testand One-Way ANOVA for mass spectrometry data (n.s. notsignificant, is
given for p-values ≥0.05; one star (*)for p-values < 0.05and ≥0.005; two stars(**) is
given forvalues < 0.005 and ≥0.0005;three stars (***)is given for values < 0.0005).
N-numbers and p-values are shown in Supplementary Data 1. Source data are
provided as a Source Data file.
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 17
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Uhrf1 is a ubiquitin ligase, has a multimodal heterochromatin
association during S-phase, plays a crucial role in DNA methylation
maintenance, contributes to global hypomethylation in the cancer
context and targets the DNA de novo methyltransferase Dnmt3A for
ubiquitin-dependent proteasomal degradation. Additionally, it was
found to be frequently overexpressed in cancer77.Remarkably,we
found that Uhrf1 physically interacts with Tet1s and is necessary for its
recruitment to constitutive heterochromatic regions, which was
mediated by the SRA domain of Uhrf1. Interestingly, though, Uhrf1 did
not ubiquitinate Tet1s. Altogether, this supports the essential role of
Uhrf1 in Tet1s accumulation at replicating heterochromatin, though,
independent of Uhrf1 ubiquitination activity.
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Nature Communications | (2022) 13:5173 18
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Fine mapping the domain required for S-phase localization of
Tet1s, yielded that the CRD is needed not only for catalytic activity
but also for the late S-phase localization, while the DSBH domain is
not required. Within the CRD, a conserved lysine was crucial for the
S-phase localization of Tet1s as this was abrogated upon mutating
the lysine to arginine (Tet1s-K852R) or glutamate (Tet1s-K852E).
Interestingly, mutations in the CRD of Tet2 have been found in
leukemia patients78. In view of the fact that we did not find Tet2 to
associate with replicating heterochromatin, we speculate that the
effect of such mutations may be rather by affecting its catalytic
activity.
Previous studies showed that lysine 852 within the CRD of all Tet
proteins is monoubiquitinated by CRL4(VprBP) and modulates Tet
catalytic activity and Tet2 chromatin binding19,20.Furthermore,the
short amino acid stretch harboring the conserved lysine, wasshown to
stabilize the DNA adjacent to the modified cytosine target, by inter-
acting with the phosphate backbone. Mutations of the conserved
lysine residue to a glutamate resulted in loss of Tet2 catalytic activity,
indicating its importance for the correct function of Tet proteins60.We
made similar observations for Tet1s by mutating lysine 852 to a glu-
tamate or an arginine, respectively, and also by inhibiting or down
regulating CRL4(VprBP). In this way, Tet1s ubiquitination at lysine 852
is abrogated and its recruitment to replicating heterochromatin
abolished. Taken together, this clearly hints to Tet1s being
ubiquitinated at lysine 852 by CRL4(VprBP), which in turn regulates
Tet1s recruitment to replicating heterochromatin. Both Tet1-CD and
Tet1-ZF-CD are able to interact with Cul4 and VprBP, and consequently
both could be ubiquitinated, but fusing the zinc finger domain to Tet1-
CD is sufficient to prevent its recruitment to replicating hetero-
chromatin by tethering it away from heterochromatin. Incidentally,
fusions of MLL with TET1 were reported in leukemia patients79.The
location of the genomic breakpoints were mapped to TET1 intron 8,
exons 9 and 12. This would retain part of the catalytic domain of TET1
but exclude the CRD domain and, hence, likely, the regulation of TET1
S-phase recruitment reported here will not play a role.
The proteins of the Tet dioxygenase family have been implicated
in crucial developmental processes, disease and in different cancer
types. Here, we propose a regulation mechanism where Uhrf1 plays an
essential role by interacting with Tet1s exclusively during late S-phase,
after the ubiquitination of a conserved lysine in the CRD by the
CRL4(VprBP) complex. Figure 10 graphically summarizes our model
for the multistep recruitment of Tet1s to heterochromatin during late
S-phase. We propose that VprBP acts as a substrate recognition com-
ponent of the E3 ubiquitin-protein ligase complex, in this case, a
substrate-specific adapter of DDB1-CUL4, which mediates ubiquitina-
tion of Tet1s. Ubiquitination of Tet1s at the conserved lysine residue in
the CRD, in turn, allows interaction with Uhrf1 and consequently
recruitment to heterochromatin during late S-phase. This is followed
Fig. 9 | 5mC and 5hmC levels at heterochromatin (LINE 1 promoter) and
euchromatic loci (Alu). A Scheme of GluMS-PCR experiments: DNA glucosylation,
MspI andHpaII digestionand PCR based5hmC/5mC detection. BCut-off of agarose
gels showing PCR products (LINE 1 protomer and Alu element) after T4-BGT
treatment and endonuclease digestion. Barplots showing densitometry measure-
ments (for PCR bands) quantifying 5mC/5hmC levels. Higher levels of 5hmC are
indicated with red edges in the barplot. CBisulfite conversion of genomic DNA
followed by PCR for amplification of LINE 1 promoter and Alu element. Unmodified
or 5caC is converted to uracil and consequently be read as a T after PCR. 5mC or
5hmC are not converted, and cannot be distinguished by this method. DTAB (Tet-
assisted bisulfite) sequencing experiments scheme.5hmC is protectedfrom further
oxidation by incubation with T4-BGT and UDP-glucose. 5mC (but not protected
5ghmC) is oxidized to caC by Tet1-CD incubation followed by bisulfite conversion
of C and 5caC and PCR. Only 5ghmC will be read as a C after PCR and sequencing.
EProcess of GFP-Tet1-CD protein purification and subsequent oxidation reaction
test using gDNA. Slot blotting of DNA before and after oxidation reaction shows
levels of 5hmC and 5caC. FBarplots showing the percentage of 5mC/5hmC at base
resolution level after bisulfite conversion of unmodified cytosines, PCR and
sequencing. Bisulfite sequencing experiments were performed for euchromatic
versus heterochromatic loci as GluMS-PCR experiments. GBarplots showing the
percentage of 5hmC at base resolution level after TAB-sequencing analysis.
Experiment was performed with gDNA treated as described in A,followedbyTet1-
CD oxidation reaction, bisulfite conversion and PCR. Barplots showing higher
percentage of 5hmC are indicated by red outlines, and those with higher percen-
tage of 5mC in Fare indicated byblue outlines. ForB,F,G, the error bars represent
the standard deviation with a 95% confidence interval. Statistical significance was
tested with a paired two-samples Wilcoxon test using R-studio. N-numbers and p-
values are shown in Supplementary Data 1. Source data are provided as a Source
Data file.
Fig. 10 | Model of Tet1s and Tet1 regulation during the ce llcycl e. Tet1 via its zinc
finger domain is tethered away from heterochromatin, which prevents spreading
of hydroxymethylation to these regions. The short isoform Tet1s lacking this
domain gets ubiquitinated by the CRL4(VprBP) complex and the modified protein
is bound by Uhrf1 and recruited to late-replicating heterochromatin. This targets
Tet1s activity to heterochromatin and results in aberrant oxidation of methylcy-
tosine in heterochromatic regions in both human and mouse cells depending on
the level of this isoform. This in turn also results in the reactivation of silenced
repetitive DNA elements like LINE 1 or major satellite repeats.
