The ATP-dependent chromatin-remodeling complexes play important roles in gene regulation by opening chromatin structures for transcriptional activators or repressors. The prototype of this type of complexes is the SWI/SNF complex, which was found in diverse organisms, including yeast, Drosophila, mouse and human. It is required for proper expression of homeotic genes and segmentation in Drosophila, and mutation in one subunit of the complex causes pediatric rhabdoid cancer in humans. We have purified several human SWI/SNF-related complexes. By microsequencing, we have identified and cloned all the subunits from the major form of the complex. Analysis revealed that BAF250a contains a DNA binding domain similar to yeast SWI1, and several LXXLL motifs, which have been previously shown to be able to interact with nuclear hormone receptors. Using transient transfection assays, we found that BAF250a in fact facilitates transcriptional activation by glucocorticoid receptor (GR). The region containing LXXLL motifs of BAF250 also interacts with GR in vitro. This work suggests that BAF250a may be a targeting subunit of hSWI/SNF, and may mediate the recruitment of the complex to DNA-bound glucocorticoid receptors. As a continuation of this project, we have cloned a novel human homolog of BAF250a, termed BAF250b. The two genes share over 60% of identity and possess same type of domain structure. We have now isolated a BAF250b-containing complex. It shares several identical subunits with BAF250a complex but also contains its own unique components. One unique subunit is ENL, a fusion partner for MLL which is a common target for chromosomal translocation in human acute leukemia. ENL is also the human ortholog of yeast SWI/SNF subunit, TFG3. We demonstrated that the resultant MLL-ENL fusion protein also assciates with a human SWI/SNF complex. Moreover, the fusion protein cooperates with SWI/SNF to activate the promoter of HOXA7, which is a downstream target of MLL and is essential for oncogenic activity of the MLL fusion proteins. Our data suggest that human SWI/SNF complexes show considerable hetergeneity, and one or more may be involved in the etiology of leukemia by functioning with MLL-fusion proteins. ? We are continuing to identify the genes that are specifically dependent on BAF, but not PBAF, for expression. Using siRNA, we were able to deplete the BAF-specific subunit, BAF250. We showed that one interferon-responsive gene, IFGM3, specifically depends on BAF but not PBAF for expression. This result demonstrates that BAF and PBAF have selectivity in mediating expression of different genes. We plan to investigate the mechanism of how BAF is targeted to IFGM3 and other genes.? Recently, we initiated a new collaborative project with Drs. Minoru Ko and Zhong Wangs labs to study the function of SWI/SNF complexes in the maintenance of pluripotency of ES cells. SWI/SNF chromatin remodeling complexes are known to be essential for early embryonic development in mice. However, the roles of these complexes in embryonic stem (ES) cells are poorly understood. One reason is that mice deficient in common components of SWI/SNF complexes die very early, before the ES cells can be established. In this project, we show that two subgroups of SWI/SNF complexes associated with BAF250a (a.k.a. Arid1a) and BAF250b (a.k.a. Arid1b) are present at high levels in undifferentiated ES cells, and their levels decrease when ES cells are induced to differentiate. We generated mouse ES cells deficient in BAF250b by gene targeting, and found that these cells have a reduced proliferation rate and an abnormal cell cycle. More importantly, they lost the self-renewal capacity of ES cells and displayed multiple markers characteristic of differentiated cells. Microarray and subsequent qRT-PCR analysis confirmed that these cells have reduced expression of several genes involved in pluripotency of ES cells, and increased expression of several differentiation genes. These data suggest that the BAF250b-associated SWI/SNF is essential for mouse ES cells to maintain its normal proliferation and undifferentiated state. ? On a related project, we collaborated with Drs. Gong and Smerdon's labs and showed that SWI/SNF complex is involved in the cellular response pathway to UV-induced DNA damage. This study uncovers another mechanism of SWI/SNF in protecting genome stability.
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