Systematic analysis of the interactome of modified chromatin
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Regulation of chromatin composition and structure is crucial for maintaining genome integrity and execution of the wide array of functions related to gene regulation and downstream cell signaling. Chemical modifications of chromatin are at the core of these processes and include methylation of the primary sequence of DNA, as well as various modifications of the proteinaceous chromatin packaging units - the histones. Recent genome-wide mapping approaches have been instrumental for characterization of the location and distribution of these marks relative to the different functional domains and gene regulatory elements in chromatin. The majority of the characterized chromatin modifications function as signaling platforms for recruitment of various protein complexes. Therefore, it is of equal importance to describe these sets of proteins for dissection of the functional consequence of their binding. More importantly, global analysis of the interactomes of functionally associated chromatin modifications might shed light on the proteins required for establishment and operation of specific chromatin domains. In this study, a novel approach for identification of chromatin modification-dependent protein binding was established. In vitro reconstituted oligonucleosomal templates with homogeneous and defined modification status were used for affinity purification from SILAC-labeled nuclear extracts. The interactomes of ten individual chromatin species were investigated and the results provided valuable insight into chromatin biology on several levels. Investigation of the nature of the subproteomes recruited by single modifications provided evidence for their functional role. Additionally, the results offer a comprehensive catalogue of candidate proteins for further dissection of specific chromatin modification molecular readout. This was exemplified here with the demonstration of the recruitment of the SWI/SNF complex to monoubiquitylated H2B-containing chromatin for regulation of transcription of a specific set of genes. Investigation of the interactome of doubly modified chromatin identified a large number of factors whose recruitment presumably depends on the cooperative action of two modifications. Furthermore, the comparative analysis of individual datasets revealed novel relationships between the different modifications. On a global scale, this resulted in the identification of a limited set of proteins that likely play an important role for the function of heterochromatin domains. Lastly, the chromatin affinity purification approach was used for developing a SILAC internal standard method for direct quantitative comparison of recruitment to different chromatin modifications and combinations thereof.Keywords:
ChIA-PET
Histone-modifying enzymes
Bivalent chromatin
Interactome
Scaffold/matrix attachment region
Epigenomics
In the nucleus of eukaryotic cells, genomic DNA associates with numerous protein complexes and RNAs, forming the chromatin landscape. Through a genome-wide study of chromatin-associated proteins in Drosophila cells, five major chromatin types were identified as a refinement of the traditional binary division into hetero- and euchromatin. These five types were given color names in reference to the Greek word chroma. They are defined by distinct but overlapping combinations of proteins and differ in biological and biochemical properties, including transcriptional activity, replication timing, and histone modifications. In this work, we assess the evolutionary relationships of chromatin-associated proteins and present an integrated view of the evolution and conservation of the fruit fly Drosophila melanogaster chromatin landscape. We combine homology prediction across a wide range of species with gene age inference methods to determine the origin of each chromatin-associated protein. This provides insight into the evolution of the different chromatin types. Our results indicate that for the euchromatic types, YELLOW and RED, young associated proteins are more specialized than old ones; and for genes found in either chromatin type, intron/exon structure is lineage-specific. Next, we provide evidence that a subset of GREEN-associated proteins is involved in a centromere drive in D. melanogaster. Our results on BLUE chromatin support the hypothesis that the emergence of Polycomb Group proteins is linked to eukaryotic multicellularity. In light of these results, we discuss how the regulatory complexification of chromatin links to the origins of eukaryotic multicellularity.
ChIA-PET
Scaffold/matrix attachment region
Euchromatin
Bivalent chromatin
Histone-modifying enzymes
Non-histone protein
Multicellular organism
ChIP-on-chip
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Citations (4)
Scaffold/matrix attachment region
ChIA-PET
Histone-modifying enzymes
PELP-1
Bivalent chromatin
SWI/SNF
Transcription coregulator
Pioneer factor
ChIP-on-chip
Histone code
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Citations (107)
Chromatin remodelers are important regulatory mechanisms that eukaryotic cells use to modify the structure of chromatin, which is made up of DNA and proteins. DNA wraps around histone proteins to make up chromatin. When these proteins are modified, the shape of the chromatin is altered. “Loosening” the chromatin structure by chromatin modifications allows for active gene expression whereas “tightening” or compaction of chromatin results in gene repression. Therefore the modifications on chromatin modulate gene expression in all eukaryotes. It has been shown that mis-regulation of chromatin remodelers contribute to various cancers. Understanding the biochemistry behind how chromatin associating proteins modify chromatin, and ultimately gene expression, can help provide insight into developing anti-cancer drugs. This study focused on characterizing AIF4, a chromatin associating protein. Since other chromatin associating proteins are known to be histone modifiers, we hypothesized that AIF4 is another chromatin remodeler. Many chromatin remodelers are found in protein complexes. These protein complexes have been shown to be important for the functions of chromatin modifying proteins. Therefore this study tested whether AIF4 interacts with other proteins. Purifying AIF4 and testing if it binds to the nucleosome will support the hypothesis that AIF4 is chromatin associating protein. Co-immunoprecipitation will be used to determine if AIF4 interacts with other proteins, indicating that AIF4 functions in a protein complex. However, we were unable to detect an interaction between AIF4 and known chromatin associating proteins. Future work will aim to determine whether AIF4 acts alone or is involved in a unique protein complex.