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Nature Communications | (2022) 13:5173 19
Content courtesy of Springer Nature, terms of use apply. Rights reserved
by oxidation of the very abundant methylcytosines within these
chromatin regions. The zinc finger domain in the full-length Tet1
protein, on the other hand, keeps the enzyme tethered away from
heterochromatin. As functional consequences of the recruitment of
the Tet1s isoform to heterochromatic regions and the consequent
spreading of 5hmC to heterochromatin, decondensation of hetero-
chromatin takes place accompanied by activation of LINE 1. Such long
interspersed DNA repeat elements are highly abundant in human
cells30, are enriched at heterochromatin34, known to be activated in
cancer cells80–83 as well as in early development84 and are normally kept
silenced by DNA methylation85 and methylcytosine binding
proteins37,43.
The reexpression of LINE 1 and satellite repeats is a commonly
observed feature of many epithelial cancer types and also observed in
clonal hematopoiesis and AML86,87. Moreover, genomic insertions
upon LINE 1 reactivation are found to be disruptive to genes, where
they are newly inserted, and this can render tumor suppressor genes
inactive. However, Tet1s recruitment to highly methylated hetero-
chromatin suggests a mechanism, relying on the targeted catalytic
activity of Tet1s and the reactivation of retrotransposable elements.
The finding that Tet1s is overexpressed in many cancer types17 makes a
co-overexpression of Uhrf1 and Tet1s a likely scenario. Speculating on
the biological significance of Tet1s targeting, we could propose a
model where the loss of DNA methylation in heterochromatin plays a
crucial role. Given the proposed role of full-length Tet1 as a tumor
suppressor88,89, our results suggest a tumor promoting role for Tet1s
by drastically changing the epigenetic landscape of a cell. Importantly,
the (patho)physiological outcome is not exclusive to cancer cells but is
regulated by the abundance of the Tet1 isoforms and their
stoichiometry.
Methods
Experimental model
We used the following cell lines:
–MCF10a (non-tumorigenic) and MCF7 (cancer), human cell lines.
–MCF7 (TET1 KO), MCF7 (TET1/TET1s KO), MCF7 (TET1 KO/
TET1s-K852R).
–C2C12 mouse myoblast.
–Mouse embryonic fibroblast MEF-PM (Dnmt1−/−,p53
−/−)andMEF-
P(p53
−/−).
–Mouse embryonic stem E14 wild-type cells and Uhrf1-deficient
cells (Uhrf1−/−).
–HEK293-EBNA human cell line.
All the references are given and details are described in Supple-
mentary Table 1.
Cell culture, transfection and treatments
C2C12 mouse myoblasts90 were cultured in Dulbecco’sModified Eagle’s
Medium (DMEM) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany;
Cat.No.: D6429) containing 20% FCS. Mouse embryonic fibroblasts
(MEF) deficient for Dnmt1 and p53 (MEF-PM, Dnmt1−/−,p53
−/−), deficient
only for p53 (MEF-P, p53−/−)49 were cultured in DMEM containing 15% or
10% FCS, respectively44. Female breast epithelial cells MCF10a were
cultured in DMEM/F12 (Sigma- Aldrich Chemie GmbH, Steinheim, Ger-
many; Cat.No.: D8900) supplemented with final concentrations of 5%
horse serum (Sigma-Aldrich Chemie GmbH, Steinheim, Germany;
Cat.No.: H0146), 20 ng/mL EGF (Sigma-Aldrich Chemie GmbH, Stein-
heim, Germany; Cat.No.: E9644), 0.5 mg/mL hydrocortisone (Sigma-
Aldrich Chemie GmbH, Steinheim, Germany; Cat.No.: H0888), 100 ng/
mL cholera toxin (Sigma-Aldrich Chemie GmbH, Stein- heim, Germany;
Cat.No.: C8052) and 10 μg/mL insulin (Sigma-Aldrich Chemie GmbH,
Steinheim, Germany; Cat.No.: I2643), and the breast cancer epithelial
cellsMCF7wereculturedinDMEM(Sigma-AldrichChemieGmbH,
Steinheim, Germany; Cat.No.: D6429) containing 10% FCS, as described
before91. Mouse embryonic E14 wild-type stem cells and therefrom
derived Uhrf1-deficient cells (Uhrf1−/−)48 were cultured under feeder-free,
2i/LIF conditions37,43 in culture dishes that were coated with 0.2 % gelatin
(Sigma-Aldrich Chemie GmbH, Steinheim, Germany, Cat.No.: G2500).
DMEM (Sigma-Aldrich Chemie GmbH, Steinheim, Germany; Cat.No.:
D6429) for embryonic stem cell culture contained 16% FCS and was, in
addition to L-glutamine and Pen/Strep, supplemented with 1x non-
essential amino acids (Sigma-Aldrich Chemie GmbH, Steinheim, Ger-
many; Cat.No.: M7145), 0.1 mM β-mercaptoethanol (Carl Roth, Karls-
ruhe,Germany,Cat.No.:4227),0.1μM PD 0325901 (Axon Medchem BV,
Groningen, The Netherlands, Cat.No.: Axon 1408), 0.3 μM CHiR 99021
(Axon Medchem BV, Groningen, The Netherlands, Cat.No.: Axon 1386)
and 1,000 U/ml LIF (Enzo Life SciencesGmbH,Lörrach,Germany,
Cat.No.: ALX-201-242). HEK293-EBNA human embryonic kidney cells
(Invitrogen; catalog # 620-07, Paisley PA49RF, UK) were cultured in
DMEM (Sigma-Aldrich Chemie GmbH, Steinheim, Germany; Cat.No.:
D6429) containing 10% FCS. All cell lines were regularly tested for
mycoplasma to ensure that they were contamination free.
C2C12, MEF, MCF7 and MCF10a cells were transfected by elec-
troporation with the AMAXA Nucleofector II system (Lonza, Cologne,
Germany), using a self-made buffer (5 mM KCl, 15 mM MgCl2, 120 mM
Na2HPO4/NaH2PO4 pH 7.2, 50 mM Mannitol)92 with default pro-
grams B032, A024, P020 or T024, respectively. Mouse embryonic
stem cells were transfected with the Neon electroporation system
(ThermoFisher scientific) according to the manufacturer’s instruc-
tions. HEK293-EBNA cells were transfected with polyethyleneimine
(PEI, Sigma-Aldrich) as previously described93.
To assess the effects of replication fork stalling on the accumu-
lation on Tet1s or Tet1-CD, C2C12 cells were transfected with mcherry-
Tet1s or Tet1-CD and miRFP-PCNA and subjected to live-cell time lapse
microscopy at 5 min time intervals. After three time points, 50 µg/mL
aphidicolin (Sigma-Aldrich, St Louis, MO, USA, Cat.No.: A0781), which
stalls the replication machinery55, or DMSO (dimethyl sulfoxide from
Sigma-Aldrich, St Louis, MO, USA, Cat.No.: 41639) were added to the
cells and imaging was continued for 30 min at the same intervals.
