Chromatin regulates gene expression and therefore plays a key role in developmental processes and in the etiology of various diseases including Cancer. Nuclear protein such as histone H1 and HMGs have been shown to bind to, and alter the properties of the chromatin fiber. Changes in the expression of these architectural proteins are linked to various developmental defects and to various diseases including cancer. However, in spite of numerous studies, the mechanism of action, and the exact cellular function of these proteins remains one of the most perplexing aspects of chromatin biology. We are using a multidisciplinary approach, including analyses of genetically modified mice, to gain a comprehensive understanding of the biological function and mechanism of action of the HMGN proteins, the only nuclear proteins that bind specifically to the nucleosome core particle, the building block of the chromatin fiber. Biochemical and cytological approaches were used to demonstrate that the binding of these proteins to chromatin alters the higher-order chromatin structure and affect the cellular transcription profile. Using immunochemical analysis and fluorescent photobleaching imaging of living cells, we demonstrated that the binding of HMGNs and H1 to chromatin is dynamic rather than static, a finding that led to new insights into the kinetics of the intranuclear organization of most nuclear proteins. By microinjecting proteins into living cells expressing tagged proteins, we demonstrated that H1 and HMGs form a network of competitive interactions on nucleosomes, a novel concept that is relevant to understanding functional redundancy among related proteins and cellular homeostatic mechanisms. By analyzing Hmgn1-/- cells and studying in vitro nucleosome reconstitution systems we found that H1 and HMGNs affect the levels of histone posttranslational modifications, thereby identifying an additional mechanism that regulates the levels of these epigenetic markers. Alterations in the levels of posttranslational modifications of histones have been linked to genetic instability and cancer. Our analyses of genetically modified mice and cells derived from these mice indicate that HMGN proteins play a role in the repair of damaged DNA and in the etiology of certain cancers. We find that loss of HMGN1 impairs the repair of both single stranded and double stranded DNA damage. The repair of the single stranded damage is impaired because the nucleotide excision repair (NER) cannot effectively access the damage sites. The repair of double stranded DNA breaks (DSB) is largely dependent on the action of the nuclear protein kinase ataxia-telangiectasia mutated (ATM). ATM regulates the activity of key molecules that affect tumorigenesis, including p53. We found that loss of HMGN1, or ablation of its ability to bind to chromatin, reduces the levels of DSB-induced ATM autophosphorylation and the activation of several ATM targets. HMGN1 alters the interaction of ATM with chromatin both prior to, and following the induction of DNA damage, and also enhances the DSB-induced acetylation of Lys14 of histone H3 (H3K14). Treatment of cells with a histone deacetylase inhibitor bypasses the HMGN1 requirement for ATM activation. Thus, HMGN1 mediate the efficient activation of ATM by optimizing its chromatin interactions both prior to and after DSBs formation. 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 following DNA damage. As chromatin binding proteins, HMGNs affect developmental processes. We found that the expression of HMGN proteins is developmentally regulated and that during development the expression of these proteins is severely down regulated. Loss of HMGN1 affects the in vitro differentiation of chondrocytes. This effect is due to misexpression of Sox-9, a key regulator of chondrocyte differentiation. HMGNs bind to the chromatin of Sox 9 gene and affect its expression. Likewise, we find that HMGN1 is expressed in the hair bulge region and loss of HMGN1 protein alters the hair cycle. We also found that HMGN1 is highly expressed in the basal layer of the epithelium, including the corneal epithelium. Loss of HMGN1 affects the organization of the corneal epithelium. In mouse embryonic cells, misexpression of HMGN affects several differentiation pathways. Our findings with genetically engineered mice and cells, taken together with our previous biochemical findings, indicate that HMGNs are fine tuners of chromatin function and that proper differentiation and proper cellular function requires regulated expression of HMGN. Cells and organism can survive without a particular HMGN; however, they are more susceptible to various stresses such as heat shock or DNA damage. We found that HMGN3, a member of the HMGN protein family, is highly and specifically expressed in the beta cells of the pancreatic islets. Knock-out mice lacking HMGN3 protein are diabetic. We elucidated the molecular mechanism leading to this phenotype and demonstrated that HMGN3 enhances the binding of the transcription factor PDX1 to the promoter of the Glut2 transporter. Loss of HMGN3 decreases the levels of Glut2 thereby impairing glucose import into beta cells, insulin secretion from these cells and glucose homeostasis of the mice. Current studies suggest that HMGN3 affects pancreas affects the differentiation of pancreas endocrine cells. NSBP1, a new member of the HMG protein family which we discovered, is highly expressed in preimplantation embryos but after implantation it is not expressed in embryonic tissues but it is highly expressed in the placenta and in trophoblast cells. The migration of trophoblast cells during and after implantation has been used as a model for cell migration during metastasis. The high expression of NSBP1 in trophoblast cells, together with recent reports in the literature that the levels of NSBP1 are elevated in prostate and breast cancer, implicate the protein in tumorigenesis. Using transgenic mice in which we specifically targeted NSBP1 expression to embryonic tissues we found that aberrant expression of NSBP1 affects the viability and phenotype of the born mice. A significant number of newborn mice are either very small or die immediately after birth. Taken together the results suggest that NSBP1 may affect the cellular phenotype. We have elicited an NSBP1-/- knock out mouse and are studying its phenotype. Cell migration is essential for various physiological processes such as embryonic development, immunity and tissue repair. The metastasis of tumor cells involves gain of migration capabilities. Thus, elucidating the fundamental mechanisms regulating cell migration has important implication to the understanding of a wide range of biological processes and may have practical applications to the development of better cancer therapies. We have discovered that cell migration involves, and is contingent on, major reorganization in the structure of the chromatin fiber. This pioneering study which is the first to report that cell migration involves changes in the structure chromatin fiber points out to an additional cellular structure that can be targeted by drugs aimed at interfering with cell mi [summary truncated at 7800 characters]
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| Lucey, Michelle M; Wang, Yan; Bustin, Michael et al. (2008) Differential expression of the HMGN family of chromatin proteins during ocular development. Gene Expr Patterns 8:433-7 |
| Gerlitz, Gabi; Livnat, Idit; Ziv, Carmit et al. (2007) Migration cues induce chromatin alterations. Traffic 8:1521-9 |
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| Yang, De; Chen, Qian; Yang, Huan et al. (2007) High mobility group box-1 protein induces the migration and activation of human dendritic cells and acts as an alarmin. J Leukoc Biol 81:59-66 |
| Postnikov, Yuri V; Belova, Galina I; Lim, Jae-Hwan et al. (2006) Chromosomal protein HMGN1 modulates the phosphorylation of serine 1 in histone H2A. Biochemistry 45:15092-9 |
| Birger, Yehudit; Davis, Janine; Furusawa, Takashi et al. (2006) A role for chromosomal protein HMGN1 in corneal maturation. Differentiation 74:19-29 |
| Ueda, Tetsuya; Postnikov, Yuri V; Bustin, Michael (2006) Distinct domains in high mobility group N variants modulate specific chromatin modifications. J Biol Chem 281:10182-7 |
| Furusawa, Takashi; Lim, Jae-Hwan; Catez, Frederic et al. (2006) Down-regulation of nucleosomal binding protein HMGN1 expression during embryogenesis modulates Sox9 expression in chondrocytes. Mol Cell Biol 26:592-604 |
| Catez, Frederic; Ueda, Tetsuya; Bustin, Michael (2006) Determinants of histone H1 mobility and chromatin binding in living cells. Nat Struct Mol Biol 13:305-10 |
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