Ikaros regulation: Study on hemolymphopoiesis

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ABSTRACT We propose to elucidate the lineage-specific mechanisms of 3D genome organization by studying the role of IKAROS in: a) regulatory loop (RL) formation supported by enhancer-based long-distance interactions and b) structural loop (SL) and insulated domain formation supported by CTCF interactions at architectural sites. Aim1. Lineage-specific regulation of genome organization principles. Here we address the functional interactions between enhancer-based RL and a novel class of dynamic structural loops (DSL) that we propose are integral to lineage segregation. Their role in lineage segregation is tested by comparative analysis of their formation after IKAROS-loss-of-function in large preB and -gain-of- function in epithelial cells. We test the hypothesis that IKAROS represses these DSL whose induction causes de-repression of extra-lineage gene profiles, notably those appropriate for epithelial cells. We examine whether this is a direct mechanism by which IKAROS and the NURD complex co-occupy a subset of CTCF sites that form DSL when either factors are removed. Alternatively we test whether IKAROS repression of DSL is indirect and relates to IKAROS’ role as an RL organizer. Aim2. Mechanisms of IKAROS regulation of super-enhancer assemblies Here we focus on the mechanisms by which IKAROS mediates assembly of super-enhancers (SE). We hypothesize that higher order interactions between IKAROS proteins bound at different chromosomal sites contributes to the formation of SE structures that entrap regulatory sites and associated factors in a confined space to mediate their function. The role of distinct IKBS and the requirement for specific IKAROS protein domains in the assembly SE is tested. We examine the role of assembled IKAROS complexes in segregating bound chromatin into regions of the nucleus with distinctive properties. Finally, we test whether the role of IKAROS in regulating SE assemblies is involved in Igk locus contraction.