To test the effects of the NEDD8 8 activating enzyme (NAE)-inhi-
bitor pevonedistat that prevents the neddylation-dependent activa-
tion of Cul-family ubiquitin ligases65 (MLN4924, MedChemExpress
Europe, Sollentuna, Sweden, Cat.No.: HY-70062) on the recruitment of
Tet1s, C2C12 were transfected with plasmids encoding fluorescently
tagged PCNA and Tet1s and 8 h later treated with 10 µM pevonedistat
or DMSO and after 5h of additional incubation subjected to live-cell
microscopy.
Dependence of 5hmC levels on Cul4 activity was addressed by
treating cells with 3 µM pevonedistat or DMSO for 24 h, followed by
fixation and 5hmC immunostaining.
For ubiquitination assays, transfected HEK293-EBNA cells were
treated with 3 µM pevonedistat or DMSO for 24 h. When using endo-
genous ubiquitin, HEK293-EBNA cells were treated with 10 µM of pro-
teasome inhibitor MG-132 (MedChemExpress Europe, Sollentuna,
Sweden,Cat.No.:HY-13259)orDMSOfor24h.
To enrich the protein lysate in cells at the S-phase stage, syn-
chronization of MCF7, MCF10a, and C2C12 cells was performed by
double thymidine arrest94 (Sigma-Aldrich) to a final concentration of
2 mM. Thymidine is used to synchronize the cells in G1/early S-phase.
MCF7 and MCF10a cells were processed for co-immunoprecipitation
5 h after release into normal growth medium. For chromatin immu-
noprecipitation (ChIP) experiments, C2C12 were processed just after
release(in G1/early S-phase) or 7 h after release into normal medium to
enrich the sample in cells at the late S-phase.
CRISPR/Cas9‑mediated genomic engineering of TET1/TET1s and
TET1s-K852R mutant generation in MCF7 cells
For the generation of TET1, TET1/TET1s knockouts, and TET1s-
K852R point mutation, specific gRNAs (Supplementary Table 4,
Supplementary Fig. 7) were cloned into a puromycin-selectable
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vector expressing both SpCas9 and gRNA (px459: F. Zhang labora-
tory). CRISPR gRNAs were designed using http://crispr.mit.edu/.
MCF7 cells were transfected with Cas9-gRNA vectors and the
homologous recombination template (for TET1s-K852R point
mutation) using the Neon electroporation system (ThermoFisher
scientific) according to the manufacturer’s instructions. Two days
after transfection, cells were plated at clonal density in DMEM
media supplemented with 2 μg/mL puromycin (Gibco). Selection
media was removed after 48 h, replaced with normal ESC media, and
colonies were allowed to grow for an additional 4–5 days. Single
colonies were transferred into 96-well plates and the plates were
duplicated after 2 days. Enrichment for mutated clones was
accomplished by amplifying the CRISPR/Cas targeted region via
PCR (oligonucleotides in Supplementary Table 4). Cell lysis in 96-
well plates and PCR on lysates were performed as previously
described95. and analyzed on 1.5% agarose gels. PCRs of positive
clones were confirmed by Sanger sequencing, and sequence align-
ment was performed with Clustal Omega (Supplementary Table 7).
Clones harboring biallelic mutations were then assessed for loss of
TET1 and/or TET1s via Western blot and immunofluorescence ana-
lysis against using monoclonal antibodies (rat anti-TET1, Supple-
mentary Table 5) as described before.
Expression constructs
Mammalian expression plasmids encoding GFP- (pc0653), mRFP-
tagged PCNA (pc1054) and miRFP-tagged PCNA (pc3385), have been
described in previous works. A GFP-tagged construct encoding the
short isoform of Tet1 (pc3901) was generated by amplifying
the respective fragment from full-length Tet1 (pc2271) and replacing
the full-length Tet1 sequence with the Tet1s sequence by restriction
with AsiSI and NotI. Mcherry-tagged Tet1s (pc3905) was generated by
replacing the sequence of Uhrf1 (pc1756) with the Tet1s sequence just
described using AsiSI and NotI restriction enzymes. GFP- or mcherry-
tagged catalytically active and inactive Tet1 catalytic domain (Tet1-CD)
constructs (pc2315, pc2547, pc2815, pc2637) as well as mcherry-tagged
catalytic domains of Tet2 and Tet3 (pc3338, pc3339) and GFP-tagged
Tet1, 2 and 3 full-length (pc2271, pc2272, pc2273) constructs have been
described before. The sequences encoding the CRD of GFP-Tet1s
(pc3904), as well as the amino acids 1–389 (pc3174) and 566-833
(pc3175) of full-length Tet1 were deleted by overlap-extension PCR96
and the amplicon obtained was used to replace the Tet1 coding
sequence in EGFP-Tet1 (pc2271). GFP-tagged Tet1-CRD (pc2334) and
Tet1-DSBH (pc2335) were generated by amplifying the respective
sequences from full-length Tet1 and replacing the full-length
Tet1 sequence by AsiSI and NotI restriction. To obtain a GFP-tagged
PBD encoding the putative Tet1-PBD (pc3918), oligo cloning was per-
formed and the DNMT1-PBD in a pEGFP-N2 backbone (pc0883) was
replaced by XmaI and EcoRI restrictions. A mcherry-tagged Tet1-ZF-CD
fusion (pc3956) was constructed by PCR-amplifying the Tet1-ZF
domain including a linker sequence from GFP-Tet1-ZF and inserting
the amplicon in the mcherry-Tet1-CD backbone by AsiSI restriction. To
mutate lysine 852 in Tet1s to glutamate (pc3915) or arginine (pc3914), a
sequence- and ligation-independent cloning approach was chosen97.
GFP- or mcherry-tagged full-length Uhrf1/Uhrf2 (pc1709, pc1756,
pc1976) and Uhrf1 single domain deletions (pc1933, pc1934, pc1935,
pc2164, pc2987) or single domains fused to GFP (pc1936, pc1937,
pc1938, pc3061, pc3063) were described in previous studies, as well as
GFP-tagged DNMT1 construct (pc1099).
Mcherry-tagged VprBP (pc2954) was cloned from murine ESC
cDNA by overlap-extension PCR96 to replace Uhrf1 (pc1756) using AsiSI
and NotI restriction enzyme sites. To obtain an GFP-tagged VprBP
(pc2953), the VprBP coding sequence was excised from the vector just
described and used to replace the TDGsequence in GFP-TDG (pc2422).
For VprBP knockdown, a lentiviral vector encoding a VprBP siRNA (5’-
CCAGATCGTGTGTTTGTTGAGCTGTCTAA-3’) under a U6 promoter
and an in frame GFP under a CMV promoter was obtained from abm-
good (pc3922).
