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![Scheme of experimental strategy. Chromatin of Drosophila control cells or cells stably expressing CENP-ACID-GFP or H3.3-GFP was digested with MNase and anti-GFP affinity purification was performed on the digested soluble chromatin. After washing steps, proteins were eluted by boiling in Laemmli buffer and separated by SDS-PAGE. Following excision from the gel and in gel tryptic digestion, peptide samples were analyzed by LC-MS/MS. (Adapted from [1])](publication/281551861/figure/fig1/AS:281005446451211@1444008327414/Scheme-of-experimental-strategy-Chromatin-of-Drosophila-control-cells-or-cells-stably.png)
Scheme of experimental strategy. Chromatin of Drosophila control cells or cells stably expressing CENP-ACID-GFP or H3.3-GFP was digested with MNase and anti-GFP affinity purification was performed on the digested soluble chromatin. After washing steps, proteins were eluted by boiling in Laemmli buffer and separated by SDS-PAGE. Following excision from the gel and in gel tryptic digestion, peptide samples were analyzed by LC-MS/MS. (Adapted from [1])
Source publication
Centromeres of higher eukaryotes are epigenetically defined by the centromere specific histone H3 variant CENP-ACID. CENP-ACID builds the foundation for the assembly of a large network of proteins. In contrast to mammalian systems, the protein composition of Drosophila centromeres has not been comprehensively investigated. Here we describe the prot...
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DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication, and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form...
Citations
... Attempts to identify proteins possibly homologous to the Constitutive Centromere Associated Network (CCAN) (Foltz et al. 2006) (Fig. 2b; Table 1) by analyzing the interactome of the immunoprecipitated dCENP-A nucleosome have failed to pinpoint proteins exclusively and constitutively present at centromeres (Barth et al. 2015(Barth et al. , 2014. Nonetheless, other factors enriched at centromeres were identified, including the histone acetyltransferase 1 (HAT1) homolog, the proteasome regulator REG, and the transcription elongation factor Spt6 (Barth et al. 2014;Bobkov et al. 2020). ...
The fruit fly Drosophila melanogaster serves as a powerful model organism for advancing our understanding of biological processes, not just by studying its similarities with other organisms including ourselves but also by investigating its differences to unravel the underlying strategies that evolved to achieve a common goal. This is particularly true for centromeres, specialized genomic regions present on all eukaryotic chromosomes that function as the platform for the assembly of kinetochores. These multiprotein structures play an essential role during cell division by connecting chromosomes to spindle microtubules in mitosis and meiosis to mediate accurate chromosome segregation. Here, we will take a historical perspective on the study of fly centromeres, aiming to highlight not only the important similarities but also the differences identified that contributed to advancing centromere biology. We will discuss the current knowledge on the sequence and chromatin organization of fly centromeres together with advances for identification of centromeric proteins. Then, we will describe both the factors and processes involved in centromere organization and how they work together to provide an epigenetic identity to the centromeric locus. Lastly, we will take an evolutionary point of view of centromeres and briefly discuss current views on centromere drive.
... This lack of information is mainly due to the fact that chromocenters are difficult to purify without disrupting their native conformation (14,15) and, like many other nuclear organelles, vary in size and shape during cell cycle and cellular differentiation (16). Although several chromocenter associated factors have been described and characterized, classi-cal affinity-MS based approaches only identified stable interactors of centromeric chromatin or the inner kinetochore complexes but failed to describe the proteomic composition of this important nuclear compartment (17)(18)(19)(20). This is very likely due to the fact that the chromocenter is held together by a multitude and rather weak interactions that do not withstand the harsh conditions used during purification. ...
... The last washing step was performed in 15 ml falcons. Nuclei isolation and extraction was performed as in (17) with modifications. All buffers were supplemented with freshly added protease inhibitors. ...
... Several groups including us have performed large scale affinity purification experiments coupled to mass spectrometry (AP-MS) using antibodies specific against dCenpA (17,19), HMR (18) or HP1a (45,46) to identify putative interactors of these proteins. However, in contrast to other well characterized nuclear protein complexes such as the nucleosome remodelers or the histone acetyltransferases (47,48), these three proteins have been refractory to classical biochemical purification. ...
During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To provide a deeper molecular insight into the composition of the chromocenter and to demonstrate the usefulness of proximity-based biotinylation as a tool to investigate those questions, we performed super resolution microscopy and proximity-based biotinylation experiments of three distinct proteins associated with the chromocenter in Drosophila. Our work revealed an intricate internal architecture of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.
... CAL1 binds to prenucleosomal CENP-A/H4 dimers, associates with the centromere and mediates CENP-A deposition both in vitro and in vivo (18,21,22). Besides CAL1, pre-nucleosomal Drosophila CENP-A can be detected in complexes with the transcription-and replication-associated histone chaperone FACT, the histone chaperone CAF1 and the histone acetyltransferase HAT1 (23)(24)(25). This situation is reminiscent of the processing of canonical H3/H4 dimers prior to chromatin assembly as these too are present in various preloading complexes (26). ...
Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.
... To identify novel factors associated with Drosophila centromeres we previously affinity-purified GFP-tagged dCENP-A containing nucleosomes and combined it with mass-spectroscopy analysis. Among the proteins enriched in dCENP-A containing chromatin was the transcription elongation factor and histone chaperone Spt6 (Barth et al. 2015). ...
Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This constitutes both an opportunity to remodel the underlying chromatin as well as the potential danger of losing epigenetic information. Centromeric transcription has been shown to be required for stable incorporation of the centromere-specific histone dCENP-A in M/G1-phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and shows enhanced association with non-phosphorylatable dCENP-A mutants compared to histone H3, while phosphomimetic residues alleviate association with Spt6. We conclude that Spt6 acts as a conserved CENP-A maintenance factor, which is required during transcription-mediated chromatin remodelling at the centromere to ensure long-term stability of epigenetic centromere identity.
... Firstly, it has been shown that ATPsyn-ε is required for normal spindle orientation in the syncytial divisions of Drosophila embryos (Kidd et al., 2005). In addition, affinity purification of CENP-A nucleosomes from Drosophila cultured cells (S2) identified ATP subunits ATPsynCF6 and ATPsyn-O (the OSCP) among 86 proteins which are specifically enriched at centromeres (Barth et al., 2015) and ATPsyn-α, ATPsynβ and ATPsyn-γ were identified amongst approximately 1800 genes associated with the centromere in 9 different purifications from Drosophila cultured cells (Barth et al., 2014). Interestingly, components of the ATP synthase complex have also been identified in protein screens to detect chromatin interacting proteins in the African clawed frog X. laevis, possibly indicating that an alternative nuclear function for ATP synthase subunits may be conserved outside of insects (Funabiki, Constanzo) ...
Recent studies in plants and Drosophila suggest that CENP-A, the
centromeric histone H3 variant and epigenetic marker of centromere
localisation may have additional roles during meiosis.
By immunoprecipitation and mass spectrometry we have identified
components of the mitochondrial ATP synthase F1 complex as potential
meiotic CENP-A interactors. Previous studies have identified a role for
ATPsyn-α and ATPsyn-β in Drosophila male fertility and in addition, male
Drosophalids express a testes specific paralogue of ATPsyn-β: ATPsyn-β-
like, which is essential for male fertility.
We report that testes specific knockdown of ATPsyn-α, ATPsyn-γ and
ATPsyn-β-like subunits results in a defect in sister chromatid centromeric
cohesion throughout meiosis I. Determination of whole tissue ATP levels
after knockdown shows that ATP levels are reduced. However loss of
cohesion severity does not correlate with ATP reduction indicating that it is likely that observed defect is not due to ATP depletion.
Knockdown of CENP-A also leads to a similar loss of sister centromere
cohesion during meiosis suggesting that a functional link between CENP-Aand the ATP synthase F1 subunits exists. Indeed, using an in vitro interactionassay we have identified that CENP-A directly interacts with ATPsyn-α andby immunofluorescence microscopy we have found that ATPsyn-α and GFPATPsyn-β-likecolocalise with CENP-A at centromeres in the germ-line.
In our model, we hypothesise that centromeric CENP-A recruits ATPsyn-α
and ATPsyn-β-like via its direct interaction with the ATPsyn-α subunit, at
the centromere these subunits promote sister centromere cohesion in a
mechanism independent of ATP production.
... Peptides derived from the trypsin digestion of the complexes were extracted, reverse phase HPLC-separated and then analyzed by tandem mass spectrometry (MS/MS) in Orbitrap LTQ spectrometer as previously described (18). Spectra were acquired and analyzed by Scaffold software, as described previously (7,19). ...
Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-I. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-I protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-I depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-I and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.
Identification of proteins that are directly or indirectly associated with a specific DNA sequence is often an important goal in molecular biology research. Proteomics of isolated chromatin fragments (PICh) is a technique used to isolate chromatin that contains homologous DNA sequence to a specific nucleic acid probe. All proteins directly and indirectly associated with the DNA sequences that hybridize to the probe are then identified by proteomics.1 We used the PICh technique to isolate chromatin associated with the centromeres of barley (Hordeum valgare), by using a 2'-deoxy-2'fluoro-ribonucleotides (2'-F RNA) probe that is homologous to the AGGGAG satellite DNA specific to barely centromeres. Proteins associated with the barley centromeric chromatin were then isolated and identified by mass spectrometry. Both alpha-cenH3 and beta-cenH3, the two centromeric histone H3 variants associated with barley centromeres, were positively identified. Interestingly, several different H2A and H2B variants were recovered in the PIChed chromatin. The limitations and future potential of PICh in plant chromatin research are discussed.
