Saïd Sif

Ikaros DNA-Binding Proteins Direct Formation of Chromatin Remodeling Complexes in Lymphocytes

The Ikaros gene family encodes zinc finger DNA-binding proteins essential for lineage determination and control of proliferation in the lymphoid system. Here, we report that, in the nucleus of a T cell, a major fraction of Ikaros and Aiolos proteins associate with the DNA-dependent ATPase Mi-2 and histone deacetylases, in a 2 MD complex. This Ikaros-NURD complex is active in chromatin remodeling and histone deacetylation. Upon T cell activation, Ikaros recruits Mi-2/HDAC to regions of heterochromatin. These studies reveal that Ikaros proteins are capable of targeting chromatin remodeling and deacetylation complexes in vivo. We propose that the restructuring of chromatin is a key aspect of Ikaros function in lymphocyte differentiation.

Reconstitution of a Core Chromatin Remodeling Complex from SWI/SNF Subunits

Protein complexes of the SWI/SNF family remodel nucleosome structure in an ATP-dependent manner. Each complex contains between 8 and 15 subunits, several of which are highly conserved between yeast, Drosophila, and humans. We have reconstituted an ATP-dependent chromatin remodeling complex using a subset of conserved subunits. Unexpectedly, both BRG1 and hBRM, the ATPase subunits of human SWI/SNF complexes, are capable of remodeling mono-nucleosomes and nucleosomal arrays as purified proteins. The addition of INI1, BAF155, and BAF170 to BRG1 increases remodeling activity to a level comparable to that of the whole hSWI/SNF complex. These data define the functional core of the hSWI/SNF complex.

Human SWI/SNF-Associated PRMT5 Methylates Histone H3 Arginine 8 and Negatively Regulates Expression of ST7 and NM23 Tumor Suppressor Genes

ABSTRACT Protein arginine methyltransferases (PRMTs) have been implicated in transcriptional activation and repression, but their role in controlling cell growth and proliferation remains obscure. We have recently shown that PRMT5 can interact with flag-tagged BRG1- and hBRM-based hSWI/SNF chromatin remodelers and that both complexes can specifically methylate histones H3 and H4. Here we report that PRMT5 can be found in association with endogenous hSWI/SNF complexes, which can methylate H3 and H4 N-terminal tails, and show that H3 arginine 8 and H4 arginine 3 are preferred sites of methylation by recombinant and hSWI/SNF-associated PRMT5. To elucidate the role played by PRMT5 in gene regulation, we have established a PRMT5 antisense cell line and determined by microarray analysis that more genes are derepressed when PRMT5 levels are reduced. Among the affected genes, we show that suppressor of tumorigenicity 7 (ST7) and nonmetastatic 23 (NM23) are direct targets of PRMT5-containing BRG1 and hBRM complexes. Furthermore, we demonstrate that expression of ST7 and NM23 is reduced in a cell line that overexpresses PRMT5 and that this decrease in expression correlates with H3R8 methylation, H3K9 deacetylation, and increased transformation of NIH 3T3 cells. These findings suggest that the BRG1- and hBRM-associated PRMT5 regulates cell growth and proliferation by controlling expression of genes involved in tumor suppression.

The p400 complex is an essential E1A transformation target.

Here, we report the identification of a new E1A binding protein complex that is essential for E1A-mediated transformation. Its core component is a SWI2/SNF2-related, 400 kDa protein (p400). Other components include the myc- and p/CAF-associated cofactor, TRRAP/PAF400, the DNA helicases TAP54alpha/beta, actin-like proteins, and the human homolog of the Drosophila Enhancer of Polycomb protein. An E1A mutant, defective in p400 binding, is also defective in transformation. Certain p400 fragments partially rescued this phenotype, underscoring the role of E1A-p400 complex formation in the E1A transforming process. Furthermore, E1A and c-myc each alter the subunit composition of p400 complexes, implying that physiological p400 complex formation contributes to transformation suppression.

Human SWI/SNF Interconverts a Nucleosome between Its Base State and a Stable Remodeled State

The human SWI/SNF complex remodels nucleosome structure in an ATP-dependent manner, although the nature of this change has not been determined. Here we show that hSWI/SNF and ATP generate an altered nucleosomal structure that is stable in the absence of SWI/SNF. This product has an altered sensitivity to digestion by DNAse, restriction enzymes, and micrococcal nuclease, and an increased affinity for GAL4. It has the same protein composition but is approximately twice the size of a normal nucleosome. Incubation of the altered nucleosome with hSWI/SNF converts this structure back to a standard nucleosome in an ATP-dependent process. These results suggest that hSWI/ SNF acts by facilitating an exchange between normal and altered, more accessible, nucleosome conformations.

Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing

Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.

mSin3A/Histone Deacetylase 2- and PRMT5-Containing Brg1 Complex Is Involved in Transcriptional Repression of the Myc Target Gene cad

ABSTRACT The role of hSWI/SNF complexes in transcriptional activation is well characterized; however, little is known about their function in transcriptional repression. We have previously shown that subunits of the mSin3A/histone deacetylase 2 (HDAC2) corepressor complex copurify with hSWI/SNF complexes. Here we show that the type II arginine-specific methyltransferase PRMT5, which is involved in cyclin E repression, can be found in association with Brg1 and hBrm-based hSWI/SNF complexes. We also show that hSWI/SNF-associated PRMT5 can methylate hypoacetylated histones H3 and H4 more efficiently than hyperacetylated histones H3 and H4. Protein-protein interaction studies indicate that PRMT5 and mSin3A interact with the same hSWI/SNF subunits as those targeted by c-Myc. These observations prompted us to examine the expression profile of the c-Myc target genes, carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase (cad) and nucleolin (nuc). We found that cad repression is altered in cells that express inactive Brg1 and in cells treated with the HDAC inhibitor depsipeptide. Using chromatin immunoprecipitation assays, we found that Brg1, mSin3A, HDAC2, and PRMT5 are directly recruited to the cad promoter. These results suggest that hSWI/SNF complexes, through their ability to interact with activator and repressor proteins, control expression of genes involved in cell growth and proliferation.

BRG-1 Is Recruited to Estrogen-Responsive Promoters and Cooperates with Factors Involved in Histone Acetylation

ABSTRACT Several factors that mediate activation by nuclear receptors also modify the chemical and structural composition of chromatin. Prominent in this diverse group is the steroid receptor coactivator 1 (SRC-1) family, which interact with agonist-bound nuclear receptors, thereby coupling them to multifunctional transcriptional coregulators such as CREB-binding protein (CBP), p300, and PCAF, all of which have potent histone acetyltransferase activity. Additionally factors including the Brahma-related gene 1 (BRG-1) that are involved in the structural remodeling of chromatin also mediate hormone-dependent transcriptional activation by nuclear receptors. Here, we provide evidence that these two distinct mechanisms of coactivation may operate in a collaborative manner. We demonstrate that transcriptional activation by the estrogen receptor (ER) requires functional BRG-1 and that the coactivation of estrogen signaling by either SRC-1 or CBP is BRG-1 dependent. We find that in response to estrogen, ER recruits BRG-1, thereby targeting BRG-1 to the promoters of estrogen-responsive genes in a manner that occurs simultaneous to histone acetylation. Finally, we demonstrate that BRG-1-mediated coactivation of ER signaling is regulated by the state of histone acetylation within a cell. Inhibition of histone deacetylation by trichostatin A dramatically increases BRG-1-mediated coactivation of ER signaling, and this increase is reversed by overexpression of histone deacetylase 1. These studies support a critical role for BRG-1 in ER action in which estrogen stimulates an ER–BRG-1 association coupling BRG-1 to regions of chromatin at the sites of estrogen-responsive promoters and promotes the activity of other recruited factors that alter the acetylation state of chromatin.

Mammalian SWI-SNF Complexes Contribute to Activation of the hsp70 Gene

ABSTRACT ATP-dependent chromatin-remodeling complexes are conserved among all eukaryotes and function by altering nucleosome structure to allow cellular regulatory factors access to the DNA. Mammalian SWI-SNF complexes contain either of two highly conserved ATPase subunits: BRG1 or BRM. To identify cellular genes that require mammalian SWI-SNF complexes for the activation of gene expression, we have generated cell lines that inducibly express mutant forms of the BRG1 or BRM ATPases that are unable to bind and hydrolyze ATP. The mutant subunits physically associate with at least two endogenous members of mammalian SWI-SNF complexes, suggesting that nonfunctional, dominant negative complexes may be formed. We determined that expression of the mutant BRG1 or BRM proteins impaired the ability of cells to activate the endogenous stress response gene hsp70 in response to arsenite, a metabolic inhibitor, or cadmium, a heavy metal. Activation of hsp70 by heat stress, however, was unaffected. Activation of the heme oxygenase 1 promoter by arsenite or cadmium and activation of the cadmium-inducible metallothionein promoter also were unaffected by the expression of mutant SWI-SNF components. Analysis of a subset of constitutively expressed genes revealed no or minimal effects on transcript levels. We propose that the requirement for mammalian SWI-SNF complexes in gene activation events will be specific to individual genes and signaling pathways.

MITF and PU.1 Recruit p38 MAPK and NFATc1 to Target Genes during Osteoclast Differentiation

Transcription factors NFATc1, PU.1, and MITF collaborate to regulate specific genes in response to colony-stimulating factor-1 (CSF-1) and receptor activator of NF-κB ligand (RANKL) signaling during osteoclast differentiation. However, molecular details concerning timing and mechanism of specific events remain ill-defined. In bone marrow-derived precursors, CSF-1 alone promoted assembly of MITF-PU.1 complexes at osteoclast target gene promoters like cathepsin K and acid 5 phosphatase without increasing gene expression. The combination of RANKL and CSF-1 concurrently increased the levels of MAPK-phosphorylated forms of MITF, p38 MAPK, and SWI/SNF chromatin-remodeling complexes bound to these target promoters and markedly increased expression of the genes. NFATc1 was subsequently recruited to complexes at the promoters during terminal stages of osteoclast differentiation. Genetic analysis of Mitf and Pu.1 in mouse models supported the critical interaction of these genes in osteoclast differentiation. The results define MITF and PU.1 as nuclear effectors that integrate CSF-1/RANKL signals during osteoclast differentiation to initiate expression of target genes, whereas a complex that includes NFATc1 may act to maintain target gene expression in differentiated cells.