Plasmids encoding a hemagglutinin-tagged ubiquitin and a major
satellite repeat recognizing pol ydactyl zinc finger (MaSat) fused to GFP
(pc1803) or a GFP-recognizing nanobody (pc2469) were described in
previous publications. To create a miRFP-tagged MaSat (pc3944),
pmiRFP670-N1 (pc3379)) was used and MaSat-GFP (pc1803) was cut
with SacI and AgeI and fused with miRFP670.
Details and references of all plasmids and oligonucleotides used
in the cloning are shown in Supplementary Tables 2, 3. Additionally,
schemes for the main Tet-X constructs are shown in Supplementary
Fig. 9. SerialCloner (Version 2.6.1) was used for plasmids design and all
constructs were verified by DNA sequencing.
Live-cell microscopy and image analysis
Live-cell time lapse, live-cell imaging and fluorescence recovery after
photobleaching (FRAP) experiments were performed with a Nikon Ti-E
microscope equipped with an UltraVIEW VoX spinning disk confocal
unit (PerkinElmer, UK), controlled by Volocity 6.3 software (Perki-
nElmer, UK), and equipped with a live-cell chamber (ACU control,
Olympus) set at 37 °C with 5% CO
2
and 60% air humidity. Z-stacks were
acquired with a ×60/1.49 NA CFI Apochromat TIRF oil immersion
objective (voxel size, 0.12 × 0.12 × 0.3–1µm; Nikon, Tokyo, Japan) or a
100x/1.49 NA CFI Apochromat TIRF oil immersion objective (voxel size,
0.071 × 0.071 × 0.5–1µm; Nikon, Tokyo, Japan) and a cooled 14-bit CCD
camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan, Cat.No.:
C9100-50). Z-stack images were analyzed using Volocity 6.3 (Perki-
nElmer, UK) and Fiji. Mid Z-planes were assembled onto videos and
annotated using Fiji98 (https://Fiji.nih.gov/ij/).
Protein accumulation at heterochromatin analysis. Hetero-
chromatin accumulation ability of ectopically expressed, fluores-
cently tagged proteins, during ongoing DNA replication, was
assessed by transfecting cells with the respective plasmids and
imaging cells live 8–12 h post-transfection. Confocal Z-stacks (voxel
size, 0.12 × 0.12 × 0.5 µm) were acquired using the aforementioned
Nikon-Ti-E setup. Z-stacks were analyzed using Fiji with a self-
written semi-automated macro routine (macro 1). For this purpose,
Z-stack images were converted to maximum Z-projections. After
this step, three circular regions with a radius of 4 pixels were chosen
in the nucleoplasm of each cell, and correspondingly, three circular
same-sized regions in heterochromatin marked by PCNA, were
measured. The ratio of the averaged signal intensities of the protein
of interest in PCNA marked pericentric heterochromatin and in the
nucleoplasm was plotted with RStudio (Version 1.1.447)99 as relative
accumulation values at replicating heterochromatin.
Fluorescence recovery after photobleaching. For FRAP experi-
ments, C2C12 cells were transfected by electroporation 8 h prior to
the experiments. FRAP analysis was essentially performed as
described before (33). Briefly, spots were chosen in nuclei of late-
replicating cells or non-replicating cells and bleached for 600 ms
with a 488 nm laser for GFP-tagged constructs (Fig. 5H) or 1 s with a
561 nm laser for mcherry-tagged proteins (Fig. 3D), both set to
100%. In late-replicating cells, replication foci marked by PCNA or
regions without PCNA accumulation were bleached. For analysis,
raw intensities of the bleached area (ROI1), a non-bleached nuclear
area (ROI2), and a background area outside the cell (ROI3) as well as
the corresponding time points were calculated using a custom Fiji
macro100. Quantitative evaluation was performed using Fiji, and
fluorescence intensity normalization and curve fitting were per-
formed with the easyFRAP software (https://easyfrap.vmnet.
upatras.gr/) as described before92 using the double normalization
method and the double term equation for the fitting procedure101.
Briefly, the mean intens ity of the bleached region (ROI1) was divided
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Nature Communications | (2022) 13:5173 21
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by the mean intensity of the ROI2 and both intensities were cor-
rected for the background levels (ROI3). T-half values were extrac-
ted from the mean exponential fitting, and plots were generated
with RStudio (Version 1.2.1335).
Fluorescent three-hybrid (F3H) assay. To address the effect of tar-
geting potential effector proteins to major satellite repeats on Tet1s/
Tet1-CD recruitment to heterochromatin, a previously described
fluorescent three-hybrid assay was adapted102. To this end, C2C12 cells
were transfected with plasmids encoding GBP-MaSat, GFP-tagged
Uhrf1 or Uhrf1 single domains or Uhrf1 single domain deletions and
mcherry-tagged Tet1-CD (Fig. 4G) and cells without GBP-MaSat where
used as control. Cells were imaged live 8–12 h post-transfection and
confocal Z-stacks (voxel size, 0.12 × 0.12 × 0.5 µm) were acquired using
the aforementioned Nikon-Ti-E setup. Z-stacks were analyzed and
mounted using Fiji. For quantification purposes, the percentage of
cells showing colocalization of GFP-tagged Uhrf1-X and mcherry-
tagged Tet1-CD in each S-phase stage was calculated with a minimum
number of 50 cells per sample.
Details about imaging systems, software and Fiji macros are
shown in Supplementary Tables 6, 7.
Immunofluorescence, microscopy and image analysis
For immunofluorescent staining of modified nucleotides in cells, Tet1
or replication associated proteins, cells were seeded on gelatin-coated
glass coverslips and fixed in 3.7% formaldehyde (Sigma-Aldrich Chemie
GmbH,Steinheim,Germany,Cat.No.:F8775)in1xPBSfor10min.After
three washing steps with PBS-T (1x PBS, 0.01% Tween-20), cells were
permeabilized with 0.5% Triton X-100 in 1x PBS for 20 min, incubated
in ice-cold 88% methanol for 5 min and washed again.
For the staining of modified nucleotides, cells were incubated
with 10 mg/mL RNaseA in 1x PBS for 30 min at 37 °C. After three more
washing steps, cells were blocked with 1% BSA in 1x PBS at 37 °C for
30 min. The primary antibody solution contained a final concentra-
tion of 0.5% BSA, 1x DNaseI reaction buffer (60 mM Tris/HCl pH 8.1,
0.66 mM MgCl
2
,1mMβ-mercaptoethanol) and 0.1 U/mL DNaseI
(Sigma-Aldrich Chemie GmbH, Steinheim, Germany, Cat.No.: D5025).
In addition, another protocol for DNA denaturation was performed
and compared with the DNaseI treatment: the cells were incubated
with 4 N HCl for 15 min at room temperature (RT), rinsed with dis-
tilled water, and placed in 100mM Tris–HCl (pH 8.5) for 10 min.
