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Inositol phosphates and core subunits of the Sin3L/Rpd3L histone deacetylase (HDAC) complex up-
regulate deacetylase activity
Ryan Dale Marcum1 and Ishwar Radhakrishnan1,*
From the 1Department of Molecular Biosciences, Northwestern University, 2205 Tech Drive, Evanston,
IL 60208-3500, USA
Running title: Inositol phosphates enhance Sin3L/Rpd3L deacetylase activity
*To whom correspondence should be addressed: Ishwar Radhakrishnan, Department of Molecular
Biosciences, Northwestern University, 2205 Tech Drive, Evanston, IL 60208-3500;
i-radhakrishnan@northwestern.edu; Tel: +1 847-467-1173; Fax: +1 847-467-6489.
Keywords: Transcription regulation, chromatin-modification, histone deacetylase (HDAC), protein-
protein interaction, cofactor-mediated protein-protein interaction, allosteric regulation, inositol phosphate
signaling; convergent evolution; gene expression; epigenetics
ABSTRACT
The constitutively nuclear histone deacetylases
(HDACs) 1, 2, and 3 erase acetyl marks on
acetyllysine residues, alter the landscape of histone
modifications, and modulate chromatin structure
and dynamics and thereby crucially regulate gene
transcription in higher eukaryotes. Nuclear HDACs
exist as at least six giant, multi-protein complexes
whose non-enzymatic subunits confer genome
targeting specificity for these enzymes. The
deacetylase activity of HDACs previously has been
shown to be enhanced by inositol phosphates,
which also bridge the catalytic domain in protein–
protein interactions with SANT domains in all
HDAC complexes except those that contain the
Sin3 transcriptional corepressors. Here, using
purified recombinant proteins, co-
immunoprecipitation and HDAC assays, pulldown
and NMR experiments, we show that HDAC1/2
deacetylase activity in one of the most ancient and
evolutionarily conserved Sin3L/Rpd3L complexes
is inducibly up-regulated by inositol phosphates,
but involves interactions with a zinc finger (ZnF)
motif in the Sin3-associated protein 30 (SAP30)
subunit that is structurally unrelated to SANT
domains, indicating convergent evolution at the
functional level. This implies that this mode of
regulation has independently evolved multiple
times and provides an evolutionary advantage. We
also found that constitutive association with another
core subunit, Rb-binding protein 4 chromatin-
binding factor (RBBP4), further enhances
deacetylase activity, implying both inducible and
constitutive regulatory mechanisms within the
same HDAC complex. Our results indicate that
inositol phosphates stimulate HDAC activity and
that the SAP30 ZnF motif performs roles similar to
that of the unrelated SANT domain in promoting
the SAP30–HDAC1 interaction and enhancing
HDAC activity.
Lysine acetylation is an abundant post-
translational histone modification found in
euchromatin that is correlated with increased
chromatin dynamics and access to the underlying
DNA template to regulatory factors, effectors, and
molecular machines (1). Histone deacetylation
reverses these effects and the chemical
transformation is mediated, in large part, by
constitutively nuclear, Class I Zn2+-dependent
HDACs 1, 2, and 3 (2,3). In mammals, these
enzymes function in the context of at least six giant,
multiprotein complexes including the
Sin3L/Rpd3L, Sin3S/Rpd3S, NuRD, CoREST,
MiDAC, and SMRT/NCoR complexes that are in
turn recruited via protein-protein interactions by
sequence-specific DNA-binding transcription
factors and/or by post-translational modifications in
the tail regions of histones (4-7). While some
progress has been made regarding how these
complexes are assembled, including the
recruitment and integration of HDACs in these
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complexes, the precise molecular role(s) of
individual subunits has been, with few exceptions,
enigmatic.
The mammalian 1.2-2 megadalton
Sin3L/Rpd3L HDAC complex regulates the
expression of a large variety of genes with roles in
cell cycle control, differentiation, metabolism and
stem cell maintenance (4,7-9). Five subunits of the
complex including Sin3A/B, SAP30/SAP30L,
Sds3/BRMS1/BRMS1L, HDAC1/2 and RBBP4/7
(paralogous subunits are distinguished by a ‘/’) are
found in a broad range of eukaryotes from yeast to
human and are thought to comprise the core
complex (10-14). Among these five subunits,
HDAC1/2 and RBBP4/7 are shared with multiple
chromatin-modifying complexes, Sin3A and Sin3B
assort into the Sin3L/Rpd3L and Sin3S/Rpd3S
complexes, respectively, while the SAP30 and Sds3
subunits (and their paralogs) are unique to the
Sin3L/Rpd3L complex.
We and others have previously shown through
biochemical and/or structural studies that the Sin3A
subunit, besides being directly recruited by
transcription factors via interactions with the PAH1
and PAH2 domains, performs a scaffolding
function by engaging in direct interactions with
HDAC1, Sds3, and SAP30 (4,7). Whereas the
interactions with HDAC1 and Sds3 is mediated by
the HDAC-interaction domain (HID; (15)), the
interaction with SAP30 occurs via the PAH3
domain, located immediately N-terminal to the HID
(16). In these studies, we also showed that Sds3
provides dimerization and putative nucleic acid
binding functions for the complex. However, the
role of SAP30 has been less obvious because,
besides a Sin3-interaction domain (SID), the
polypeptide contains only one other domain that
harbors a unique zinc finger (ZnF) motif that we
and others proposed could bind to negatively-
charged molecules through a highly conserved,
positively-charged surface (17,18). Indeed,
elucidation of the precise molecular roles of the
Sin3, Sds3, and SAP30 polypeptides has been
hampered, due in large part, to the lack of broadly
distributed and well-characterized protein-protein
interaction domains within these proteins.
