Nucleosomal binding proteins, HMGN, is a family of chromatin architectural proteins that are expressed in all vertebrate nuclei. Although previous studies have discovered that HMGN proteins have ...important roles in gene regulation and chromatin accessibility, whether and how HMGN proteins affect higher order chromatin status remains unknown. We examined the roles that HMGN1 and HMGN2 proteins play in higher order chromatin structures in three different cell types. We interrogated data generated in situ, using several techniques, including Hi-C, Promoter Capture Hi-C, ChIP-seq, and ChIP-MS. Our results show that HMGN proteins occupy the A compartment in the 3D nucleus space. In particular, HMGN proteins occupy genomic regions involved in cell-type-specific long-range promoter-enhancer interactions. Interestingly, depletion of HMGN proteins in the three different cell types does not cause structural changes in higher order chromatin, i.e., in topologically associated domains (TADs) and in A/B compartment scores. Using ChIP-seq combined with mass spectrometry, we discovered protein partners that are directly associated with or neighbors of HMGNs on nucleosomes. We determined how HMGN chromatin architectural proteins are positioned within a 3D nucleus space, including the identification of their binding partners in mononucleosomes. Our research indicates that HMGN proteins localize to active chromatin compartments but do not have major effects on 3D higher order chromatin structure and that their binding to chromatin is not dependent on specific protein partners.
Efficient and correct responses to double-stranded breaks (DSB) in chromosomal DNA are crucial for maintaining genomic stability and preventing chromosomal alterations that lead to cancer. The ...generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signalling network of the cellular response to DSB. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both before and after DSB formation, thereby optimizing its activation. Loss of HMGN1 or ablation of its ability to bind to chromatin reduces the levels of ionizing radiation (IR)-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys 14 of histone H3 (H3K14) in an HMGN1-dependent manner and treatment of cells with histone deacetylase inhibitors bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation after DNA damage.
Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by ...post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.
Although human papillomavirus was identified as an aetiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating ...gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major subnetworks upregulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal human papillomavirus decline in women with invasive cancer who were previously unable to clear the virus.
High mobility group N1 protein (HMGN1), a nucleosomal-binding protein that affects the structure and function of chromatin, is encoded by a gene located on chromosome 21 and is overexpressed in Down ...syndrome, one of the most prevalent genomic disorders. Misexpression of HMGN1 affects the cellular transcription profile; however, the biological function of this protein is still not fully understood. We report that HMGN1 modulates the expression of methyl CpG-binding protein 2 (MeCP2), a DNA-binding protein known to affect neurological functions including autism spectrum disorders, and whose alterations in HMGN1 levels affect the behavior of mice. Quantitative PCR and Western analyses of cell lines and brain tissues from mice that either overexpress or lack HMGN1 indicate that HMGN1 is a negative regulator of MeCP2 expression. Alterations in HMGN1 levels lead to changes in chromatin structure and histone modifications in the MeCP2 promoter. Behavior analyses by open field test, elevated plus maze, Reciprocal Social Interaction, and automated sociability test link changes in HMGN1 levels to abnormalities in activity and anxiety and to social deficits in mice. Targeted analysis of the Autism Genetic Resource Exchange genotype collection reveals a non-random distribution of genotypes within 500 kbp of HMGN1 in a region affecting its expression in families predisposed to autism spectrum disorders. Our results reveal that HMGN1 affects the behavior of mice and suggest that epigenetic changes resulting from altered HMGN1 levels could play a role in the etiology of neurodevelopmental disorders.
Background: HMGN1 is a chromatin-binding protein that modulates the cellular transcription profile.
Results: Mice that either overexpress or lack HMGN1 have altered behavioral phenotypes. HMGN1 is a negative regulator of MeCP2, a protein involved in neurodevelopmental disorders.
Conclusion: Misregulation of HMGN1 protein levels lead to behavioral changes in mice.
Significance: HMGN1 is an epigenetic factor that contributes to the development of neurodevelopmental disorders.
Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription ...factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark enhances the interaction of architectural protein with chromatin regulatory sites and identify determinants that facilitate the localization of HMGN proteins at regulatory sites to modulate cell-type specific gene expression.
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