Then, cells were washed with PBS again. The primary antibody mix
was incubated at 37 °C for 70 min and afterwards washed three times
with PBS-TE (PBS-T + 100 mM EDTA). Cells that were immunostained
against Tet1, PCNA and VprBP were blocked in 1% BSA for 30 min after
fixation and incubated with primary antibodies diluted in 1% BSA for
60 min at room temperature. Antibodies against 5mC103 and 5hmC,
5fC or 5caC (all Active Motif, La Hulpe, Belgium), as well as char-
acteristics of antibodies against Tet110, PCNA and secondary anti-
bodies, as well as dilutions used, are described in Supplementary
Table 5. After incubation with the primary antibodies, cells were
washed three times with PBS-T. For the detection of the primary
antibodies, cells were incubated with fluorescently tagged secondary
antibodies diluted in 1% BSA. Alexa Fluor 488-conjugated goat anti-
rabbit IgG (H + L) (1:500), Alexa Fluor 488-conjugated goat
anti-mouse IgG (H + L) (1:500), Alexa Fluor 594-conjugated goat anti-
rabbit IgG (H + L) (1:250; ThermoFisher Scientific, Invitrogen, Carls-
bad CA, USA, Cat.No.: R37117), Cy5-conjugated donkey anti-mouse
IgG (H + L) (1:250; The Jackson Laboratory, Bar Harbor, ME, USA,
Cat.No.: 715-715-150). After an incubation of 45 min at room tem-
perature, cells were washed three times with PBS-T, counterstained
with 10 mg/ml DAPI (4’,6-diamidino-2-phenylindole, Sigma-Aldrich
Chemie GmbH, Steinheim, Germany, Cat.No.: D9542) and mounted in
Mowiol®4-88 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany,
Cat.No.: 81381).
In situ quantification of cytosine modification levels. To address the
endogenous levels of 5mC, 5hmC, 5fC and 5caC in heterochromatic
domains of MCF10a or MCF7 cells, cells were immunostained against
5mC and 5hmC, 5fC or 5caC and counterstained with DAPI (Fig. 1B).
Confocal Z-stacks with a Z-step length of 0.5 µmwereacquiredwiththe
described Nikon Ti-E spinning disk setup: maximum Z-projections
were generated and circular ROIs with a radius of 5 pixelswere created
around DAPI-dense regions (Supplementary Fig. 1B). To quantify
cytosine modifications levels in pericentric heterochromatin and the
nucleoplasm, C2C12 cells were transfected with GFP/mcherry-tagged
Tet1-variants or GFP (pc0713) alone and immunostained against 5hmC,
5fC or 5caC and counterstained with DAPI. Cells were imaged with the
before described confocal spinning disk setup and analyzed with Fiji.
For bothMCF and C2C12 cells, images were analyzed with a self-written
semi-automated macro routine for Fiji (macro 3). In brief, maximum
Z-projections were generated and binary nuclear masks were created
of the respective 16-bit images, based on the DAPI/5mC signal. For this,
images were thresholded with the triangle method and gray values
above 3000 were considered for the mask. A second binary mask for
C2C12 or MCF heterochromatin was created with the triangle thresh-
old method for all pixels with intensities above 7000. Based on these
two masks a third mask for the nucleoplasm was calculated and the
respective mean cytosine modification levels were measured and
plotted with RStudio (Version 1.1.447).
High-content microscopy. Endogenous Tet1/Tet1s, 5mC and 5hmC
levels and levels of ectopically expressed GFP- or mcherry-tagged
proteins were measured with the Operetta high-content screening
system (Perkin Elmer, UK) in wide-field mode, equipped with a Xenon
fiber optic light source and a 20x/0.45 NA long working distance or a
×40/0.95 NA objective. For excitation and emission, following filter
combinations were used, 360-400 nm and 410-480 nm for DAPI or
AMCA, 460-490 nm and 500–550 nm for GFP or Alexa-488 as well as
560–580 nm and 590–640 nm for TexasRed or Alexa-594. Fluores-
cence intensity levels were quantified with the Harmony software
(Version 3.5.1, PerkinElmer, UK) (Supplementary Table 7). For the
analysis of cells that were transfected with GFP or mcherry fusion
encoding plasmids and stained against 5mC, 5hmC, 5fC, 5caC or Tet1/
Tet1s and counterstained with DAPI, cell nuclei were first identified
according to their GFP/mcherry fluorescence and evaluated for mor-
phological properties like roundness and size. These parameters as
well as the mean and sum nuclear fluorescence intensities of DAPI,
GFP/mcherry and Alexa-594/488-labeled antibodies were calculated in
cells fitting these morphological criteria. For further analysis of cyto-
sine modifications levels, sum nuclear Alexa-594/488 intensities were
divided by the sum nuclearDAPI intensity, to compensate for potential
cell cycle-dependent fluctuations and these normalized values were
grouped according to the mean GFP/mcherry intensities of the
respective cells. GFP levels below 50 arb. units (Arbitrary Units) and
mcherry levels below 100 arb. units were considered as background.
The Alexa values of cells above the background level were normalized
by dividing the respective values by the averaged intensity of cells
below the background levels. For further analysis of Tet1/Tet1s levels,
sum nuclear Alexa intensities in MCF7, MCF10a and C2C12 cells were
divided by the average sum nuclear Alexa intensity in non-transfected
C2C12 (showing the lowest levels of Tet proteins). In all immuno-
fluorescence experiments, cells above GFP or mcherry background
levels were grouped in different expression level groups according to
their mean GFP or mcherry fluorescence levels: low (50-100 AU), mid
(100-500 AU), and high (500-1000 AU) for GFP, and low (100-500 AU),
mid (500-1000 AU), and high (1000-5000 AU) for mcherry (Supple-
mentary Fig. 2C). The nuclei of MCF10a and MCF7 cells that were
simultaneously stained for 5mC and 5hmC, 5fC or 5caC and counter-
stained with DAPI, were identified by their DAPI staining and further
grouped according to morphological criteria. The mean and sum
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved
nuclear fluorescence intensities of DAPI, Alexa-488-labeled 5mC and
Alexa-594 labeled 5hmC were calculated in cells fitting these criteria.
For normalization, the sum nuclear Alexa-488 and Alexa-594 levels of
cells that were only stained with the secondary antibodies, were
averaged to obtain a background fluorescence value. The sum nuclear
fluorescence values of Alexa-488/594 incells thatwere incubated with
primary and secondary antibody were each normalized to the
respective average background fluorescence and further normalized
to the DAPI fluorescence intensity. Exported measurement results
were further analyzed and plotted with RStudio (Version 1.1.447)99.
Quantitative analyses of the 3D nuclear landscape.C2C12cellswere
transfected with Tet1 or Tet1s, and 12 h after were fixed and immu-
nostained against PCNA. DNA was counterstained with DAPI as
described above. Leica SP5 II confocal microscope equipped with an
HCX PL APO ×100/1.44 oil objective was used for imaging of Z-stacks
and Fiji was used for image processing. RStudio was used for image
analysis with Nucim and statistics61. We assessed seven different
chromatin compaction levels in individual cell nuclei using DAPI as
a proxy for local differences in chromatin compaction. The tools
are freely available in open-source R packages “nucim”and
“bioimagetools”.
