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.
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.
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