Structural And Functional Studies Of Human Swi/snf Chromatin-remodeling
人类 Swi/snf 染色质重塑的结构和功能研究
基本信息
- 批准号:10467892
- 负责人:
- 金额:$ 127.74万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:ARID1A geneAbnormal CellAcute leukemiaAgingAlanineAntibodiesBDNF geneBindingBiochemistryBiological AssayBrainBrain-Derived Neurotrophic FactorCell CycleCell Differentiation processCell physiologyCellsCharacteristicsChildhoodChimeric ProteinsChromatinChromatin ModelingChromatin Remodeling FactorChromatin StructureChromosomal translocationChromosome CondensationCollaborationsComplexCpG dinucleotideDNADNA BindingDNA Binding DomainDNA DamageDNA-Binding ProteinsDataDefectDifferentiated GeneDrosophila genusEmbryonic DevelopmentEnzymesEtiologyExhibitsGene ExpressionGene Expression RegulationGene SilencingGene TargetingGenesGenetic TranscriptionGenome StabilityGlucocorticoid ReceptorGoalsHistone DeacetylationHomeobox GenesHomologous GeneHumanIn VitroInterferonsKDM1A geneKnock-inKnockout MiceMaintenanceMalignant NeoplasmsMass Spectrum AnalysisMediatingMental RetardationMetabolicMethyl-CpG-Binding Protein 2Microarray AnalysisMultiprotein ComplexesMusMutateMutationMyocardiumNeuronsNuRD complexNuclear Hormone ReceptorsOncogenicOrganismOrthologous GenePaperPathway interactionsPatientsPhenotypePhosphorylationPhosphorylation SitePlayProcessPublicationsPublished CommentPublishingQuantitative Reverse Transcriptase PCRReactionRegulationRepressionRett SyndromeRoleSWI/SNF Family ComplexSWI1SeizuresSiteSmall Interfering RNAStem cell pluripotencyStructureSubgroupThe SunTranscription CoactivatorTranscription RepressorTranscriptional ActivationTransfectionUV induced DNA damageUndifferentiatedWorkYeastscancer typechromatin remodelingembryonic stem cellgene repressiongenetic informationgirlshistone demethylasehuman diseasein vivoknockout geneleukemialocomotor deficitmutantnervous system disordernovelpluripotencypromoterprototyperecruitresponseself-renewal
项目摘要
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.
项目成果
期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Uncovering early response of gene regulatory networks in ESCs by systematic induction of transcription factors.
通过系统诱导转录因子,揭示了ESC中基因调节网络的早期反应。
- DOI:10.1016/j.stem.2009.07.012
- 发表时间:2009-10-02
- 期刊:
- 影响因子:23.9
- 作者:Nishiyama A;Xin L;Sharov AA;Thomas M;Mowrer G;Meyers E;Piao Y;Mehta S;Yee S;Nakatake Y;Stagg C;Sharova L;Correa-Cerro LS;Bassey U;Hoang H;Kim E;Tapnio R;Qian Y;Dudekula D;Zalzman M;Li M;Falco G;Yang HT;Lee SL;Monti M;Stanghellini I;Islam MN;Nagaraja R;Goldberg I;Wang W;Longo DL;Schlessinger D;Ko MS
- 通讯作者:Ko MS
Interplay of BAF and MLL4 promotes cell type-specific enhancer activation.
- DOI:10.1038/s41467-021-21893-y
- 发表时间:2021-03-12
- 期刊:
- 影响因子:16.6
- 作者:Park YK;Lee JE;Yan Z;McKernan K;O'Haren T;Wang W;Peng W;Ge K
- 通讯作者:Ge K
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Weidong Wang其他文献
Weidong Wang的其他文献
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{{ truncateString('Weidong Wang', 18)}}的其他基金
Developing proinsulin misfolding inhibitors for beta cell protection and diabetes treatment
开发用于 β 细胞保护和糖尿病治疗的胰岛素原错误折叠抑制剂
- 批准号:
10529960 - 财政年份:2022
- 资助金额:
$ 127.74万 - 项目类别:
Developing proinsulin misfolding inhibitors for beta cell protection and diabetes treatment
开发用于 β 细胞保护和糖尿病治疗的胰岛素原错误折叠抑制剂
- 批准号:
10665748 - 财政年份:2022
- 资助金额:
$ 127.74万 - 项目类别:
Preclinical Validation of PPARg Acetylation Inhibitors for Diabetes Prevention and Treatment
PPARg 乙酰化抑制剂预防和治疗糖尿病的临床前验证
- 批准号:
10580851 - 财政年份:2021
- 资助金额:
$ 127.74万 - 项目类别:
Cytoprotection and the mechanism of action of a natural product Khellin against ER stress
天然产物 Khellin 对抗 ER 应激的细胞保护和作用机制
- 批准号:
9974514 - 财政年份:2018
- 资助金额:
$ 127.74万 - 项目类别:
Cytoprotection and the mechanism of action of a natural product Khellin against ER stress
天然产物 Khellin 对抗 ER 应激的细胞保护和作用机制
- 批准号:
10285537 - 财政年份:2018
- 资助金额:
$ 127.74万 - 项目类别:
Preclinical Development of Khellin Analogs for Anti-Diabetic Therapy
用于抗糖尿病治疗的 Khellin 类似物的临床前开发
- 批准号:
9336063 - 财政年份:2016
- 资助金额:
$ 127.74万 - 项目类别:
Preclinical Development of Khellin Analogs for Anti-Diabetic Therapy
用于抗糖尿病治疗的 Khellin 类似物的临床前开发
- 批准号:
9353780 - 财政年份:2016
- 资助金额:
$ 127.74万 - 项目类别:
Identification and Characterization of human Rsc Chromatin-Remodeling Complex
人类 Rsc 染色质重塑复合物的鉴定和表征
- 批准号:
6431445 - 财政年份:
- 资助金额:
$ 127.74万 - 项目类别:
Identification/characterization Complex-Bloom Syndrome
复杂布卢姆综合症的鉴定/表征
- 批准号:
7132305 - 财政年份:
- 资助金额:
$ 127.74万 - 项目类别:
Characterization Complex Involved In Rothmund-Thomson
Rothmund-Thomson 涉及的表征复合体
- 批准号:
7132306 - 财政年份:
- 资助金额:
$ 127.74万 - 项目类别:
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