Recent studies have unexpectedly suggested
that Class I HDACs 1, 2, and 3 are regulated by
inositol phosphates derived from membrane lipids
(19-21). Besides promoting interactions between
HDAC1/3 and SANT-domain bearing proteins via
a conserved surface located near the enzyme active
site, certain inositol phosphates were shown in
these studies to significantly enhance the activity of
the enzyme. However, unlike in the NuRD and
SMRT HDAC complexes, where these inositol
phosphate-dependent interactions have been
described, none of the subunits in the Sin3L/Rpd3L
complex harbor SANT domains. Here, we show
that the SAP30 ZnF motif, which is structurally
unrelated to SANT domains, performs similar roles
in promoting the interaction between SAP30 and
HDAC1 while concomitantly enhancing enzyme
activity. We further show that the histone H3/H4-
interacting subunit RBBP4 also enhances the basal
deacetylase activity of HDAC1 through
constitutive interactions with the catalytic domain.
Results
SAP30 Engages with HDAC1 in the Sin3L/Rpd3L
Complex
Since the SAP30 ZnF motif was previously
shown to bind to molecules enriched in negatively
charged moieties, we surmised that the SAP30
subunit might have a role akin to the SANT
domains of MTA1 and SMRT corepressors in the
NuRD and SMRT complexes (19-21). Specifically,
we asked whether SAP30, and the ZnF motif in
particular, could directly interact with HDAC1. In
co-immunoprecipitation (co-IP) experiments with
HA-tagged HDAC1 and Flag-tagged SAP30, the
wild-type proteins exhibited a robust interaction
(Fig. 1A). However, mutation of C112, a key Zn2+-
coordinating residue, to alanine in SAP30, that we
previously showed precluded proper folding of the
ZnF (17), failed to efficiently immunoprecipitate
HDAC1, implying that a properly folded ZnF motif
was necessary for a robust interaction with
HDAC1. We then asked whether full-length
HDAC1 or a construct lacking a large section of the
unstructured C-terminal tail, but retaining the
catalytic domain, was sufficient for the interaction.
Both constructs were efficiently
immunoprecipitated by SAP30 (Supp. Fig. S1A),
implying that the catalytic domain of HDAC1
served as the primary site of interaction.
To test whether the SAP30 ZnF motif was
sufficient to engage HDAC1 in direct interactions,
we expressed and purified the zinc finger and
conducted pulldown assays with immobilized Flag-
tagged HDAC1. Although no binding was detected
for the His6-tagged SAP30 ZnF when presented
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alone (Fig. 1B), inclusion of inositol 1,4,5,6-
tetrakisphosphate (InsP4) resulted in a robust
interaction between the two proteins. However, the
interaction was abrogated when HDAC1 was
presented with SAP30 ZnF pre-treated with EDTA,
even though the binding reaction was supplemented
with InsP4. This again confirmed that the
interaction was specific and that a properly folded
zinc finger was critical for interactions with
HDAC1. Previous studies involving HDAC1/3 and
SANT domains indicated that a minimum of four
phosphate groups at strategic positions in the
inositol ring were necessary for efficient
interactions (21). We tested whether inositol 1,4,5-
triphosphate (InsP3) and inositol 1,2,3,4,5,6-
hexakisphosphate (InsP6) could similarly promote
interactions between the SAP30 ZnF and HDAC1
over a range of inositol phosphate concentrations.
Whereas, InsP6 promoted a robust interaction at
high micromolar concentrations, by contrast, InsP3
did so poorly, even at 100 µM concentration (Fig.
1D). This implies that the same trends noted
previously involving inositol phosphate-mediated
protein-HDAC interactions (21), also apply to the
SAP30-HDAC1 interaction.
We then asked whether SAP30
immunoprecipitates harbored deacetylase activity
in cells. Flag-tagged wild-type SAP30 or the
C112A mutant were co-expressed with HDAC1,
and following immunoprecipitation, eluted from
the anti-Flag resin (Supp. Fig. S1B). Deacetylase
activity assays demonstrated ~4-fold increased
activity in the wild-type immunoprecipitates
compared to the C112A mutant (Fig. 1C).
However, addition of InsP6 did not lead to any
significant increase in deacetylase activity, likely
due to the presence of endogenous InsPs in the
immunoprecipitates. Collectively, these results
demonstrate that SAP30 can directly and
independently recruit deacetylase activity via
interactions with HDAC1 in a manner that is
dependent on an intact ZnF in mammalian cells.
To gain insights into the SAP30 ZnF interaction
with inositol phosphates at the molecular level, we
performed titrations with 15N-labeled protein and
InsP6. The addition of increasing amounts of InsP6
induced relatively small, yet readily discernible
changes in the NMR spectrum with only a subset of
resonances showing significant perturbations (Fig.
1E). The resonances ‘shifted’ as a function of added
ligand, indicative of fast dissociation kinetics for
the complex. Indeed, the modest stability of the
complex was confirmed by the equilibrium
dissociation constant of 250 ± 11 µM obtained
through non-linear least squares fitting of the
binding isotherms (Fig. 1E). Mapping the chemical
shift changes on to the structure of the SAP30 ZnF
motif reveals a contiguous surface with the most
significant perturbations (Supp. Fig. S2). This
surface overlaps with the conserved, basic surface
formed by residues in the α1 and α2 helices that
was previously anticipated to be functionally
relevant (17). However, NMR titration experiments
to probe whether the same surface is also involved
in HDAC1 interactions was precluded by the
limited solubility of the HDAC.
