The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components

Soldi, Monica; Bonaldi, Tiziana

doi:10.1074/mcp.m112.024307

Cited by 56 publications

(71 citation statements)

References 106 publications

(118 reference statements)

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“…The mESC chromatin proteomic profiling data produced a catalog of chromatin-associated proteins, which includes a large number of known and candidate chromatin regulators and transcriptional regulators. Our results overlap and confirm chromatin-associated proteins identified in other MS studies (8,12) but also provide a larger catalog of chromatin proteins associated with a larger variety of chromatin types. We confirmed that several of the candidates (PHIP, UBR7, USP48, and PUS7) were indeed associated with appropriate histone-marked segments of the genome as detected by chromatin proteomic profiling.…”

Section: Discussionsupporting

confidence: 89%

“…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).…”

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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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Section: Discussionsupporting

confidence: 89%

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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“…The minimum score required for the phosphorylation site was 30 to obtain reliable results. Protein phosphorylation was quantified using the peptideextracted ion chromatograms embedded in MaxQuant (22). Student's t test was used for statistical comparisons.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

Wang

Lin

Huang

et al. 2015

Molecular & Cellular Proteomics

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One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. Molecular & Cellular Proteomics 14: 10.1074/mcp.M114.045468, 1104-1112, 2015.Austin (1) and Chang (2) discovered that sperm must reside in the female genital tract for a specific period of time to acquire the ability to fertilize an egg and named this process "capacitation." During capacitation, several biochemical changes occur, including enhancement of tyrosine phosphorylation (3), increased intracellular Ca 2ϩ and cAMP levels (4), hyperactivated motility (5), and increased membrane plasma permeability (6). Mature sperm are highly differentiated and specialized cells, there is almost no transcription, and the genomic ribosome is inactive (5). Therefore, regulation of proteins at the level of post-translational modification is expected to play important roles in sperm functions. In mammalian sperm, phosphorylated proteins, protein kinases, and phosphatases are reported to function in sperm motility, capacitation, and acrosome reaction (7, 8). Tyrosine phosphorylation and dephosphorylation are required for sperm to reach, bind, penetrate, and fuse with the oocyte (5). Tyrosinephosphorylated proteins have been found in human (9), monkey (10), rat (11), and mouse (12) sperm. The sperm tail is the main location of protein tyrosine phosphorylation, and tyrosine phosphorylation of the sperm tail is related to hyperactivated motility (13). However, the mechanism of protein tyrosine phosphorylation regulation in sperm capacitation is not well studied. With high throughput ability, proteomics has been used to characterize phosphorylation in sperm. For the human sperm, Ficarro et al. (14) used two-dimensional polyacrylamide gel electrophoresis (PAGE), anti-phosphotyrosine antibody labeling, and tandem mass spectrometry (MS/MS) to identify tyrosine phosphoproteins during capacitation. They identified...

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“…Moreover, D 3 -acetylation offers two additional advantages that facilitate the discernment of isobaric modified peptides: first, the alkylation occurs only on unmodified and mono-methylated lysines, but not on di-and tri-methylated residues; as such modified peptides bearing the same total number of modifications but in different arrangements are differentially decorated by distinct set of D 3 -acetyl groups that produce unambiguous m/z shifts. Second, distinct patterns of D 3 -alkylation cause slightly different retention times in liquid chromatography on reverse phase column, which generate an additional level of separation for isobaric modified peptides 21 .…”

Section: Representative Resultsmentioning

confidence: 99%

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Soldi

Bonaldi

2014

JoVE

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Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locusspecific structural and functional configurations. Video LinkThe video component of this article can be found at

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The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components

Cited by 56 publications

References 106 publications

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

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

Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

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