Chromatin proteins captured by ChIP–mass spectrometry are linked to dosage compensation in Drosophila

Wang, Charlotte I.; Alekseyenko, Artyom A.; LeRoy, Gary; Elia, Andrew E. H.; Gorchakov, Andrey A.; Britton, Laura Mae P.; Elledge, Stephen J.; Kharchenko, Peter V.; Garcia, Benjamin A.; Kuroda, Mitzi I.

doi:10.1038/nsmb.2477

Cited by 105 publications

(107 citation statements)

References 81 publications

(123 reference statements)

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“…S7E). These findings of MSL3 enrichment for active PTMs such as H4K16 acetylation and H3K79 methylation using the on-bead method are consistent with an earlier report by our group using the in-gel method (25).…”

Section: Stage-tip Desalting Stage-tip Desaltingsupporting

confidence: 82%

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Zee

Alekseyenko

McElroy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.chromatin complexes | affinity pulldown | liquid chromatography-mass spectrometry | BioTAP-XL | histone modifications C hromatin encompasses the subset of proteins and RNAs in complex with DNA to form the chromosomes within eukaryotic nuclei. As the primary protein constituents of chromatin, histones organize genomic DNA into nucleosomes and display an extensive array of posttranslational modifications (PTMs) (1). It is increasingly evident that specific histone PTMs are selectively recognized by specific nonhistone proteins that are themselves regulators of many nuclear events such as epigenetic silencing (2, 3). Consequently, defects in these chromatin components often manifest in a number of human diseases (4).As specific interactions between modified histones and nonhistone proteins help distinguish complexes participating in distinct processes, the identification of protein and modification-dependent interactions provides key insights into chromatin biology. Various chromatography and pulldown methods have been developed to identify binding partners. In a typical native pulldown experiment, nuclei from hypotonically lysed cells are digested with micrococcal nuclease (MNase). Extracted chromatin is subjected to immunoprecipitation to isolate the desired complexes. If using a tag such as FLAG, complexes are then competitively eluted and identified. Several drawbacks to native pulldowns as described here are that MNase leaves behind a substantial insoluble nuclear pellet and degrades associating RNAs, that salt extraction of the digested nucleosomes risks dissociation of the tagged bait from its interacting partners, and that most tags do not have sufficient affinity for their respective antibody to permit highly stringent washes. The result is often an incomplete picture of the chromatin...

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Section: Stage-tip Desalting Stage-tip Desaltingsupporting

confidence: 82%

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Zee

Alekseyenko

McElroy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Mass spectrometry (MS) can identify large populations of proteins present in specific preparations (8-10) but does not reveal how these proteins occupy specific portions of the genome. A recently developed approach combined ChIP with MS (ChIP-MS) to identify protein complexes that are associated with other proteins known to occupy sites in the genome (11)(12)(13). Here we adapt this approach to profile the proteins associated with chromatin containing specific histone modifications across the genome of mouse embryonic stem cells (mESCs).…”

mentioning

confidence: 99%

Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions

Xiong

Dadon

Abraham

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

123

112

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More than a thousand proteins are thought to contribute to mammalian chromatin and its regulation, but our understanding of the genomic occupancy and function of most of these proteins is limited. Here we describe an approach, which we call "chromatin proteomic profiling," to identify proteins associated with genomic regions marked by specifically modified histones. We used ChIP-MS to identify proteins associated with genomic regions marked by histones modified at specific lysine residues, including H3K27ac, H3K4me3, H3K79me2, H3K36me3, H3K9me3, and H4K20me3, in ES cells. We identified 332 known and 114 novel proteins associated with these histone-marked genomic segments. Many of the novel candidates have been implicated in various diseases, and their chromatin association may provide clues to disease mechanisms. More than 100 histone modifications have been described, so similar chromatin proteomic profiling studies should prove to be valuable for identifying many additional chromatin-associated proteins in a broad spectrum of cell types.chromatin | genomics | proteomics T here are more than 1,000 transcription factors, cofactors, and chromatin regulators encoded in the mammalian genome, but we have limited understanding of the genomic occupancy and function of most of these (1-4). Understanding how these proteins interact with specific active and repressed portions of the genome would provide clues to their functions in global gene control, but limitations inherent in widely used genomic and proteomic technologies make acquiring this information laborious and expensive. Chromatin immunoprecipitation coupled to sequencing (ChIP-seq) can reveal the sites that a specific protein occupies in the genome (5-7) but is laborious and is limited by the availability of antibodies specific to candidate genome-binding proteins. Mass spectrometry (MS) can identify large populations of proteins present in specific preparations (8-10) but does not reveal how these proteins occupy specific portions of the genome. A recently developed approach combined ChIP with MS (ChIP-MS) to identify protein complexes that are associated with other proteins known to occupy sites in the genome (11-13). Here we adapt this approach to profile the proteins associated with chromatin containing specific histone modifications across the genome of mouse embryonic stem cells (mESCs). These chromatin modifications mark regions of the genome where specific transcriptional activities occur, thus implicating the associated proteins in these activities.

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“…The Chironomus tentans homolog of Rump, CtHrp59, was shown to associate with a subset of actively transcribed regions on polytene chromosomes, dependent on the presence of RNA, suggesting that chromatin association might be due to the binding to nascent transcripts (Kiesler et al, 2005). Rump has also been recently identified by mass spectrometry in the chromatinassociated fraction of the dosage compensation complex (Wang et al, 2013). Because our high resolution ChIP-seq profiling of Rump was performed in a female cell line, the sites we observed cannot be due to interaction with the dosage compensation complex.…”

Section: Rump Is a Multifunctional Proteinmentioning

confidence: 99%

The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner

King

Matzat

Dale

et al. 2014

Journal of Cell Science

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Chromatin insulators are DNA-protein complexes that are situated throughout the genome that are proposed to contribute to higherorder organization and demarcation into distinct transcriptional domains. Mounting evidence in different species implicates RNA and RNA-binding proteins as regulators of chromatin insulator activities. Here, we identify the Drosophila hnRNP M homolog Rumpelstiltskin (Rump) as an antagonist of gypsy chromatin insulator enhancer-blocking and barrier activities. Despite ubiquitous expression of Rump, decreasing Rump levels leads to improvement of barrier activity only in tissues outside of the central nervous system (CNS). Furthermore, rump mutants restore insulator body localization in an insulator mutant background only in non-CNS tissues. Rump associates physically with core gypsy insulator proteins, and chromatin immunoprecipitation and sequencing analysis of Rump demonstrates extensive colocalization with a subset of insulator sites across the genome. The genome-wide binding profile and tissue specificity of Rump contrast with that of Shep, a recently identified RNA-binding protein that antagonizes gypsy insulator activity primarily in the CNS. Our findings indicate parallel roles for RNA-binding proteins in mediating tissue-specific regulation of chromatin insulator activity.

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Chromatin proteins captured by ChIP–mass spectrometry are linked to dosage compensation in Drosophila

Cited by 105 publications

References 81 publications

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions

The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner

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