Structural And Functional Studies Of Human Swi/snf Chromatin-remodeling

atp依赖性染色质重塑复合物通过打开染色质结构以激活或抑制转录因子，在基因调控中发挥重要作用。这类复合物的原型是SWI/SNF复合物，它在多种生物中被发现，包括酵母、果蝇、小鼠和人类。它是果蝇同源基因的正确表达和分割所必需的，而该复合体的一个亚基突变导致人类儿童横纹肌样癌。我们纯化了几个人类SWI/ snf相关复合物。通过微测序，我们已经鉴定并克隆了该复合体主要形式的所有亚基。分析显示，BAF250a含有一个类似于酵母SWI1的DNA结合域，以及几个LXXLL基序，这些基序先前已被证明能够与核激素受体相互作用。通过瞬时转染实验，我们发现BAF250a实际上促进了糖皮质激素受体（GR）的转录激活。BAF250含有LXXLL基序的区域在体外也与GR相互作用。这项研究表明，BAF250a可能是hSWI/SNF的靶向亚基，并可能介导复合物向dna结合的糖皮质激素受体的募集。作为该项目的延续，我们克隆了一种新的BAF250a的人类同源物，命名为BAF250b。这两个基因具有60%以上的同一性和相同类型的结构域结构。值得注意的是，我们分离出了一个含baf250b的复合物。它与BAF250a复合体有几个相同的亚基，但也含有自己独特的成分。一个独特的亚基是ENL，是MLL的融合伙伴，是人类急性白血病染色体易位的共同目标。ENL也是酵母SWI/SNF亚基TFG3的人类同源基因。我们证明了MLL-ENL融合蛋白与人类SWI/SNF复合物相关。此外，融合蛋白与SWI/SNF协同激活HOXA7的启动子，HOXA7是MLL的下游靶点，对MLL融合蛋白的致癌活性至关重要。我们的数据表明，人类SWI/SNF复合物表现出相当大的异质性，一个或多个可能通过mll融合蛋白的功能参与白血病的病因学。

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. Notably, we have 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 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. We initiated a 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. We are continuing to collaborate with Dr. Zhong Wang's group to investigate the roles of SWI/SNF in cardiac muscles. 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. We studied the regulation of MeCP2, a methyl DNA binding protein involved in Rett syndrome. Rett syndrome is a neurological disorder and one of the most common causes of mental retardation in girls. In up to 80% of the patients, the defects lie in mutation of the MeCP2 gene. MeCP2 consists of a DNA binding domain specific for methylated CpG dinucleotide, and a transcriptional repression domain. It could therefore bind methylated DNA through its binding domain and then silence gene expression through its repression domain. Indeed, MeCP2 has been shown to function as a transcriptional repressor both in vivo and in vitro. One previous publication suggested that MeCP2 functions through its stable association with SWI/SNF chromatin remodeling complex. We immunopurified MeCP2 and SWI/SNF complex, and found that there is no detectable association between these molecules. A previous study has shown that MeCP2 becomes hyperphosphorylated at S421 when neurons are induced to undergo depolarization. This phosphorylation inhibits MeCP2 DNA-binding activity, which correlates with increased transcription of BDNF, an important regulator for neuronal function. We used mass spectrometry to identify 5 phosphorylation sites in MeCP2 purified from normal mouse brain, and 2 additional sites in MeCP2 from mice induced to undergo seizure. We have also successfully generated one phosphorylation site-specific antibody for Ser80, and were able to confirm phosphorylation at Ser80 in vivo. In collaboration with Dr. Y. Suns lab at UCLA, we find that although the total level of MeCP2 phosphorylation is increased during depolarization of neurons, the level of phosphorylation at Ser80 is actually decreased. Furthermore, we find that mutating Ser80 to alanine (S80A) reduces BDNF gene expression in cultured neurons derived from either normal mice or MeCP2-null mice. These data suggest an important role of phosphorylation at Ser80 in MeCP2 function. By chromatin-IP, we found that an S80A mutation increased the level of MeCP2 bound to the BDNF promoter in vivo, which correlates with the observation that the same mutation reduced BDNF transcription. Through collaboration with Drs. Qiang Chang and Rudy Jaenisch (MIT), a knock-in mutant that substitutes Ser80 with alanine has been generated in mice. These mice exhibit locomotor deficits, a phenotype observed in Rett syndrome patients. The data provide in vivo evidence for the functional significance of MeCP2 phosphorylation. We are continuing to collaborate with the above labs to investigate the how MeCP2 phosphorylation is regulated. Our lab discovered the NURD complex many years ago. This complex has both ATP-dependend chromatin remodeling and histone deacetylation activities. Recently, a Cell paper claims that LSD1, a histone demethylase, is part of the NURD complex. We and several other labs re-examined this issue and found that there is no detectable association between LSD1 and NURD complex. We published our findings as a Comment to the Cell article.