Christian Muchardt

Purification and biochemical heterogeneity of the mammalian SWI-SNF complex.

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1‐associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2‐SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47‐associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co‐purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI‐SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4‐VP16 to a nucleosomal template similar to the yeast SWI‐SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI‐SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI‐SNF complex may be tailored to the needs of a differentiated cell type.

The human SWI/SNF subunit Brm is a regulator of alternative splicing.

The SWI/SNF (mating-type switch/sucrose nonfermenting) complex involved in chromatin remodeling on promoters has also been detected on the coding region of genes. Here we show that SWI/SNF can function as a regulator of alternative splicing. We found that the catalytic subunit Brm favors inclusion of variant exons in the mRNA of several genes, including E-cadherin, BIM, cyclin D1 and CD44. Consistent with this, Brm associates with several components of the spliceosome and with Sam68, an ERK-activated enhancer of variant exon inclusion. Examination of the CD44 gene revealed that Brm induced accumulation of RNA polymerase II (RNAPII) with a modified CTD phosphorylation pattern on regions encoding variant exons. Altogether, our data suggest that on genes regulated by SWI/SNF, Brm contributes to the crosstalk between transcription and RNA processing by decreasing RNAPII elongation rate and facilitating recruitment of the splicing machinery to variant exons with suboptimal splice sites.

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri‐implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild‐type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types.

Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1α

In mammalian cells, as in Schizosaccharomyces pombe and Drosophila, HP1 proteins bind histone H3 tails methylated on lysine 9 (K9). However, whereas K9‐methylated H3 histones are distributed throughout the nucleus, HP1 proteins are enriched in pericentromeric heterochromatin. This observation suggests that the methyl‐binding property of HP1 may not be sufficient for its heterochromatin targeting. We show that the association of HP1α with pericentromeric heterochromatin depends not only on its methyl‐binding chromo domain but also on an RNA‐binding activity present in the hinge region of the protein that connects the conserved chromo and chromoshadow domains. Our data suggest the existence of complex heterochromatin binding sites composed of methylated histone H3 tails and RNA, with each being recognized by a separate domain of HP1α.

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.

Histone modifications induced by a family of bacterial toxins

Upon infection, pathogens reprogram host gene expression. In eukaryotic cells, genetic reprogramming is induced by the concerted activation/repression of transcription factors and various histone modifications that control DNA accessibility in chromatin. We report here that the bacterial pathogen Listeria monocytogenes induces a dramatic dephosphorylation of histone H3 as well as a deacetylation of histone H4 during early phases of infection. This effect is mediated by the major listerial toxin listeriolysin O in a pore-forming-independent manner. Strikingly, a similar effect also is observed with other toxins of the same family, such as Clostridium perfringens perfringolysin and Streptococcus pneumoniae pneumolysin. The decreased levels of histone modifications correlate with a reduced transcriptional activity of a subset of host genes, including key immunity genes. Thus, control of epigenetic regulation emerges here as an unsuspected function shared by several bacterial toxins, highlighting a common strategy used by intracellular and extracellular pathogens to modulate the host response early during infection.

When the SWI/SNF complex remodels...the cell cycle.

Mammalian cells contain several chromatin-remodeling complexes associated with the Brm and Brg1 helicase-like proteins. These complexes likely represent the functional homologs of the SWI/SNF and RSC complexes found in Saccharomyces cerevisiae. The mammalian chromatin-remodeling complexes are involved in both activation and repression of a variety of genes. Several lines of evidence also indicate that they play a specific role in the regulation of cell growth. Brm is down-regulated by ras signaling and its forced re-expression suppresses transformation by this oncogene. Besides, the Brg1 gene is silenced or mutated in several tumors cell lines and a Brg1-associated complex was recently found to co-purify with BRCA1, involved in breast and ovarian cancers. Finally, the gene encoding SNF5/Ini1, a subunit common to all mammalian SWI/SNF complexes, is inactivated in rhabdoid sarcomas, a very aggressive form of pediatric cancer. The current review will address observations made upon inactivation of Brm, Brg1 and SNF5/Ini1 by homologous recombination in the mouse, as well as the possible implication of these factors in the regulation of the Retinoblastoma pRb-mediated repression of the transcription factor E2F.

RB and c-Myc Activate Expression of the E-Cadherin Gene in Epithelial Cells through Interaction with Transcription Factor AP-2

ABSTRACT E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. We recently reported that inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers including E-cadherin. Here we show that both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 was demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibited its binding to both RB and AP-2. Taken together, our results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc.

Tethering of HP1 proteins to chromatin is relieved by phosphoacetylation of histone H3

Histone H3 lysine 9 methylation is associated with long‐term transcriptional repression through recruitment of heterochromatin protein 1 (HP1) proteins. These proteins are believed to promote the formation of dense chromatin structures interfering with DNA accessibility. During the G2 phase of the cell cycle, HP1 proteins are delocalized from foci of pericentromeric heterochromatin, while a wave of H3 serine 10 phosphorylation is initiated within these regions. Here, we show that in vivo phosphorylation of serine 10 in G2 can occur on histone tails methylated on lysine 9. Unexpectedly, this modification favours rather than prevents HP1 binding to chromatin. Dissociation of HP1 from the methylated histone H3 tails is observed only after a third modification by acetylation of lysine 14, which occurs in prophase. We propose that phosphoacetylation of histone H3 could be a general mechanism allowing the cell to overcome HP1‐mediated transcriptional repression.

The PRC1 Polycomb group complex interacts with PLZF/RARA to mediate leukemic transformation

Ectopic repression of retinoic acid (RA) receptor target genes by PML/RARA and PLZF/RARA fusion proteins through aberrant recruitment of nuclear corepressor complexes drives cellular transformation and acute promyelocytic leukemia (APL) development. In the case of PML/RARA, this repression can be reversed through treatment with all-trans RA (ATRA), leading to leukemic remission. However, PLZF/RARA ectopic repression is insensitive to ATRA, resulting in persistence of the leukemic diseased state after treatment, a phenomenon that is still poorly understood. Here we show that, like PML/RARA, PLZF/RARA expression leads to recruitment of the Polycomb-repressive complex 2 (PRC2) Polycomb group (PcG) complex to RA response elements. However, unlike PML/RARA, PLZF/RARA directly interacts with the PcG protein Bmi-1 and forms a stable component of the PRC1 PcG complex, resulting in PLZF/RARA-dependent ectopic recruitment of PRC1 to RA response elements. Upon treatment with ATRA, ectopic recruitment of PRC2 by either PML/RARA or PLZF/RARA is lost, whereas PRC1 recruited by PLZF/RARA remains, resulting in persistent RA-insensitive gene repression. We further show that Bmi-1 is essential for the PLZF/RARA cellular transformation property and implicates a central role for PRC1 in PLZF/RARA-mediated myeloid leukemic development.