Condensin-mediated genome organization and transcriptional regulation

描述（由申请人提供）： 细胞核内特定位点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，并且调节这些事件的机制也是未知的。在有丝分裂开始时染色质的有效组织和浓缩需要称为浓缩蛋白的保守复合物家族。我们的研究揭示了果蝇凝聚素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的机制，组织和定位的全球染色质结构有助于协调本地的转录事件在体内。dCAP-D3直接靶标和dCAP-D3/Condensin II介导的基因簇调控所必需的序列的鉴定将通过ChIP-seq分析完成，然后与我们先前的微阵列数据进行比较。还将对靶基因簇处dCAP-D3结合位点缺陷的转基因系中的基因转录进行分析。为了鉴定dCAP-D3在体内调节基因簇的能力所必需的相关蛋白质，将使用dsRNA在体内耗尽凝聚蛋白II亚基，然后测试dCAP-D3调节的基因簇的转录物水平是否受到影响。IP/质谱分析将鉴定新的体内dCAP-D3结合伴侣，还将研究它们对dCAP-D3介导的基因簇转录调控的贡献。将通过检测DNA成环事件和双色DNA-FISH结合免疫荧光的试验，检查dCAP-D3调节基因簇的高阶染色质结构和亚核定位。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本地转录控制之间的机械联系。这一贡献将是重要的，因为它将进一步我们的三维方面的转录调控，发生在一个活的有机体的理解。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，dCAP-D3/Condensin II调节转录的机制的阐明将潜在地揭示许多人类疾病的治疗干预的新途径。

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.