Condensin-mediated genome organization and transcriptional regulation

凝缩蛋白介导的基因组组织和转录调控

• 批准号: 8346031
• 负责人: Michelle S Longworth
• 金额: $ 29.83万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8346031
• 关键词: Address; Adult; Affect; Automobile Driving; Binding; Binding Sites; Biological Assay; Brain; Cell Nucleus; Cell physiology; Cells; ChIP-seq; Characteristics; Chromatin; Chromatin Loop; Chromatin Structure; Chromosome Structures; Chromosome Territory; Chromosomes; Co-Immunoprecipitations; Color; Complex; Coupled; DNA; DNA Sequence; Data; Defect; Development; Double-Stranded RNA; Drosophila genus; Drosophila melanogaster; Elements; Event; Family; Gene Cluster; Gene Expression; Genes; Genetic Materials; Genetic Transcription; Genome; Genomic Instability; Goals; Health; Higher Order Chromatin Structure; Human; Immunofluorescence Immunologic; In Vitro; Interphase; Knowledge; Lead; Life; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mitosis; Multiprotein Complexes; Nuclear; Nuclear Lamina; Nuclear Pore; Organism; Orthologous Gene; Physical condensation; Positioning Attribute; Proteins; Regulation; Research; Role; Syndrome; Testing; Therapeutic Intervention; Tissues; Transcript; Transcriptional Regulation; Transgenic Organisms; Work; Yeasts; condensin; fly; human disease; in vivo; interest; knock-down; member; mutant; novel; programs; research study; tumor

DESCRIPTION (provided by applicant): Project Summary The reorganization of specific loci within the nucleus is often tightly coupled with the timely activation of specific programs of transcription, but the complexes driving these changes are not well characterized and mechanisms that regulate these events are unknown. Efficient organization and condensation of chromatin at the beginning of mitosis requires a conserved family of complexes called Condensins. Our research has uncovered a novel role for the Drosophila Condensin II subunit, dCAP-D3 in regulating the transcription of clusters of functionally-related genes. Our long-term goal is to identify the mechanisms by which the organization and positioning of the global chromatin structure helps to coordinate local transcription events in vivo. Identification of dCAP-D3 direct targets and sequences necessary for dCAP-D3/Condensin II mediated gene cluster regulation will be accomplished through ChIP- seq analyses followed by comparison to our previous microarray data. Analysis of gene transcription in transgenic lines deficient for dCAP-D3 binding sites at target gene clusters will also be performed. To identify associated proteins necessary for dCAP-D3's ability to regulate gene clusters in vivo, dsRNAs will be used to deplete Condensin II subunits in vivo and then test whether transcript levels of dCAP-D3 regulated gene clusters are affected. IP/Mass spectrometry analyses will identify novel in vivo dCAP-D3 binding partners and their contributions to dCAP-D3-mediated transcriptional regulation of gene clusters will also be studied. The higher order chromatin structure and sub-nuclear localization of dCAP-D3 regulated gene clusters will be examined by performing assays which detect DNA looping events and two color DNA-FISH combined with immunofluorescence. The contribution of the proposed research will be to provide previously unknown, mechanistic links between the global organization of chromatin domains and local transcriptional control. This contribution will be significant because it will further our understanding of the three-dimensional aspects of transcriptional regulation that occur inside a living organism. Since our preliminary data suggests that the ability of CAP-D3 to regulate gene clusters is conserved in human cells, mechanisms of dCAP-D3-mediated transcriptional regulation might also be conserved. Misregulation of global chromatin organizers is implicated in developmental syndromes and cancers. Therefore, elucidation of the mechanisms by which dCAP-D3/Condensin II regulates transcription will potentially uncover new avenues for therapeutic intervention in a number of human diseases. PUBLIC HEALTH RELEVANCE: Project Narrative The complexes and mechanisms which organize the global structure of DNA to control more localized programs of gene expression are not well characterized. We propose to use the model organism of the fruit fly, Drosophila melanogaster, to identify conserved mechanisms by which the Condensin II complex, an important global organizer of DNA regulates the transcription of functionally related clusters of genes inside of a living organism. Since misregulation of global DNA organizers can lead to genomic instability resulting in developmental syndromes and tumor development, our proposed studies will potentially uncover new avenues for therapeutic intervention in a number of human diseases.

描述（由申请人提供）： 项目摘要 细胞核内特定基因座的重组通常与特定转录程序的及时激活紧密相关，但驱动这些变化的复合物尚未得到很好的表征，调节这些事件的机制也未知。有丝分裂开始时染色质的有效组织和浓缩需要一个称为浓缩蛋白的保守复合物家族。我们的研究发现了果蝇凝缩蛋白 II 亚基 dCAP-D3 在调节功能相关基因簇转录中的新作用。我们的长期目标是确定整体染色质结构的组织和定位有助于协调体内局部转录事件的机制。 dCAP-D3 直接靶标和 dCAP-D3/Condensin II 介导的基因簇调控所需序列的鉴定将通过 ChIP-seq 分析来完成，然后与我们之前的微阵列数据进行比较。还将对目标基因簇上缺乏 dCAP-D3 结合位点的转基因系中的基因转录进行分析。为了鉴定 dCAP-D3 体内调节基因簇能力所需的相关蛋白，将使用 dsRNA 消耗体内 Condensin II 亚基，然后测试 dCAP-D3 调节基因簇的转录水平是否受到影响。 IP/质谱分析将鉴定新型体内 dCAP-D3 结合伴侣，并且还将研究它们对 dCAP-D3 介导的基因簇转录调控的贡献。 dCAP-D3 调节基因簇的高级染色质结构和亚核定位将通过检测 DNA 环事件和两色 DNA-FISH 与免疫荧光相结合的检测来检查。拟议研究的贡献将是提供染色质结构域的全局组织和局部转录控制之间以前未知的机制联系。这一贡献将是意义重大的，因为它将进一步加深我们对生物体内发生的转录调控的三维方面的理解。由于我们的初步数据表明 CAP-D3 调节基因簇的能力在人类细胞中是保守的，因此 dCAP-D3 介导的转录调节机制也可能是保守的。整体染色质组织者的失调与发育综合征和癌症有关。因此，阐明 dCAP-D3/Condensin II 调节转录的机制将有可能为许多人类疾病的治疗干预开辟新途径。 公共卫生相关性： 项目叙述 组织 DNA 整体结构以控制更多局部基因表达程序的复合物和机制尚未得到很好的表征。我们建议使用果蝇（果蝇）的模式生物来确定Condensin II复合体（一种重要的全球DNA组织者）调节生物体内功能相关基因簇转录的保守机制。由于全球 DNA 组织者的失调可能导致基因组不稳定，从而导致发育综合征和肿瘤发展，因此我们提出的研究将有可能揭示许多人类疾病治疗干预的新途径。