Details about imaging systems, software and Fiji macros are
shown in Supplementary Tables 6, 7.
Co-immunoprecipitation and western blotting
Co-immunoprecipitations were essentially performed as described
before104. In brief, HEK293-EBNA cells growing in 100 mm dishes were
PEI-transfected and harvested by centrifugation 48 h later at 90%
confluence. The cell pellet was washed with ice-cold 1x PBS and pel-
leted again. Thesupernatantwas discarded and the pellet resuspended
in 200 µL lysis buffer (20mM Tris-HCl pH 8, 150mM NaCl, 0.5 mM
EDTA, 0.5% NP-40) supplemented with Pepstatin A (1 µM; Sigma-
Aldrich, St. Louis, MO, USA), PMSF (10 µM, Sigma-Aldrich, St. Louis,
MO, USA) and AEBSF (1 mM, AppliChem, Darmstadt, Germany). Cells
were homogenized with a syringe (21G needles, 20 strokes) and
incubated on ice for 30 min with repeated vortexing in between.
Lysates were then cleared by centrifugation for 15 min at 13,000gand
4 °C. 15% of the lysate was used as input and the rest was incubated
with GFP-binder beads produced as described before53 on a rotator at
4 °C for 90 min. Afterwards, the beads were washed 3 times with
500 µL washing buffer. Input and bound fraction were boiled at 95 °C
in 4x SDS loading buffer (200 mM Tris/HCl pH 6.8, 400 mM DTT, 8%
SDS, 0.4% bromophenol blue and 40% glycerol), separated on a 6%
SDS-PA (sodium dodecyl sulfate–polyacrylamide) gels and transferred
onto nitrocellulose membranes (GE Healthcare, Munich, Germany).
Membranes were blocked with 3% low fat milk in 1x PBS for 30 min and
subsequently incubated with the primary antibodies diluted in a
blocking buffer for 2 h at room temperature. After washing with 1x PBS
supplemented with 0.01% Tween-20, the membrane was incubated
with the respective secondary antibodies. For the detection of GFP- or
RFP-tagged proteins, rat monoclonal anti-GFP (ChromoTek, Planegg-
Martinsried, Germany) and rat monoclonal anti-RFP105 were used as
primary antibodies. HA-tagged ubiquitin used for the ubiquitination
assay was detected with the mouse monoclonal antibody anti-HA tag
(clone 12CA5) directed against a nonapeptide sequence derived from
the influenza hemagglutinin protein106. Antibodies details are sum-
marized in Supplementary Table 5.
For western blot and endogenous co-immunoprecipitation,
MCF7, MCF10a and C2C12 cells growing in 100 mm dishes were pro-
cessed for protein extraction and cell lysates were prepared as
described above. For endogenous co-immunoprecipitation of Tet1/
Tet1s in MCF7 and MCF10a cells were synchronized at late G1/early S,
15% of the cell lysate was used as input and the rest was incubated as
with Pierce™Protein G agarose beads (ThermoFisher Scientific)
preincubated with antibodies against Tet1/Tet1s or MIN (attP synthetic
peptide) as negative control for immunoprecipitation, for 1 h. For
detection of the different proteins, the following primary antibodies
were used: rabbit anti-VprBP polyclonal antibody (Proteintech, USA),
rabbit anti-Cul4B polyclonal antibody (Sigma-Aldrich), mouse anti-
Cul4 (H-11) monoclonal antibody (Santa Cruz Biotechnology) and Tet1/
Tet1s were detected with monoclonal rat antibody (clone 4H7)10.As
secondary antibodies, horseradish peroxidase (HRP) conjugated goat
anti-rat IgG (Jackson; West Grove, PA, USA) (1:500), sheep anti-mouse
IgG (Amersham Pharmacia Biotech, United Kingdom), and goat anti-
rabbit IgG (Sigma-Aldrich, United States) were used (1:5000). Toimage
the membranes the Amersham AI600 Imager wasused (GE Healthcare,
Chicago, II, USA). Cut-outs of the membranes were made for a better
composition of the figures. Details about antibodies and imaging sys-
tems are shown in Supplementary Tables 5, 6. Uncropped and
unprocessed scans of all of the blots are available in the Source Data
file. The full images and replicates are provided with the data sets
uploaded to https://doi.org/10.48328/tudatalib-594.3.
Chromatin immunoprecipitation
C2C12 were transfected with different GFP-tagged constructs and
after synchronization were fixed with 1% formaldehyde for 10 min at
room temperature. The crosslink was quenched with 125 mM glycine
(5 min at room temperature). Nuclei were isolated after mild lysis in
hypotonic buffer (10 mM HEPES pH 8, 1.5 mM MgCl2, 60 mM KCl)
and 20 strokes in a tight dounce homogenizer. Chromatin was
sheared in the sonication buffer (0.5% SDS 10 mM EDTA, 50 mM
Tris–HCl pH 8.1). Fragmentation of chromatin was carried out by
ultrasound treatment using a Branson 250 Sonifier (4 × 30 sec at 20%
power with a 1 min break on ice in between shearings) obtaining a
shearing distribution from 1000bp-300bp with most of the DNA
concentrated in the 500 bp range. For each transfection, chromatin
from one p100 plate at 80% confluency was immunoprecipitated by
incubation with GFP-binder beads produced as described before53 on
a rotator overnight at 4 °C. Afterwards, cell debris were pelleted at
8000gand 4 °C for 10 min, and the supernatant was taken as
unbound or input fraction. The beads were washed three times and
collected by centrifuging at 8000gand 4 °C for 5 min. The chromatin
collected (ChIP sample) was then reverse-crosslinked in the presence
of 200 mM NaCl at 65 °C for at least 5 h, followed by RNase A
(50 μgml
−1) treatment for 30 min at 37 °C and proteinase K (100 μg
ml−1) treatment for 3 h at 50 °C. DNA elution was carried out in 1%
SDS, 100 mM NaHCO3, in a rotary shaker at room temperature for
30 min. Pure DNA was isolated using the Qiagen PCR purification kit,
and was used to perform real-time quantitative polymerase chain
reaction of major satellite repeats. The input sample was essentially
prepared following the same protocol.
Genomic DNA preparation
For the preparation of genomic DNA (gDNA), MCF cells, as well as
C2C12 mouse myoblast were pelleted (10 min, 200g,4°C)andincu-
bated overnight at 50 °C in TNES buffer (10 mM Tris; pH 7.5, 400 mM
NaCl, 10mMEDTA, 0.6% SDS) supplemented with 1 mg/ml Proteinase K
(Carl Roth, Karlsruhe, Germany)107. RNA was removed by the addition
of 0.6 mg/ml RNase A (Qiagen, Hilden, Germany) for 30 min at 37 °C.
gDNA was extracted by the addition of 6 M NaCl at a final concentra-
tion of 1.25 M and vigorous shaking107. After centrifugation (15 min,
11,000g, RT), gDNA was precipitated from the supernatant by the
addition of 100% ice cold ethanol followed by incubation at –20 °C for
1 h and subsequent centrifugation (10 min, 11,000g,4°C).Aftera
washing step in 70% ethanol, gDNA was air dried and solved in ddH
2
O.
