Chromosome movements that regulate tissue-specific gene expression

National Institute On Aging

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Abstract

The 3-dimensional chromatin configuration of IgH alleles ensures 1) utilization of a diverse repertoire of variable (VH) gene segments and 2) class switch recombination (CSR) to express different heavy chain isotypes during immune responses. Each of these critical processes is initiated by specialized enzymes that target the IgH locus in the context of this 3D structure. The RAG recombinase operates during B cell development and activation-induced deaminase (AID) initiates CSR. The scaffolding proteins CTCF and YY1 have been implicated in establishing 3D configuration of IgH alleles. During FY19 we found that: 1)The E enhancer and its associated YY1 binding site did not affect CSR in primary murine B cells. 2)YY1 protein interacted with MCM proteins that are involved in initiating DNA replication. The domains of YY1 required were identified and ongoing studies are aimed at determining whether direct YY1/MCM interactions specify isotype preference during CSR. 3)We introduced point mutations into the YY1 binding site of the IgH E enhancer to create delmE1 mice using CRISPR/Cas9 technology. These mice were backcrossed to C57Bl/6 RAG2-deficient mice, and bone marrow pro-B cells used to analyze germline IgH transcription and 3D structure. ChIP assays were used to identify changes in the epigenetic state of the mutated locus. 4)delmE1 mice on a RAG-sufficient background were used to measure the effects of loss of YY1 binding to E on VDJ recombination and the generation of a diverse VH repertoire.