SAP30 ZnF and HDAC1 Interact via Conserved
Surfaces
To determine the role of individual residues in
promoting the formation of the SAP30 ZnF-
HDAC1 complex, we mutated several basic
residues located in the α1 and α2 helices that were
implicated in inositol phosphate binding by our
NMR studies and evaluated them by pulldown
assays. To maximize disruption, the residues were
mutated to glutamic acid either in isolation or in
combination with another residue. Rather
unexpectedly, most of the mutations that were
screened produced only modest or no effects on
binding to HDAC1 (Fig. 2A). The sole exception
was R88E that either in isolation or in combination
with R123E completely abrogated the interaction,
implying that the R88 side chain is most likely
involved in engaging with phosphate moieties in
inositol phosphates.
We then asked whether other residues in the
general vicinity of R88 in the three-dimensional
structure were also important for the interaction. In
this regard, we mutated Q120 and two serines (S84
and S86) in the loop segment preceding helix α1
along with Y110 located in another loop segment
preceding α2 in these interactions. Whereas the
Q120E mutant bound to HDAC1 just like the wild-
type protein, both the S84E,S86E double mutant as
well as the Y110A mutant showed complete loss of
binding to HDAC1 (Fig. 2B). Consistent with the
other results, deletion of the SAP30 ZnF C-terminal
unstructured tail segment (spanning residues 124-
131), which is located distally from R88 and
harbors multiple basic residues that showed
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chemical shift perturbations in InsP6 titrations, had
little or no effect on HDAC1-binding activity.
To define the complementary protein surface
involved in the formation of the ternary complex,
we mutated basic residues in HDAC1 that were
previously shown to bind to inositol phosphates
including K31, R270, and R306 to glutamic acid
both individually and in combination (21). The
individual mutations significantly reduced SAP30
ZnF binding activity, while the triple mutant
completely abrogated binding (Fig. 2C). We also
mutated residues near these residues including Y23
and H33 that comprise the binding site in the
MTA1-InsP6-HDAC1 complex. While the Y23A
mutant bound SAP30 ZnF comparably as the wild-
type protein, binding by the H33A mutant was
significantly diminished, implying that residues
beyond those involved in binding inositol
phosphates were important for the stability of the
ternary complex.
Collectively, our mutagenesis studies reveal
that R88, S84, S86, and Y110 side chains in SAP30
ZnF and H33, K31, R270, and R306 in HDAC1 are
critical for the stability of the ternary complex. In
both proteins, the residues define contiguous
surfaces for interactions with inositol phosphates
and HDAC1 (Fig. 2D). To gain more detailed
insights into the mode of interaction involving the
two proteins and InsP6, we performed multi-body
molecular docking of all three components using
HADDOCK with those residues found to be critical
for the interaction designated as ‘active’ residues
for docking (22). The resulting docked structures
were clustered based on the fraction of common
inter-molecular contacts (>0.75). Twenty-eight
structures from the largest cluster (out of 100
structures used for the final refinement in explicit
solvent) with no NOE violations >0.3 Å and
favorable energies were selected for analysis.
While the backbone level precision of the
ensemble for ordered regions is reasonable (0.71 Å
from the average structure), the precision at the side
chain level for interfacial residues is significantly
lower since the restraints were sparse and side
chains at the interface were allowed to move during
the docking (Fig. 3A). Nevertheless, certain
features of the complex could be readily and
consistently detected. InsP6 adopts the chair-like
conformation in previously reported structures and
is sandwiched between the two proteins. One face
of the ‘ring’ of phosphates in InsP6 engages in
electrostatic and/or hydrogen bonding interactions
with K33, R270, and R306 of HDAC1, as noted
previously in the case of the NuRD complex ((21);
Fig. 3B & 3C). Arg88 is the sole side chain in
SAP30 ZnF engaging in salt bridging interactions
with the other face of the phosphate ring. The
hydroxyl groups of S84, S86, and Y110 all engage
different phosphates in hydrogen bonding
interactions. The short side chain of the serine
residues brings the polypeptide backbone in close
proximity to the phosphates, allowing it to engage
in hydrogen bonding interactions. The side chain of
Y110 inserts into a hydrophobic pocket formed by
the side chains of H33, I305, and Y336. Additional
hydrophobic interactions are observed, albeit less
consistently, while other types of favorable
interactions involving interfacial residues are
plausible, even though they are less common in the
ensemble. Notwithstanding these issues, the size of
the protein-protein interface is small, amounting to
a little under 300 Å2, on average, implying that the
primary affinity determinants stem from
interactions with the inositol phosphate moiety.
Indeed, every residue in both proteins involved in
these interactions is invariant over a broad range of
species (Supp. Fig. S3), suggesting strong
evolutionary pressure to preserve these
interactions.
