Interaction of HP1 and Brg1/Brm with the Globular Domain of Histone H3 Is Required for HP1-Mediated Repression

The heterochromatin-enriched HP1 proteins play a critical role in regulation of transcription. These proteins contain two related domains known as the chromo- and the chromoshadow-domain. The chromo-domain binds histone H3 tails methylated on lysine 9. However, in vivo and in vitro experiments have shown that the affinity of HP1 proteins to native methylated chromatin is relatively poor and that the opening of chromatin occurring during DNA replication facilitates their binding to nucleosomes. These observations prompted us to investigate whether HP1 proteins have additional histone binding activities, envisioning also affinity for regions potentially occluded by the nucleosome structure. We find that the chromoshadow-domain interacts with histone H3 in a region located partially inside the nucleosomal barrel at the entry/exit point of the nucleosome. Interestingly, this region is also contacted by the catalytic subunits of the human SWI/SNF complex. In vitro, efficient SWI/SNF remodeling requires this contact and is inhibited in the presence of HP1 proteins. The antagonism between SWI/SNF and HP1 proteins is also observed in vivo on a series of interferon-regulated genes. Finally, we show that SWI/SNF activity favors loading of HP1 proteins to chromatin both in vivo and in vitro. Altogether, our data suggest that HP1 chromoshadow-domains can benefit from the opening of nucleosomal structures to bind chromatin and that HP1 proteins use this property to detect and arrest unwanted chromatin remodeling

ras transformation is associated with decreased expression of the brm/SNF2alpha ATPase from the mammalian SWI-SNF complex.

The brm and BRG-1 proteins are mutually exclusive subunits of the mammalian SWI-SNF complex. Within this complex, they provide the ATPase activity necessary for transcriptional regulation by nucleosome disruption. Both proteins were recently found to interact with the p105Rb tumor suppressor gene product, suggesting a role for the mammalian SWI-SNF complex in the control of cell growth. We show here that the expression of brm, but not BRG-1, is negatively regulated by mitogenic stimulation, and that growth arrest of mouse fibroblasts leads to increased accumulation of the brm protein. The expression of this protein is also down-regulated upon transformation by the ras oncogene. Re-introduction of brm into ras transformed cells leads to partial reversion of the transformed phenotype by a mechanism that depends on the ATPase domain of the protein. Our data suggest that increased levels of brm protein favour the withdrawal of the cell from the cycle whereas decreased expression of the brm gene may facilitate cellular transformation by various oncogenes

The hbrm and BRG-1 proteins, components of the human SNF/SWI complex, are phosphorylated and excluded from the condensed chromosomes during mitosis.

In yeast, the SNF/SWI complex is believed to regulate transcription by locally altering the chromatin structure. At the present time, three human homologues of yeast SNF/SWI proteins have been characterized: hbrm and BRG-1, homologues of SNF2/SWI2, and hSNF5, a homologue of SNF5. We show here that, during mitosis, hbrm and BRG-1 are phosphorylated and excluded from the condensed chromosomes. In this phase of the cell cycle, the level of hbrm protein is also strongly reduced, whereas the level of BRG-1 remains constant. The mitotic phosphorylation of hbrm and BRG-1 is found not to disrupt the association of these proteins with hSNF5 but correlates with a decreased affinity for the nuclear structure in early M phase. We suggest that chromosomal exclusion of the human SNF/SWI complex at the G2-M transition could be part of the mechanism leading to transcriptional arrest during mitosis

Growth inhibition by the mammalian SWI–SNF subunit Brm is regulated by acetylation

In mammalian cells, the SWI–SNF chromatin-remodeling complex is a regulator of cell proliferation, and overexpression of the catalytic subunit Brm interferes with cell cycle progression. Here, we show that treatment with histone deacetylase (HDAC) inhibitors reduces the inhibitory effect of Brm on the growth of mouse fibroblasts. This observation led to the identification of two carboxy-terminal acetylation sites in the Brm protein. Mutation of these sites into non-acetylatable sequences increased both the growth-inhibitory and the transcriptional activities of Brm. We also show that culture in the presence of HDAC inhibitors facilitates the isolation of clones overexpressing Brm. Removal of the HDAC inhibitors from the growth medium of these clones leads to downregulation of cyclin D1. This downregulation is absent in cell transformed by oncogenic ras