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 FY21 we accomplished the following: - we had previously generated mice with a deletion in a DNase I hypersensitive site that falls near a previously determined region that folds the 2Mb VH domain into discrete 99kb domains. The effects of this deletion on VH repertoire and B cell development are being tested. - the collaboration to carry out high resolution microscopic analysis of the chromatin configuration of the VH domain using multiple short oligonucleotide probes was delayed due to loss of personnel from the project. - we temporarily put on hold the recruitment of Gal4- or TetDBD- fusion proteins to the Em enhancer to focus on aging studies. - we extended epigenetic studies of B cell aging in several directions. First, to test the hypothesis a subset of age-associated changes were caused by down-regulation of Ezh2, we inhibited enzymatic activity of the PRC2 complex in primary bone marrow pro-B cells from RAG2-deficient mice and assayed the effects on gene expression and histone modifications. Ezh2 inhibition recapitulated effects on expression of myeloid-specific transcription factors in young pro-B cells. Anti-H3K27me3 ChIP-Seq revealed reduced bivalency at promoters of Cebp genes. Second, we completed analysis of the effects of age on WT pro-B cells; up-regulation of the myeloid program was evident in these cells as well as had been previously noted by us in RAG-deficient pro-B cells. Third, we carried out single cell RNA-Seq using WT and RAG-deficient proB cells and WT pre-B cells. Analysis is ongoing. - to determine which aspects of chromatin changes were caused by down-regulation of the B cell program, especially Ebf1, we carried out Hi-C with WT, Ebf1+/-, and Ebf1+/-Pax5+/- pro-B cells expanded in culture. Sequencing and analysis is ongoing.