Inositol phosphates, SAP30 and RBBP4 Enhance
HDAC1 Deacetylase Activity
Since certain inositol phosphates enhance
deacetylase activity in other HDAC-containing
multiprotein complexes, we asked whether this was
also the case for the Sin3L/Rpd3L complex. To
answer this question, HDAC1 deacetylase activity
assays using a model acetylated peptide substrate in
end-point format were conducted in the presence of
excess SAP30 ZnF (23). Whereas InsP4, InsP5, and
InsP6 enhanced deacetylase activity two- to three-
fold, albeit at high inositol phosphate
concentrations (>100 µM), InsP3 showed no effect
(Fig. 4A). The deacetylase activity was unaffected
by InsP4 in the absence of SAP30 ZnF. These
results thus imply that a minimum of four
phosphates in inositol phosphates was critical for
the enhancement of HDAC1 deacetylase activity.
To characterize the effects of HDAC1 and
SAP30 ZnF mutations on deacetylase activity, we
measured the kinetic parameters of the enzyme
using the same model substrate above. These assays
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were conducted in the presence of 100 µM InsP6.
Whereas the inclusion of SAP30 ZnF only had little
effect on the Km, the kcat was enhanced almost two-
fold (Fig. 4B; Table 1). The HDAC1 R270E mutant
exhibited diminished kcat, although the catalytic
efficiency (kcat/Km ratio) was unchanged from the
wild-type enzyme. The inclusion of SAP30 ZnF led
to only a slight enhancement in both kcat and kcat/Km
for this HDAC1 mutant. Similarly, the SAP30 ZnF
R88E,R123E mutant produced little or no increase
in these parameters relative to that measured for the
enzyme alone. Collectively, these results indicate
that the enhancement of HDAC1 deacetylase
activity emanates from stable formation of the
SAP30 ZnF-InsP6-HDAC1 ternary complex.
Since RBBP4 is known to interact with
HDAC1 constitutively, we asked whether it might
affect HDAC1 deacetylase activity. Compared to
HDAC1 alone, the inclusion of RBBP4 enhanced
kcat almost two-fold, although the Km also increased
significantly to only modestly increase the catalytic
efficiency (Fig. 4C; Table 1). The addition of
SAP30 ZnF further enhanced kcat while diminishing
Km to produce a robust surge in kcat/Km. However,
the effects of RBBP4 and SAP30 ZnF on HDAC1
deacetylase activity appear to be additive rather
than synergistic. In contrast to the enhancement in
deacetylase activity produced by RBBP4 and
SAP30 ZnF individually and in combination,
inclusion of Sin3A HID, which can also associate
constitutively with HDAC1, produced no such
effect (Supp. Fig. S4).
Since enhancements in deacetylase activity
occurred at somewhat high concentrations of
inositol phosphates, presumably reflecting a
modest affinity interaction between SAP30 ZnF
and HDAC1, we asked whether the enhancements
might be more physiologically relevant in the
context of the Sin3L/Rpd3L complex. Since the
interaction between Sin3 and SAP30 is of
nanomolar affinity and the structure of this complex
is known (16), we co-expressed and co-purified a
fusion protein of SAP30 and Sin3A spanning the
ZnF and SID domains in the former and the HID
domain of the latter with HDAC1. Since Sin3A
HID and HDAC1 can associate constitutively, we
conducted activity assays as a function of inositol
phosphate concentration. Whereas InsP3 caused a
modest increase in deacetylase activity at high
concentrations (>100 µM), InsP4, InsP5, and InsP6
produced several-fold enhancements even at low
micromolar concentrations (Fig. 4D; Table 2),
which is well within the intracellular concentrations
measured for these inositol phosphates (24),
implying that these cofactors most likely have a role
in regulating the deacetylase activity of HDAC1.
Discussion
The Sin3L/Rpd3L complex is ancient and one
of the most broadly distributed HDAC complexes
in eukaryotes with half a dozen subunits conserved
from yeast to human (14,25). In yeast, Rpd3, the
ortholog of mammalian HDAC1/2, is found in two
complexes Rpd3L and Rpd3S (26). Although Rpd3
has been reported to be regulated by inositol
phosphates and shares a conserved inositol
phosphate-binding surface with its orthologs from
a variety of species (27), the identity of the subunit
in these HDAC complexes involved in inositol
phosphate-based regulation has remained elusive,
until now. Our studies establish that this role is
performed by the evolutionarily conserved SAP30
subunit in the mammalian Sin3L/Rpd3L complex.
The involvement of the SAP30 zinc finger motif in
these interactions is unexpected because it shares
no overt sequence or structural similarity with
SANT domains that were previously implicated in
this role (19-21). Interestingly, the ZnF motif is
narrowly distributed and is found in only one other
protein in higher eukaryotes – that of the SAP30
paralog, SAP30L (28), which is also found in a
subset of Sin3L/Rpd3L complexes, targeting the
complex to the nucleolus (29). The human paralogs
share 79% identity and 89% similarity in this region
with all the residues deemed to be critical for
engaging with inositol phosphates or HDAC1 by
our studies invariant in the two proteins. Thus,
SAP30L is expected to play similar roles as SAP30
in regulating the deacetylase activity of HDACs 1
and 2 in these complexes.
Since inositol phosphate-based activation of
HDAC activity was first described for the human
HDAC3-containing SMRT complex (20), this
paradigm has been extended for the human
HDAC1/2 containing-NuRD (19,21), and now, to
the mammalian Sin3L/Rpd3L complexes (Fig. 5).
