Recombinant Histone H2A-H2B Dimers

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Recombinant Histone H2A-H2B dimers Cat. No. C23010010 Purity: Purified using FPLC,> 98% purity as determined by Source: E. coli SDS-PAGE Lot #: 001 Storage buffer: 20 mM Tris-Cl pH 7.9, 1 M NaCl, 1mM EDTA, Size: 50 µg/ 50 µl 0.5 mM PMSF and 1 mM DTT. Concentration: 1 µg/µl Storage: Store at -80°C; guaranteed stable for 2 years from Specificity: Human date of receipt when stored properly. Precautions: This product is for research use only. Not for use in diagnostic or therapeutic procedures. Description: Dimer of full length N-terminal Flag tagged recombinant histone H2A and N-terminal His tagged recombinant histone H2B, produced in E. coli. Protein description Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. Quality control Figure 1. SDS page of the Recombinant Histone H2A-H2B dimers. The position of the proteins of interest is indicated on the right; the marker (in kDa) is shown on the left. 1 Figure 2. ESi-TOF analysis of Recombinant Histone H2A-H2B dimers. Diagenode sa. BELGIUM | EUROPE Diagenode Inc. USA | NORTH AMERICA Last update: September 7, 2015 LIEGE SCIENCE PARK 400 Morris Avenue, Suite 101 Rue Bois Saint-Jean, 3 Denville, NJ 07834 - USA 4102 Seraing (Ougrée) - Belgium Tel: +1 862 209-4680 Tel: +32 4 364 20 50 Fax: +1 862 209-4681 Fax: +32 4 364 20 51 [email protected] [email protected] [email protected] 2 [email protected].

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Datasheet for Histone H2B Human, Recombinant (M2505; Lot 0031404)
Supplied in: 20 mM Sodium Phosphate (pH 7.0), Quality Control Assays: Ionization-Time of Flight Mass Spectrometry). The Histone H2B 300 mM NaCl and 1 mM EDTA. SDS-PAGE: 0.5 µg, 1.0 µg, 2.0 µg, 5.0 µg, average mass calculated from primary sequence Human, Recombinant 10.0 µg Histone H2B Human, Recombinant were is 13788.97 Da. This confirms the protein identity Note: The protein concentration (1 mg/ml, 73 µM) loaded on a 10–20% Tris-Glycine SDS-PAGE gel as well as the absence of any modifications of the is calculated using the molar extinction coefficient and stained with Coomassie Blue. The calculated histone. 1-800-632-7799 for Histone H2B (6400) and its absorbance at molecular weight is 13788.97 Da. Its apparent [email protected] 280 nm (3,4). 1.0 A units = 2.2 mg/ml N-terminal Protein Sequencing: Protein identity 280 molecular weight on 10–20% Tris-Glycine SDS- www.neb.com was confirmed using Edman Degradation to PAGE gel is ~17 kDa. M2505S 003140416041 Synonyms: Histone H2B/q, Histone H2B.1, sequence the intact protein. Histone H2B-GL105 Mass Spectrometry: The mass of purified Histone H2B Human, Recombinant is 13788.5 Da Protease Assay: After incubation of 10 µg of M2505S B r kDa 1 2 3 4 5 6 7 as determined by ESI-TOF MS (Electrospray Histone H2B Human, Recombinant with a standard 250 4.0 mixture of proteins for 2 hours at 37°C, no 100 µg 1.0 mg/ml Lot: 0031404 150 13788.5 100 proteolytic activity could be detected by SDS- RECOMBINANT Store at –20°C Exp: 4/16 80 PAGE.
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Histone Variants: Deviants?
Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Histone variants: deviants? Rohinton T. Kamakaka2,3 and Sue Biggins1 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA; 2UCT/National Institutes of Health, Bethesda, Maryland 20892, USA Histones are a major component of chromatin, the pro- sealing the two turns of DNA. The nucleosome filament tein–DNA complex fundamental to genome packaging, is then folded into a 30-nm fiber mediated in part by function, and regulation. A fraction of histones are non- nucleosome–nucleosome interactions, and this fiber is allelic variants that have specific expression, localiza- probably the template for most nuclear processes. Addi- tion, and species-distribution patterns. Here we discuss tional levels of compaction enable these fibers to be recent progress in understanding how histone variants packaged into the small volume of the nucleus. lead to changes in chromatin structure and dynamics to The packaging of DNA into nucleosomes and chroma- carry out specific functions. In addition, we review his- tin positively or negatively affects all nuclear processes tone variant assembly into chromatin, the structure of in the cell. While nucleosomes have long been viewed as the variant chromatin, and post-translational modifica- stable entities, there is a large body of evidence indicat- tions that occur on the variants. ing that they are highly dynamic (for review, see Ka- makaka 2003), capable of being altered in their compo- Supplemental material is available at http://www.genesdev.org. sition, structure, and location along the DNA. Enzyme Approximately two meters of human diploid DNA are complexes that either post-translationally modify the packaged into the cell’s nucleus with a volume of ∼1000 histones or alter the position and structure of the nucleo- µm3.
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The Mechanism of Cross-Talk Between Histone H2B Ubiquitination and H3 Methylation by Dot1l
bioRxiv preprint doi: https://doi.org/10.1101/501098; this version posted December 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The mechanism of cross-talk between histone H2B ubiquitination and H3 methylation by Dot1L Evan J. Worden, Niklas Hoffmann, Chad Hicks and Cynthia Wolberger* Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD USA *Corresponding author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/501098; this version posted December 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Methylation of histone H3, lysine 79 (H3K79), by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B at lysine 120 (H2B-Ub), and is a well-characterized example of histone modification cross-talk that is conserved from yeast to humans. The mechanism by which H2B-Ub stimulates Dot1L to methylate the relatively inaccessible histone core H3K79 residue is unknown. The 3.0 Å resolution cryo-EM structure of Dot1L bound to ubiquitinated nucleosome reveals that Dot1L contains binding sites for both ubiquitin and the histone H4 tail, which establish two regions of contact that stabilize a catalytically competent state and positions the Dot1L active site over H3K79.
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H2A Ubiquitylated Mononucleosomes Next-Generation Substrates for Deubiquitylation Enzyme (DUB) Assays
H2A Ubiquitylated Mononucleosomes Next-Generation Substrates for Deubiquitylation Enzyme (DUB) Assays Next-Generation DUB Assay Substrates are here. Get results that matter. • Enabling access to DUB targets that require nucleosome substrates in vitro • Proper substrates for DUB inhibitor development • Unmatched quality control for results you can trust Histone monoubiquitylation (ub1) acts as a critical signaling center that regulates cascades of downstream epigenetic enzymes to modify gene transcription. The physiological substrate for chromatin-targeting DUBs is the nucleosome (Nuc), the basic repeating unit of chromatin (comprised of histone proteins wrapped by DNA). Current high-throughput screening (HTS) DUB assays use unnatural modified or diubiquitin conjugates as substrates, which poorly mimic endogenous targets in vivo. In collaboration with Boston Biochem, EpiCypher is delivering ubiquitylated nucleosome substrates for drug screening and chromatin biology research. FIGURE 1 Ub Ub Schematic representation of mononucleosoms assembled from recombinant human histones Ub expressed in E. coli (two each of histones H2A, H2B, H3 and H4). H2A H2A Approximately 50% of the nucleosomes are monoubiquitylated on histone H2A lysine 118, while the other 50% are monoubiquitylated on both histone H2A lysine 118 and histone H2A lysine 119 (multi-mono- ubiquitylated). Next Generation Deubiquitylation Enzyme (DUB) Assay Substrates EpiCypher has developed recombinant mononucleosomes carrying monoubiquitylation on H2A. These ubiquitylated nucleosomes are generated enzymatically using the RING1B/BMI1 ubiquitin ligase complex. The resulting product is highly pure (>95% of nucleosomes are ubiquitylated) and consists of nucleosomes monoubiquitylated at H2A lysine 118/119 (Figure 1; the physiological target of RING1B/BMI1 in vivo). FIGURE 2 Deubiquitylation Assay Data: Mononucleosomes H2A Ubiquityl, Recombinant Human, Biotinylated (1 μg) were employed in a deubiquitylation (DUB) assay using no enzyme (Lane 1), USP5 (Lane 2) or USP16 (Lane 3) and run on an SDS PAGE gel.