After chromatin immunoprecipitation (ChIP), isolated gDNA from
C2C12 cells was fragmented (<2000 bp) by sonication. The con-
centration of gDNA was measured on a TECAN infinite M200 plate
reader (Tecan Group Ltd., Maennedorf, Switzerland).
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 23
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Real-time quantitative polymerase chain reaction of major
satellite repeats
For C2C12 mouse myoblasts, equal amounts of DNA (0.5 ng) wereused
for real-time PCR with Platinum SYBR Green qPCR SuperMix-UDG w/
ROX (Invitrogen, Paisley PA4 9RF, UK) on a StepOne-Plus Real-Time
PCR System (Applied Biosystems, Darmstadt, Germany) according to
the manufacturer’s instruction. UDG was inactivated for 2 min at 50 °C
and DNA wasdenatured for 10min at 95 °C. Cycle parameters were set
to 40 cycles of 15 s at 95 °C and 45 s at 60 °C. Specificity of amplifica-
tion products was confirmed by melting curve analysis. DNA levels
were normalized to Gapdh and calculated using the comparative CT
method. Primers for quantitative real-time PCR contained the follow-
ing sequences: Gapdh forward: 5-CCA TACATACAGGTT TCT CCA G-3,
Gapdh reverse: 5-CTG GAA AGCTGT GGC GTG ATG G-3,MajSat for-
ward: 5-GGC GAG AAA ACT GAA AAT CAC G-3, MajSat reverse (20):
5-AGG TCC TTC AGT GTG CAT TTC-3108.
UHPLC‑MS/MS analysis of DNA samples
Isolation of genomic DNA was performed according to earlier pub-
lished work109. The amount of DNA was calculated after photo-
metrically determining the DNA concentration (Implen
NanoPhotometer, N60, Version NPOS 4.2e build 14900), before being
digested to nucleosides by Nucleoside Digestion Mix (M0649S) kit
(New England BioLabs Inc.) according to the manufacturer’sinstruc-
tions. 1 μg of genomic DNA was used, to which we spiked a heavy
labeled nucleoside mix. This mix was prepared from heavy labeled
nucleosides with the final concentrations of [15N2,D2]-, [D3]-mdC 51.0
pmol, hmdC 7.7 pmol, [15N2]-fdC 0.04557 pmol, [15N2]-cadC 0.04301
pmol and [15N5]-8-oxo-dG 0.109 pmol. A final volume of 50 μLwas
then incubated at 37 °C for 1.5 h. Before submitting the samples to
mass spectrometry, all samples were filtered by using an AcroPrep
Advance 96 filter plate 0.2 μm Supor (Pall Life Sciences). The injection
volume amounted to 39 μL. Data were processed according to earlier
published work110. Quantitative UHPLC-MS/MS analysis of the digested
DNA samples was performed using an Agilent 1290 UHPLC system
equipped with a UV detector and an Agilent 6490 triple quadrupole
mass spectrometer. Natural nucleosides were quantified with the stable
isotope dilution technique. For the concurrent analysis of all nucleo-
sides in one single analytical run, the source-dependent parameters
were as follows: gas temperature 80 °C, gas flow 15 L/min (N2), nebulizer
30 psi, sheath gas heater 275 °C, sheath gas flow 15 L/min (N2), capillary
voltage 2,500 V in the positive ion mode, capillary voltage −2250 V in
the negative ion mode and nozzle voltage 500 V. The fragmentor vol-
tage was 380 V/ 250 V. Delta EMV was setto500Vforthepositivemode.
Chromatography was performed with a Poroshell 120 SB-C8 column
(Agilent, 2.7 μm, 2.1 mm × 150 mm) at 35 °C using a gradient of water
and MeCN, each containing 0.0085% (v/v) formic acid, at a flow rate of
0.35 mL/min: 0 →4min; 0→3.5% (v/v) MeCN; 4 →6.9 min; 3.5 →5%
MeCN; 6.9 →7.2 min; 5 →80% MeCN; 7.2 →10.5 min; 80% MeCN;
10.5 →11.3 min; 80 →0% MeCN; 11.3 →14 min; 0% MeCN111.Concentra-
tions of DNA modifications were calculated using integrated values from
ion chromatogram peaks (Supplementary Data 2).
DNA glucosylation, MspI and HpaII digestion and PCR based
5hmC and 5mC detection (GluMS-PCR)
To detect 5hmC in the different MCF cell lines, gDNA was extracted
and measured for concentration and purity as described above. 10 µg
of gDNA were treated with or without 0.18 µMofT4phage
b-glucosyltransferase (T4-BGT)112 in a final volume of 300 µLsupple-
mented with 1x NEB cut smart buffer (NEB) and 1mM of UDP-glucose
(Sigma-Aldrich) for 18 h at 37 °C. Then 3 µg of glucosylated or mock
treated DNA was used for digestion with 100 units of MspI (NEB) at
37 °C for 18 h in a final volume of 50 µL, which was followed by treat-
ment with 20 µg of proteinase K (PK, Carl Roth GmbH) for 30 min at
50 °C. Following proteolysis, PK was inactivated for 10 min at 98 °C.
The MspI-resistant fraction was amplified using PCR with primers
flanking the MspI site (Supplementary Table 4, primers). After PCR, the
relative amounts of 5hmC were analyzed as described previously66.To
detect 5mC at position 482 in L1 5’UTR before Tet1 transfection, the
gDNA was treated with or without T4-BGT as described above. The
gDNA was further treated with MspI and HpaII (50 units each, NEB) or
mock for 18 h at 37 °C. The PCR reactions were performed as described
above, and quantification of PCR products was done by analytic gel
densitometry67 using Fiji: After electrophoresis, a digital image of the
gel was taken and densitometric readings obtained. The total pixel
density for each lane was determined by drawing a rectangle around
the bands and measuring the area of the intensity peaks. Each mea-
surement was normalized by the non-digested control.
Bisulfite sequencing and TAB (Tet-assisted bisulfite) sequencing
Bisulfite DNA sequencing analyses were performed as previously
described113,114.Briefly, genomic DNA from MCF cells was isolated as
described before. Next, 0.5 mi crograms of genomic DNA were bisulfite
converted using an EpiTect Bisulfite kit (Qiagen, Hilden, Germany)
following manufacturer’s instructions. To determine the DNA methy-
lation and hydroxymethylation status of the LINE 1 protomor and
Alu repeats, we performed PCR sequencing using primers in Supple-
mentary Table 4. To this end, 300–500 ng of converted genomic DNA
were used in a 50ml PCR reaction as follows: 2min at 95 °C, 35 cycles
of 30 s at 94°C followed by 30 s at 54 °C and 60 s at 72 °C, and a final
extension of 10min at 72 °C. Amplified products were visualized as
single bands in agarose gels and cloned in pCR4-TOPO TA Vector
optimized for sequencing (ThermoFisher Scientific) and at least 20
individual clones were sequenced for each sample. The unique
sequence in each clone was analyzed using QUMA at http://quma.cdb.
riken.jp/115. Next, the percent of methylated and hydroxymethylated
CpG sites was calculated by comparison to a consensus sequence from
untreated DNA.