Despite differences in the identities of the proteins
involved and the structural motifs used for
engagement with inositol phosphates, each one of
these complexes rely on a minimum of four
phosphate groups in these cofactors for efficient
engagement and activation. Interestingly, whereas
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InsP3 has minimal impact on the deacetylase
activity in each of these complexes, InsP4, InsP5,
and InsP6 potentiate the deacetylase activity to
similar extents. The same three basic residues in the
catalytic domain are involved in engaging with the
cofactors in all three complexes, although the
protein-protein interaction surface extends beyond
these residues to varying extents. In all three cases,
engagement with the cofactor and the catalytic
domain results in tangible enhancement in
deacetylase activity. Although a structural basis for
the enhancement is not readily apparent from the
crystal structures, molecular dynamics simulations
have suggested that the effect could be due to
allosteric modulation of protein motions within the
loops comprising the active site (30).
Interestingly, the level of enhancement of
deacetylase activity differs for each complex but is
most critically dependent on the identity of the
HDAC. Whereas the deacetylase activity of
HDAC3 in the SMRT complex is potentiated
substantially by inositol phosphates (~100-fold;
(21)), that of HDAC1 in the NuRD and
Sin3L/Rpd3L complexes is comparably modest (2
to 3-fold; (19)). Although the baseline catalytic rate
and efficiency for HDAC1 is twice as that of
HDAC3, the cofactor-mediated activation for the
latter is much more substantial ((23); note that the
baseline activity of HDAC3 corresponds to when
SMRT is present, since the enzyme is otherwise
inactive). Despite the seemingly modest inositol
phosphate-mediated enhancement in deacetylase
activity for HDAC1, mutations in the inositol
phosphate-binding site causes a substantial
decrease in cell viability (31), highlighting a critical
role played by these small molecule cofactors and
HDAC-associating subunits in multi-protein
complexes.
In all three complexes characterized thus far,
HDACs are recruited and integrated into these
complexes through constitutive associations (by
SMRT, MTA1/2, and Sin3A/B in the SMRT,
NuRD and Sin3L/Rpd3L complexes, respectively),
and therefore, even in the absence of inositol
phosphates, all three complexes retain significant
deacetylase activity. The role of inositol phosphates
thus appears to be in turbocharging the activity of
these enzymes. Since these HDAC complexes have
well-documented roles in regulating the cell cycle
(4-7) and since InsP4, InsP5, and InsP6 levels
fluctuate, peaking in G1 (24), this would suggest
that these cofactors have evolved to function as
facile, molecular signals to tune HDAC activity.
A less appreciated role for inositol phosphates
is that they add another point of contact between the
HDAC and the interacting subunit, contributing to
the tight association and integration of the HDAC
subunits in these complexes. Unlike the MTA1
subunit in the NuRD complex that engages with
HDAC1 both constitutively and inducibly via
structurally distinct domains, association with
HDAC1 in the Sin3L/Rpd3L complex is more
distributed with specific domains of separate
polypeptides Sin3A and SAP30 performing these
roles. Interestingly, RBBP4, which is shared by
both of these complexes and engages in constitutive
interactions with HDAC1 also enhances
deacetylase activity. Structural studies of this
interaction are needed to understand the basis for
this activation.
Its evolutionary conservation notwithstanding,
SAP30 is a subunit unique to the Sin3L/Rpd3L
complex, which raises questions regarding the
identity of the corresponding subunit in the
Sin3S/Rpd3S complex. Since the latter complex
has a different role in transcription elongation in
actively transcribed genes, it is possible that a need
for turbocharging deacetylase activity does not
exist for this complex. Alternatively, since SAP30
ZnF and the SANT domains have independently
evolved to enhance HDAC activity, it is plausible
that additional motifs might yet exist. Indeed,
although SAP30 is evolutionarily conserved, the
SAP30 ortholog in yeast lacks the highly conserved
ZnF motif found in a broad range of organisms
from fly to human (17). Since the inositol
phosphate-binding site is conserved in yeast Rpd3,
we surmise that a yet another structural motif either
within SAP30 or in a different subunit of the Rpd3L
complex likely targets this site, suggesting that this
mechanism of HDAC regulation has independently
evolved multiple times and provides an
evolutionary advantage to the organism.
Experimental Procedures
Protein Expression and Purification
Escherichia coli BL21(DE3) was transformed
with pMCSG7 vector encoding SAP30 ZnF (aa 64-
131) and grown at 37 °C until it reached an OD600nm
of 0.6. Cultures were transferred to 16 °C and
induced with 1 mM isopropyl-β-D-
thiogalactopyranoside for 16 h prior to harvesting.
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Cells were suspended in 50 mM Tris-HCl buffer
(pH 8.0) containing 50 mM NaCl, 1 mM Tris-(2-
carboxy-ethyl)-phosphine (TCEP) hydrochloride, 1
mM phenylmethylsulfonyl fluoride (PMSF), 1 µM
leupeptin, 1 mM pepstatin and 0.1% Triton X-100
and lysed by sonication. After centrifugation the
lysate was loaded onto a HiTrap SP cation
exchange column (GE Healthcare). The protein was
eluted using a gradient from 0.05 to 1 M NaCl over
5 column volumes. Fractions were screened by
SDS-PAGE and those containing SAP30 ZnF were
combined and incubated with tobacco etch virus
(TEV) protease overnight to remove the His6 tag at
4 °C; this step was skipped in the case where His6-
tagged protein was required. Further purification
was achieved by reversed phase HPLC using a
linear gradient of 0.1% trifluoracetic acid (TFA)
and 0.1% TFA with 80% acetonitrile. Fractions
were screened by matrix assisted laser
desorption/ionization (MALDI) and lyophilized.
15N labeled samples were purified using the same
method except that cells were grown in 15N
ammonium sulfate containing M9 minimal media.