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The Role of Histone H2av Variant Replacement and Histone H4 Acetylation in the Establishment of Drosophila Heterochromatin
The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin Jyothishmathi Swaminathan, Ellen M. Baxter, and Victor G. Corces1 Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. Here we show that the histone H2A variant of Drosophila melanogaster, H2Av, localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. H2Av accumulation or histone H4 Lys 12 acetylation is not affected by mutations in Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin and requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place. [Keywords: Chromatin; silencing; transcription; histone; nucleus] Received September 8, 2004; revised version accepted November 4, 2004. The basic unit of chromatin is the nucleosome, which is guchi et al. 2004). The role of histone variants, and spe- made up of 146 bp of DNA wrapped around a histone cially those of H3 and H2A, in various nuclear processes octamer composed of two molecules each of the histones has been long appreciated (Wolffe and Pruss 1996; Ah- H2A, H2B, H3, and H4.
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Transcriptional Regulation by Histone Ubiquitination and Deubiquitination
Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press PERSPECTIVE Transcriptional regulation by histone ubiquitination and deubiquitination Yi Zhang1 Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599, USA Ubiquitin (Ub) is a 76-amino acid protein that is ubiqui- The fact that histone ubiquitination occurs in the largely tously distributed and highly conserved throughout eu- monoubiquitinated form and is not linked to degrada- karyotic organisms. Whereas the extreme C-terminal tion, in combination with the lack of information regard- four amino acids are in a random coil, its N-terminal 72 ing the responsible enzymes, prevented us from under- amino acids have a tightly folded globular structure (Vi- standing the functional significance of this modification. jay-Kumar et al. 1987; Fig. 1A). Since its discovery ∼28 Recent identification of the E2 and E3 proteins involved years ago (Goldknopf et al. 1975), a variety of cellular in H2B ubiquitination (Robzyk et al. 2000; Hwang et al. processes including protein degradation, stress response, 2003; Wood et al. 2003a) and the discovery of cross-talk cell-cycle regulation, protein trafficking, endocytosis sig- between histone methylation and ubiquitination (Dover naling, and transcriptional regulation have been linked et al. 2002; Sun and Allis 2002) have set the stage for to this molecule (Pickart 2001). Ubiquitylation is pro- functional analysis of histone ubiquitination. In a timely posed to serve as a signaling module, and the informa- paper published in the previous issue of Genes & Devel- tion transmitted by this tag may depend on the nature of opment, Shelley Berger and colleagues (Henry et al.
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A Mutation in Histone H2B Represents a New Class of Oncogenic Driver
Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/2159-8290.CD-19-0393 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver Richard L. Bennett1, Aditya Bele1, Eliza C. Small2, Christine M. Will2, Behnam Nabet3, Jon A. Oyer2, Xiaoxiao Huang1,9, Rajarshi P. Ghosh4, Adrian T. Grzybowski5, Tao Yu6, Qiao Zhang7, Alberto Riva8, Tanmay P. Lele7, George C. Schatz9, Neil L. Kelleher9 Alexander J. Ruthenburg5, Jan Liphardt4 and Jonathan D. Licht1 * 1 Division of Hematology/Oncology, University of Florida Health Cancer Center, Gainesville, FL 2 Division of Hematology/Oncology, Northwestern University 3 Department of Cancer Biology, Dana Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School 4 Department of Bioengineering, Stanford University 5 Department of Molecular Genetics and Cell Biology, The University of Chicago 6 Department of Chemistry, Tennessee Technological University 7 Department of Chemical Engineering, University of Florida 8 Bioinformatics Core, Interdisciplinary Center for Biotechnology Research, University of Florida 9 Department of Chemistry, Northwestern University, Evanston IL 60208 Running title: Histone mutations in cancer *Corresponding Author: Jonathan D. Licht, MD The University of Florida Health Cancer Center Cancer and Genetics Research Complex, Suite 145 2033 Mowry Road Gainesville, FL 32610 352-273-8143 [email protected] Disclosures: The authors have no conflicts of interest to declare Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/2159-8290.CD-19-0393 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
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Histones H3 and H4 Are Components of Upstream Activation Factor Required for the High-Level Transcription of Yeast Rdna by RNA Polymerase I
Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13458–13462, December 1997 Biochemistry Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I JOHN KEENER*, JONATHAN A. DODD*, DOMINIQUE LALO, AND MASAYASU NOMURA† Department of Biological Chemistry, University of California, Irvine, CA 92697-1700 Contributed by Masayasu Nomura, October 16, 1997 ABSTRACT RNA polymerase I (Pol I) transcription in consisting of Rrn6p, Rrn7p, and Rrn11p; refs. 3, 9, and 10), the yeast Saccharomyces cerevisiae is greatly stimulated in vivo Rrn3p (5), and Pol I are required. In addition to these factors, and in vitro by the multiprotein complex, upstream activation upstream activation factor (UAF) and TATA box-binding factor (UAF). UAF binds tightly to the upstream element of the protein, as well as the upstream element, are required for a rDNA promoter, such that once bound (in vitro), UAF does not high level of transcription from the yeast rDNA promoter (4, readily exchange onto a competing template. Of the polypep- 11). Purified UAF previously was shown to contain three tides previously identified in purified UAF, three are encoded genetically defined subunits, Rrn5p, Rrn9p, and Rrn10p, and by genes required for Pol I transcription in vivo: RRN5, RRN9, two uncharacterized subunits, p30 and p18 (4). and RRN10. Two others, p30 and p18, have remained unchar- DNA in the eukaryotic nucleus is organized as chromatin, acterized. We report here that the N-terminal amino acid consisting mostly of regularly repeating nucleosomes in which sequence, its mobility in gel electrophoresis, and the immu- DNA is wrapped around an octameric structure of core noreactivity of p18 shows that it is histone H3.
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The Histone Variant Composition of Centromeres Is Controlled by The
ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3347–3359 doi:10.1242/jcs.148189 RESEARCH ARTICLE The histone variant composition of centromeres is controlled by the pericentric heterochromatin state during the cell cycle Ekaterina Boyarchuk1,2,3,4,5,*, Dan Filipescu1,2,3,4,5,*, Isabelle Vassias1,2,3,4,5, Sylvain Cantaloube1,2,3,4,5 and Genevie`ve Almouzni1,2,3,4,5,` ABSTRACT to the cohesion of sister chromatids (Alonso et al., 2010; Bernard et al., 2001; Guenatri et al., 2004). Centric chromatin is marked by Correct chromosome segregation requires a unique chromatin the histone H3 variant CenH3 (CENP-A in vertebrates), which is environment at centromeres and in their vicinity. Here, we address interspersed with H3 nucleosomes carrying H3K4me2 and how the deposition of canonical H2A and H2A.Z histone variants is H3K36me2/3 marks (Bergmann et al., 2012; Sullivan et al., controlled at pericentric heterochromatin (PHC). Whereas in 2004). The surrounding PHC domains lack CenH3, but are euchromatin newly synthesized H2A and H2A.Z are deposited characterized by the presence of canonical H3, H3.3 and several throughout the cell cycle, we reveal two discrete waves of H2A variants, including H2A.Z (reviewed in Boyarchuk et al., deposition at PHC – during mid to late S phase in a replication- 2011). PHC domains also display an enrichment for constitutive dependent manner for H2A and during G1 phase for H2A.Z. This heterochromatin marks such as DNA methylation, H3K9me2/3 and G1 cell cycle restriction is lost when heterochromatin features are H4K20me3, as well as a depletion of acetylated histones and altered, leading to the accumulation of H2A.Z at the domain.
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CHROMATIN a Ubiquitin Crowbar Opens Chromatin Monoubiquitylation of Histone H2B Is Found to Disrupt Condensation of Chemically Defined Chromatin Fibers
news & views CHROMATIN A ubiquitin crowbar opens chromatin Monoubiquitylation of histone H2B is found to disrupt condensation of chemically defined chromatin fibers. A novel fluorescence-based assay is used in concert with analytical ultracentrifugation to uncover the synergistic roles of histone acetylation and ubiquitylation on chromatin dynamics. Craig L Peterson ukaryotic genomes must be condensed the lack of methodologies for generating in groups with less chemical biology into compact chromatin structures large quantities of recombinant, site- expertise5. More recently, they developed Eto fit within the limited confines specifically ubiquitylated protein. Previous a simplified method for large-scale of the nucleus. Consequently, these analyses of simple histone marks, such as ubiquitylation of H2B, using a site-specific 6 condensed structures limit access to histone acetylation, used expressed protein disulfide linkage (uH2BSS; Fig. 1). With this the underlying DNA and are inherently ligation (EPL) to generate semi-synthetic method, it is then a rather simple procedure repressive to essential nuclear functions, histones4. Ubiquitylation is a more difficult to reconstitute recombinant, homogeneous such as transcription, DNA repair and modification, however, as it involves nucleosomal arrays in which each replication. Given that these processes formation of a branched polypeptide nucleosome contains two copies of uH2B. function efficiently within a chromatin (Fig. 1a). Previously, Muir and colleagues Fierz et al.1 have now applied this environment, it should come as no surprise devised sophisticated EPL techniques for approach to determine the effect of H2B that mechanisms exist within cells that generating uH2B, but the yields were low ubiquitylation on the folding dynamics of ensure that condensed chromatin structures and the method unlikely to be recapitulated nucleosomal arrays.