ChIA-PET
ChIP-on-chip
Histone-modifying enzymes
Scaffold/matrix attachment region
Bivalent chromatin
ChIP-sequencing
Histone code
Non-histone protein
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Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation.
ChIA-PET
Bivalent chromatin
Scaffold/matrix attachment region
Multicellular organism
Histone-modifying enzymes
ChIP-sequencing
ChIP-on-chip
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Citations (320)
In cells, chromatin is folded into a 30 nm fibre. Recent genome-wide studies have shown that DNaseI-sensitive sites are present in both transcribed and non-transcribed genes and are enriched in the gene-dense regions of the human genome. The distribution of open chromatin has also been shown to correlate with gene density rather than transcription. In this review it is suggested that open chromatin corresponds to a 30 nm fibre interspersed with discontinuities, and that blocks of openchromatin might facilitate gene transcription, but are neither necessary nor sufficient. The nature of these discontinuities is not known but could correspond to alterations in chromatin fibre structure caused by irregular nucleosome positioning, nucleosome remodelling activities, variant histones or the binding of specific transcription factors.
ChIA-PET
Histone-modifying enzymes
Scaffold/matrix attachment region
ChIP-sequencing
Transcription
Bivalent chromatin
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Citations (28)
ChIA-PET
CTCF
Bivalent chromatin
Histone-modifying enzymes
Scaffold/matrix attachment region
Histone code
ChIP-sequencing
Cite
Citations (1)
Abstract In the nucleus of eukaryotic cells, genomic DNA associates with numerous protein complexes and RNAs, forming the chromatin landscape. Through a genome-wide study of chromatin-associated proteins in Drosophila cells, five major chromatin types were identified as a refinement of the traditional binary division into hetero- and euchromatin. These five types are defined by distinct but overlapping combinations of proteins and differ in biological and biochemical properties, including transcriptional activity, replication timing and histone modifications. In this work, we assess the evolutionary relationships of chromatin-associated proteins and present an integrated view of the evolution and conservation of the fruit fly D. melanogaster chromatin landscape. We combine homology prediction across a wide range of species with gene age inference methods to determine the origin of each chromatin-associated protein. This provides insight into the emergence of the different chromatin types. Our results indicate that the two euchromatic types, YELLOW and RED, were one single activating type that split early in eukaryotic history. Next, we provide evidence that GREEN-associated proteins are involved in a centromere drive and expanded in a lineage-specific way in D. melanogaster . Our results on BLUE chromatin support the hypothesis that the emergence of Polycomb Group proteins is linked to eukaryotic multicellularity. In light of these results, we discuss how the regulatory complexification of chromatin links to the origins of eukaryotic multicellularity.
ChIA-PET
Scaffold/matrix attachment region
Euchromatin
Histone-modifying enzymes
Bivalent chromatin
Multicellular organism
Non-histone protein
ChIP-on-chip
ChIP-sequencing
Cite
Citations (0)
More than four decades ago, it was shown that RNA stably associates with chromatin. These studies indicated that chromatin-associated RNAs (caRNA) might be involved in the organization of chromatin structure. However, it is only recently that pools of chromatin-associated RNAs were characterized and functional studies were initiated. In Drosophila cells, an RNP complex consisting of snoRNAs and Decondensation factor 31 (Df31) is stably tethered to chromatin, mediated by the RNA- and histone-binding activities of Df31. Biochemical and functional characterizations suggest a structural role of this complex in chromatin organization. The binding of the Df31-snoRNA complex to chromatin results in the opening and the maintenance of accessible higher order structures of chromatin. We suggest that different classes of chromatin-associated RNPs are required for the targeted opening of higher order structures of chromatin, enabling the activation of DNA-dependent processes such as transcription.
Bivalent chromatin
ChIA-PET
Scaffold/matrix attachment region
Histone-modifying enzymes
ChIP-sequencing
Transcription
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Citations (10)
ChIA-PET
ChIP-on-chip
Scaffold/matrix attachment region
Bivalent chromatin
ChIP-sequencing
Histone-modifying enzymes
Proteome
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Citations (60)
ChIA-PET
Histone-modifying enzymes
Transcription coregulator
Scaffold/matrix attachment region
Transcription preinitiation complex
Bivalent chromatin
Pioneer factor
Transcription
ChIP-on-chip
Cite
Citations (92)