Point mutations in the SAP30 ZnF expression
construct were introduced using the QuikChange
mutagenesis protocol (Agilent) and the mutants
were produced using the same procedure as the
wild-type protein. All mutations were confirmed by
DNA sequencing.
Full-length human HDAC1 was cloned into a
pcDNA3.1 vector with a C-terminal Flag tag and
TEV protease cleavage site. Flag-tagged HDAC1
was expressed in non-adherent HEK293F cells
following a previously established protocol (32).
Cells were harvested after 48 h of expression and
the cell pellet was resuspended in lysis buffer (50
mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM
potassium acetate, 1 mM EDTA, 1 mM PMSF, 1
µM leupeptin, 1 mM pepstatin, 0.2% Triton X-100,
5% glycerol) and sonicated. After centrifugation,
lysates were pre-cleared by incubation with
Sepharose 4B resin (Sigma-Aldrich). Clarified
lysates were incubated with anti-Flag M2 affinity
resin (Sigma-Aldrich) for 1 h at 4 °C. The resin was
then washed with lysis buffer, high salt buffer (50
mM Tris-HCl, pH 7.5, 300 mM NaCl, 50 mM
potassium acetate, 1 mM EDTA, 5% glycerol), and
rinse buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 50 mM potassium acetate, 1 mM EDTA),
incubated overnight at 4 °C with TEV protease and
the flow-through containing untagged HDAC1 was
collected. HDAC1 elutions were combined and
dialyzed against 50 mM Tris pH 7.5, 100 mM NaCl,
50 mM KCl, 1 mM TCEP and then 50 mM Tris pH
7.5, 100 mM NaCl, 50 mM KCl, 1 mM TCEP with
10 µM ZnCl2. HDAC1 was co-expressed and co-
purified with RBBP4 using the same protocol as
that for HDAC1 alone.
HA-tagged HDAC1 was co-expressed with a
C-terminally Flag-tagged SAP30 (aa 64-131)-
Sin3A (aa 532-766) fusion construct sub-cloned
into the pcDNA3.1 vector. The complex was
purified by suspending the cell pellet in lysis buffer
and followed by sonication. Lysates were
centrifuged and pre-cleared with Sepharose 4B
resin and incubated with anti-Flag M2 affinity resin
(Sigma-Aldrich) for 1 h at 4 °C. The resin was
washed with lysis, high salt, and rinse buffers after
which it was incubated with 10 µM Sds3 SID (201-
234) for 1 h at 4 °C. The resin was washed with
rinse buffer and the complex was eluted using 150
µg/ml Flag peptide. The sample was immediately
dialyzed in the same manner as HDAC1 alone.
Co-immunoprecipitation Experiments
HEK293T cells were transfected using calcium
phosphate with the indicated expression plasmids
and collected 48 h post-transfection. Cells were
lysed in 20 mM HEPES, pH 7.9, 150 mM KCl, 5%
glycerol, 10 µM zinc acetate, 0.2% NP-40, 1 mM
PMSF, 1 µM leupeptin, 1 mM pepstatin. Cell
extracts were incubated for 1 h at 4 °C with anti-
Flag M2 affinity resin (Sigma-Aldrich). The resin
was washed five times with high-salt buffer (20
mM HEPES, pH 7.9, 300 mM KCl, 5% glycerol, 10
µM zinc acetate, 0.2% NP-40) and samples were
boiled with SDS-PAGE loading buffer. Proteins
were then resolved using SDS-PAGE, transferred
to nitrocellulose, and then probed with primary
antibodies, either anti-Flag (Sigma-Aldrich,
#F3165, 1:500 dilution) or anti-HA (Sigma-
Aldrich, #H3663, 1:500 dilution) after which anti-
mouse HRP-conjugated secondary antibody
(Thermo Fisher Scientific, #OB617005, 1:1000
dilution) was used. The blot was imaged using West
Pico chemiluminescent substrate (Thermo
Scientific, #34080) and a Syngene Pxi
chemiluminescent imager. Mutations in the Flag-
tagged, full-length human SAP30 and HA-tagged
human HDAC1 constructs were introduced using
the QuikChange protocol (Agilent); all mutations
were confirmed by DNA sequencing. Wild-type
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and mutant SAP30-HDAC1 complexes were
immunoprecipitated and eluted using excess Flag
peptide (ApexBio) for deacetylase activity assays.
Pulldown Experiments
HDAC1 was expressed and purified as
described above except that it was left bound to the
anti-Flag M2 affinity resin. SAP30 ZnF wild-type
and mutants were expressed as previously
described and pellets were resuspended in pulldown
buffer (20 mM HEPES, pH 7.9, 100 mM NaCl, 50
mM potassium acetate, 5% glycerol, 0.1% NP-40,
1 mM PMSF, 1 µM leupeptin, 1 mM pepstatin) and
lysed by sonication. Cell lysates were incubated
with HDAC1-bound anti-Flag resin in the presence
of 100 µM InsP4 or InsP6 for 30 min. The resin was
washed five times with pulldown buffer and boiled
in SDS-PAGE loading buffer. Samples were
resolved by SDS-PAGE, transferred to
nitrocellulose, stained with either anti-Flag or anti-
His (Thermo Fisher, MA121315, 1:1000 dilution)
antibody. Blots were visualized in the same manner
as described above for the co-IP experiments.
