Condensin-mediated genome organization and transcriptional regulation

描述(由申请人提供)： 核内特定位点的重组通常与特定转录程序的及时激活密切相关，但驱动这些变化的复合体还没有得到很好的表征，调节这些事件的机制也不清楚。在有丝分裂开始时，染色质的有效组织和凝聚需要一个保守的复合体家族，称为凝集素。我们的研究发现了果蝇凝集素II亚单位dCAP-D3在调节功能相关基因簇转录方面的新作用。我们的长期目标是确定全球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。识别dCAP-D3直接靶标和dCAP-D3/COMPAIN II介导的基因簇调控所需的序列将通过芯片序列分析完成，然后与我们之前的微阵列数据进行比较。还将分析在目标基因簇上缺乏dCAP-D3结合位点的转基因品系的基因转录。为了确定dCAP-D3‘S在体内调节基因簇的能力所必需的相关蛋白，我们将利用dsRNAs在体内耗尽凝集素II亚基，然后检测dCAP-D3调控的基因簇的转录水平是否受到影响。IP/MS分析将在体内鉴定新的dCAP-D3结合伙伴，并研究它们在dCAP-D3介导的基因簇转录调控中的作用。DCAP-D3调控基因簇的高阶染色质结构和亚核定位将通过检测DNA环事件和双色DNA-FISH结合免疫荧光来检测。这项拟议的研究的贡献将是提供以前未知的、染色质结构域的全球组织和局部转录控制之间的机械联系。这一贡献将是重大的，因为它将促进我们对发生在活的有机体内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，dCAP-D3介导的转录调控机制也可能是保守的。全球染色质组织者的错误调控与发育综合征和癌症有关。因此，阐明dCAP-D3/凝聚素II调节转录的机制可能会为治疗许多人类疾病开辟新的途径。

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.