Tet-assisted bisulfite (TAB) sequencing was performed as descri-
bed in previous studies44,116. Briefly, GFP-Tet1-CD was purified using
GBP beads from Sf9 insect cells infected with the recombinant bacu-
lovirus coding for mouse Tet1-CD with N-terminal GFP-tag (pc2838)44.
Afterwards, control DNA samples from GluMS-PCR experiments, in
which 5hmC was converted to 5ghmCby incubation with T4-BGT, were
incubated in an oxidation reaction during 4 hwith recombinant Tet1-
CD,at37°CinTetoxidationbuffer(10MFe(NH4)2(SO4)2.6H2O,
100 mM NaCl, 50 mM HEPES (pH 8), 1.2mM adenosine triphosphate
(ATP), 2.5 mM dithiothreitol (DTT), 1 mM a-ketoglutarate (aKG) and 2
mM L-ascorbic acid). Following Tet1-CD incubation, the reaction was
stopped by the addition of 20 g of proteinase K at 50 °C for 2 h.
Bisulfite treatment, PCR, cloning, and sequence analysis was per-
formed as described above.
Slot blotting
The catalytic activity of the purified proteins was verified by per-
forming an oxidation reaction with genomic DNA from MCF7 and
MCF10a cells. gDNA s amples were denatured at 99 °C for 10 min and
placed quickly on ice for 5 min. Denatured gDNA was mixed with ice
cold 20× saline–sodium citrate (SSC) buffer at a final concentration
of 4.8× SSC and blotted on a nitrocellulose membrane (Bio-Rad
Laboratories, Hercules, CA, USA), which was pre-equilibrated in 20×
SSC. After air-drying, the membrane was blocked with 3% milk in
PBST (PBS containing 0.1% Tween) for 30 min at room temperature
(RT), followed by incubation with either mouse rabbit 5mC (1:1000)
or rabbit anti 5caC (1:1000, Active Motif, La Hulpe, Belgium) anti-
bodies for 2 h at RT. The membrane was washed 3 times for 10 min
with PBST, before it was incubated with secondary antibody anti-
rabbit IgG Cy3 (The Jackson Laboratory, Bar Harbor, ME, USA) for
1 h at RT and imaged with the AI600 (GE Healthcare, Chicago, II,
USA). The amount of DNA loaded for each sample was verified by
Article https://doi.org/10.1038/s41467-022-32799-8
Nature Communications | (2022) 13:5173 24
Content courtesy of Springer Nature, terms of use apply. Rights reserved
staining the membrane with Methylene blue for 5 min (Sigma-
Aldrich).
For protein samples, purified mouse and human TET1 was spotted
directly onto the nitrocellulose membrane, which were incubated in
blocking buffer (5% (w/v) non-fat dry milk in PBS for 1 h at room tem-
perature. Anti-Tet1/Tet1s primary antibody was used undiluted and
incubated overnight at 4°C, followed by three washes in PBS/0.1%
Tween-20. Subsequently, membranes were incubated for 1 h at room
temperature with secondary antibody anti-rat IgG Cy3 (The Jackson
Laboratory, Bar Harbor, ME, USA) and imaged as described above. The
amount of protein loaded for each sample was determined with Pierce
660 nm protein assay (Thermo Scientific). Antibodies details are
summarized in Supplementary Table 5.
Homology modeling
A structure homology model of Tet1s was generated using the auto-
mated SWISS-MODEL homology modeling server pipeline117.Tothis
end, an atomic-resolution model of Tet1s was constructed based on
amino acids 579–1980 of Tet1 (NP_001240786.1) using the crystal
structure of Tet260 as template. The generated model was visualized
with UCSF Chimera (https://www.cgl.ucsf.edu/chimera/). Software is
indicated in Supplementary Table 7.
Statistics and reproducibility
Data visualization and statistical analysis were performed using RStudio
(versions V1.2.1335, V1.2.5033 and 1.1.447), https://rstudio.com/). In all
figures showing boxplots, the box represents 50% of the data, starting in
the first quartile (25%) and ending in the third (75%). The line inside
represents the median. The whiskers represent the upper and lower
quartile. Outliers are excluded and defined as 1.5 times the interquartile
range. Barplots show the average value of the distribution and the
whiskers represent the standard deviation with a 95% confidence
interval. Bar and line plots show normalized averaged values, and error
bars show the respective standard deviation. Line intensity profile plots
represent the fluorescence intensities along the distance of the selected
arrow segment. For the statistics, independent two-group comparison
was made for all conditions with Wilcoxon-Mann-Whitney or One-Way
ANOVA tests. Related to this, n.s., not significant, is given for p-values >
or equal to 0.05; one star (*) is given for p-values < 0.05 and > or equal to
0.005; two stars (**) is given for values < 0.005 and > or equal to 0.0005;
three stars (***) is given for values < 0.0005; between the top of two
boxes subjected to comparison. All statistical values (number (#) of cells
(N), mean, median, standard deviation (SD), standard error of the mean
(SEM), 95% confidence interval (CI) and p-values are summarized in
Supplementary Data 1. No statistical method was used to predetermine
sample size. Investigators were not blinded during the experiments and
when assessing the outcome. All cells analyzed showed the reported
behavior of the representative images shown in the respective figures.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The data that support this study are available from the corresponding
authors upon reasonable request. All data sets have been deposited
and are available at https://doi.org/10.48328/tudatalib-594.3.Source
data are provided with this paper.
Code availability
Code used in this study for assessment of DAPI intensity classes
(Supplementary Fig. 5G) is available under https://bioimaginggroup.
github.io/nucim/ and was published in ref. 61. All custom written R
scripts and ImageJ macros are available (https://doi.org/10.48328/
tudatalib-594.3).
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Acknowledgements
The authors thank Anne Lehmkuhl and Manuela Milden for excellent
technical assistance. We are indebted to Alexander Löwer for providing
us with MCF10a and MCF7 cell lines. We thank Alexander Rapp, Heinrich
Leonhardt and Weihua Qin for experimental advice and scientificdis-
cussions. This research was funded by the Deutsche For-
schungsgemeinschaft (DFG, German Research Foundation) grants CA
198/10-1 Project number 326470517 and CA 198/16-1 Project number
425470807 to M.C.C.
Author contributions
M.A., F.D.H., A.Z., F.S., S.Z., C.R., A.K.L., and T.C. performed experiments.
M.A., F.D.H. and A.Z. analyzed data. M.A., F.D.H., and M.C.C. conceived
the project, M.A. and F.D.H. generated final figures and M.A., F.D.H., and
M.C.C. wrote the manuscript. All authors revised, commented and
agreed on the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Competing interests
The authors declare no competing interests.
Additional information
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Florian D. Hastert or M. Cristina Cardoso.
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