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From Chromatin Compaction to Copper Homeostasis a Disse
UNIVERSITY OF CALIFORNIA Los Angeles The eukaryotic histones H2A and H3: From chromatin compaction to copper homeostasis A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular Biology by Oscar A. Campos 2018 © Copyright by Oscar A. Campos 2018 ABSTRACT OF THE DISSERTATION The eukaryotic histones H2A and H3: From chromatin compaction to copper homeostasis by Oscar A. Campos Doctor of Philosophy in Molecular Biology University of California, Los Angeles, 2018 Professor Siavash K. Kurdistani, Chair In eukaryotes, genome size varies disproportionately relative to nuclear size such that larger genomes are generally more compact than smaller genomes. We found that the histone H2A N- terminal domain (NTD) and the histone H2B C-terminal domain have co-evolved with genome size, and that the co-evolving residues contribute to the differential chromatin compaction in organisms as diverse as the budding yeast and humans. Interestingly, histones appear to predate eukaryotes and evolved from archaeal histone-like proteins. The structural simplicity of the ancestral histones raises doubts as to whether they participated in the types of chromatin regulatory functions histones perform today. We therefore asked whether histones performed a different function in the early eukaryotes. In the nucleosome structure, the interface region of the two histone H3 proteins forms a potential transition metal coordination site. Interestingly, the appearance of eukaryotes roughly coincided with the accumulation
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Histone Monoubiquitination in Chromatin Remodelling: Focus on the Histone H2B Interactome and Cancer
cancers Review Histone Monoubiquitination in Chromatin Remodelling: Focus on the Histone H2B Interactome and Cancer Deborah J. Marsh 1,2,* , Yue Ma 1 and Kristie-Ann Dickson 1 1 Translational Oncology Group, Faculty of Science, School of Life Sciences, University of Technology Sydney, Ultimo, NSW 2007, Australia; [email protected] (Y.M.); [email protected] (K.-A.D.) 2 Kolling Institute, Faculty of Medicine and Health, Northern Clinical School, University of Sydney, Camperdown, NSW 2006, Australia * Correspondence: [email protected]; Tel.: +61-2-9514-7574 Received: 17 October 2020; Accepted: 17 November 2020; Published: 20 November 2020 Simple Summary: Post-translational modifications (PTM) of histone tails represent epigenomic regulation of the chromatin landscape, influencing gene expression and the response to DNA damage. This review focusses on cancer-associated roles of ubiquitin as a histone PTM, specifically in conjunction with an E3 ubiquitin ligase cascade that results in the addition of a single ubiquitin (monoubiquitination) to histone H2B at lysine 120 (H2Bub1). H2Bub1 has roles in chromatin accessibility important for transcriptional elongation, the DNA damage response, cellular proliferation and developmental transitions, including in stem cell plasticity. It has been implicated in inflammation and tumour progression, with examples of its loss associated with a worse prognosis for patients with some cancers. Many factors involved in the H2Bub1 interactome are well known cancer-associated proteins, including p53, BRCA1 and components of the SWI/SNF remodelling complex. Increased knowledge of H2Bub1 and its interactome offers new opportunities for therapeutic targeting of malignancy. Abstract: Chromatin remodelling is a major mechanism by which cells control fundamental processes including gene expression, the DNA damage response (DDR) and ensuring the genomic plasticity required by stem cells to enable differentiation.
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