NMR Experiments
NMR data were acquired on a 600 MHz Agilent
DD2 NMR spectrometer at 25 °C. Dry, lyophilized
15N-SAP30 ZnF was dissolved in 50 mM Tris-d11
acetate-d4 buffer (pH 6.0) and 0.2% NaN3 with
equimolar ZnCl2 added. The inositol phosphates
InsP4 (Cayman Chemicals) and InsP6 (Sigma-
Aldrich) used for the titrations were dissolved in the
same buffer and used without additional
purification. Data processing and analysis was
performed using Felix (Felix NMR) and
NMRFAM-Sparky (33).
HDAC1 Deacetylase Activity Assays
A model peptide substrate containing an
aminocoumarin (AMC) moiety was used for the
deacetylase assays. The Ac-Gly-Ala-Lys(Ac)-
AMC peptide was purchased from Bachem
Americas and used in a slightly modified protocol
described previously (23). Reactions were
performed in a 384-well plate at room temperature
with 1 nM HDAC1 in 50 mM HEPES, pH 7.4, 100
mM KCl, 0.001% Tween-20, 5% DMSO, and 200
nM trypsin. Fluorescence data were acquired at
room temperature for 1 h on a Biotek Synergy 4
microplate reader with excitation and emission
wavelengths set to 360 and 440 nm, respectively.
Data were fitted with R using non-linear least
squares fitting. Endpoint assays were conducted in
a similar manner as above but using a fixed
concentration of 60 µM of the AMC peptide.
Steady-state fluorescence in these assays was
measured after 1 h of equilibration.
Molecular Modeling
The previously determined NMR structure of
SAP30 ZnF (PDB code: 2KDP; (17)) was docked
with the crystal structure of HDAC1 bound to InsP6
(PDB code: 5ICN; (21)) using HADDOCK version
2.2 (22). Active residues for docking were assigned
based on the results of pulldown experiments with
mutant proteins. These included S84, S86, R88, and
Y110 in SAP30 ZnF and K31, H33, R270, and
R306 in HDAC1, besides the InsP6 moiety. To
sample a broader range of conformations, the first
ten conformers of SAP30 ZnF in the NMR
ensemble were used in the calculations. Force field
parameters for InsP6 were generated for CNS using
the PRODRG server (34); these were supplemented
with additional chirality restraints. One thousand
structures were calculated during the rigid body
docking phase; the 200 best structures were used for
semiflexible docking, of which the 100 best
structures were refined in explicit solvent. Default
parameters were used except the distance restraints
were weighted more significantly (0.1, 0.5, 1.0 for
the three phases of the docking calculation).
Distance restraints between specific InsP6
phosphate groups and K31, R270, and R306 to
target distances observed in the crystal structure
were enforced throughout the calculation to ensure
the InsP6 moiety was anchored to HDAC1 (21).
Analysis of the resulting structures were conducted
using PLIP (35), LIGPLOT (36), and MONSTER
(37).
Acknowledgements: We would like to thank Gregory David (New York University) for critical reading of
this manuscript. We are grateful to the Robert Lurie Comprehensive Cancer Center at Northwestern for
supporting structural biology research.
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FOOTNOTES
This work was supported by grants from the American Heart Association (14GRNT20170003 and
17GRNT33680167) to I.R. R.D.M. was supported by pre-doctoral fellowships from the National Institute
of General Medical Sciences (T32 GM008382) and the American Heart Association (16PRE27260041).
The abbreviations used are: HDAC, histone deacetylase; HID, HDAC interaction domain; SID, Sin3
interaction domain; InsP, inositol phosphate; co-IP, co-immunoprecipitation; TCEP, Tris-(2-carboxy-
ethyl)-phosphine; PMSF, phenylmethylsulfonyl fluoride; TEV, tobacco etch virus; MALDI, matrix assisted
laser desorption/ionization; TFA, trifluoroacetic acid; AMC, aminocoumarin.
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Table 1. Kinetic parameters for wild-type and mutant HDAC1 enzyme activity*
Protein(s)
Km, µM
kcat, s-1
kcat/Km, M-1 s-1
HDAC1
HDAC1+SAP30 ZnF$
HDAC1 R270E
HDAC1 R270E+SAP30 ZnF$
HDAC1+SAP30 ZnF R88E,R123E$
HDAC1+RBBP4
HDAC1+RBBP4+SAP30 ZnF
$
13.98 ± 1.08
12.37 ± 0.68
10.24 ± 0.70
9.65 ± 1.25
17.36 ± 3.93
18.92 ± 1.63
11.78 ± 1.62
2.88 ± 0.07
4.75 ± 0.08
2.12 ± 0.05
2.46 ± 0.11
3.19 ± 0.24
4.89 ± 0.14
6.35 ± 0.25
2.06 × 105
3.84 × 105
2.07 × 105
2.55 × 105
1.84 × 105
2.58 × 105
5.39 × 10
5
*uncertainties correspond to 95% confidence intervals
$experiments with SAP30 ZnF proteins conducted in the presence of 30 µM ZnF and 100 µM D-myo-
inositol 1,2,3,4,5,6-hexakisphosphate (InsP6)
Table 2. EC50 parameters for HDAC1 activation by inositol phosphates*
Inositol phosphate
EC50, µM
D-myo-inositol 1,4,5-triphosphate (InsP
3
)
D-myo-inositol 1,4,5,6-tetrakisphosphate (InsP4)
D-myo-inositol 1,3,4,5,6-pentakisphosphate (InsP5)
D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6)
n.d.
