The three-dimensional organization of chromatin within the nucleus supports long-range interactions between linearly distant portions of the genome. The chromatin loops help control gene expression and are important in cellular health and development. Overall, genome organization includes both Topologically Associating Domains (TADs) and specific enhancer-promoter interactions. This organization is mediated by insulator proteins which cluster at TAD boundaries, and may bind in varying combinations at enhancers. Insulator sites may also depend on the local chromatin context including nucleosome occupancy and long non-coding RNA (lncRNA). Nucleosome remodeler components such as ISWI specifically localize to insulator binding sites, and insulator proteins are thought to prefer nucleosome free regions. Additionally lncRNA such as hsr?, has been implicated in recruiting nucleosome remodeler components to chromatin. It is the purpose of this proposal to investigate the role of nucleosome remodelers and hsr? lncRNA in insulator protein binding to chromatin and in chromatin looping. This will be done by testing the following hypothesis: ISWI binds chromatin dependent on hsr? lncRNA, and alters nucleosome occupancy so that insulator proteins may bind. This hypothesis will be tested using the following aims:
Aim1 : Determine the role of ISWI in insulator function by mapping nucleosomes genome-wide in nucleosome remodeler (ISWI) and insulator protein knockdown lines. Additionally, the dependency on insulator proteins for ISWI to bind chromatin, and vice versa, will be tested by a series of ChIP-seq experiments using ISWI and insulator protein antibodies in the appropriate knockdown lines.
Aim2 : Determine the relationship between hsr? lncRNA, ISWI, and Insulators by testing localization of hsr? lncRNA by ChiRP-seq. Dependence of hsr?'s chromatin interaction on ISWI will also be tested in this manner. ChIP-seq will also be performed for ISWI and insulator proteins in hsr? depletion lines to determine the dependency of chromatin binding on lncRNA.
Aim3 : Determine the effect of hsr? lncRNA on chromatin organization by using enhancer blocking and HI-C assays. Established enhancer blocking assays will be used to test if hsr? exhibits enhancer blocking activity at the same loci as ISWI. HI-C in hsr? depletion line will be used to detect how hsr? affects chromatin organization genome-wide.
Genomes are composed of thousands of genes requiring precise expression control in order to maintain cellular integrity, development, and health. Long range expression control via chromatin folding often occurs between enhancers and promoters which in turn may be regulated by nucleosome remodelers such as ISWI, and by long non-coding RNA such as hsr?. This proposal uses the Drosophila model system to study the role of nucleosome positioning and long non-coding RNA in the control of long range chromatin looping.
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