5.18 ± 1.03
5.19 ± 0.88
6.07 ± 0.57
*uncertainties correspond to standard deviations from three independent measurements
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Figure 1. SAP30 physically associates via the ZnF domain with the catalytic domain of HDAC1. (A)
Domain map of SAP30. Co-immunoprecipitation (Co-IP) of HA-tagged HDAC1 by Flag-tagged, wild-type
and the C112A mutant of SAP30. (B) Pulldown assays conducted with immobilized Flag-tagged HDAC1
and His6-tagged SAP30 ZnF in the presence or absence of D-myo-inositol 1,4,5,6-tetrakisphosphate (InsP4)
and EDTA. (C) Endpoint activity assays with wild-type and C112A SAP30 immunoprecipitates, with and
without the addition of 100 µM InsP6.; error bars represent standard deviations (n=3). (D) Pulldown assays
conducted as in panel C, except in the presence or absence of varying amounts of InsP3 or InsP6. (E) 1H-
15N correlated spectrum of a 250 µM sample of 15N-SAP30 ZnF in the absence (magenta) and presence
(cyan) of eight equivalents of InsP6 (left panel). Expanded plot showing changes in peak position for V122
in the same titration (top right) and binding isotherms deduced from changes in chemical shifts for selected
residues (bottom right). In panels A, B, and D, molecular weights (in KDa) are shown on the right-hand
side of each blot.
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Figure 2. Mapping residues at the interfaces of the SAP30 ZnF-InsP6-HDAC1 complex via mutagenesis
and interaction assays. Pulldown assays conducted with immobilized Flag-tagged HDAC1 and His6-tagged
SAP30 ZnF single-site mutants of candidate basic residues involved in engaging with inositol phosphates
(A) or other solvent-exposed conserved residues (B). The protein labeled ∆124-131 corresponds to SAP30
ZnF that has a C-terminal deletion; the construct spans residues 64-123. (C) Pulldown assays with
immobilized Flag-tagged wild-type or mutant HDAC1 and His6-tagged SAP30 ZnF. All pulldown
experiments were conducted in the presence of InsP6. (D) Mutations mapped onto the surface of SAP30
ZnF (right) and HDAC1 (left), purple indicates mutations that lead to loss of binding in pulldown
experiments and green indicates no change was observed. In panels A, B, and C, molecular weights (in
KDa) are shown on the right-hand side of each blot.
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Figure 3. A structural model for the SAP30 ZnF-InsP6-HDAC1 complex obtained via molecular docking.
SAP30 ZnF backbone and side chain carbon atoms are colored in cyan while HDAC1 backbone and side
chain carbon and InsP6 carbon atoms are colored in tan; non-carbon atoms are shown in Corey-Pauling-
Koltun (CPK) colors. (A) The ensemble of 28 docked structures deemed to be consistent with the functional
studies described in Figure 2. (B) A close-up view of the interface showing the side chains of interacting
residues in the representative structure of the ensemble. (C) Stabilizing, inter-molecular hydrogen bonding
interactions (green lines) involving the side chains of essential residues in the two proteins and InsP6 in the
representative model in 3D (left panel) and in 2D (right panel). Dashed lines in the latter representation
indicate hydrogen bonds while semicircles with radiating spokes identify residues involved in inter-
molecular hydrophobic contacts. Carbon atoms in the 2D representation are colored in black while the bond
colors are in cyan for SAP30 ZnF, tan for HDAC1, and orange for InsP6.
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Figure 4. Enhancement in the catalytic activity of HDAC1 by SAP30 and RBBP4. (A) Changes in the level
of HDAC1 deacetylase activity measured in an end-point assay format as a function of inositol phosphate
concentration in the presence of 30 µM SAP30 ZnF. Changes in activity are reported relative to the level
of activity measured in the absence of inositol phosphates. The data shown in gray and denoted ‘InsP4 (
SAP30 ZnF)’ measured deacetylase activity in the presence of InsP4 but in the absence of SAP30 ZnF. (B)
Deacetylase assays conducted using wild-type or mutant SAP30 ZnF and/or wild-type or mutant HDAC1
in the presence of 100 µM InsP6. (C) Changes in the kinetic parameters in deacetylase assays of HDAC1
conducted in the absence or presence of RBBP4, SAP30 ZnF and 100 µM InsP6. (D) Changes in the level
of deacetylase activity measured in an end-point assay format as a function of inositol phosphate
concentration in the presence of stoichiometric amounts of HDAC1 and SAP30-Sin3A fusion protein.
Changes in activity are reported relative to the level of activity measured in the absence of inositol
phosphates.
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Figure 5. Structural comparison of various HDAC complexes. (A) Crystal structure of the MTA1-InsP6-
HDAC1 complex (PDB accession code: 5ICN), (B) structural model of the SAP30 ZnF-InsP6-HDAC1
complex from molecular docking, and (C) crystal structure of the SMRT DAD-InsP4-HDAC3 complex
(PDB accession code: 4A69). Note the parallels in InsP and HDAC engagement by the MTA1 and SMRT
subunits featuring structurally similar SANT domains, whereas the mode of InsP and HDAC engagement
by the structurally unrelated SAP30 ZnF domain is unique, implying convergent evolution at the functional
level.
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Ryan Dale Marcum and Ishwar Radhakrishnan
(HDAC) complex up-regulate deacetylase activity
Inositol phosphates and core subunits of the Sin3L/Rpd3L histone deacetylase
published online July 29, 2019J. Biol. Chem.
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