Chromosome movements that regulate tissue-specific gene expression

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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 FY20 we accomplished the following: - we 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 distal domain requires Pax 5 for its generation and the proximal domain is presumable determined by CTCF (but this remains untested for now). The deleted site is at the boundary between proximal and distal domains. The effects of this deletion on VH repertoire and B cell development are being tested. - we initiated a collaboration to carry out high resolution microscopic analysis of the chromatin configuration of the VH domain using multiple short oligonucleotide probes. - we recruited YY1 fusion proteins to the TetO/Gal4 substituted Em region to identify the role of Y1 in generating a diverse VH repertoire. - we had found that the 3D chromatin structure of the IgH locus was disrupted in pro-B cells from old mice. In following up this observation we carried out ChIP-Seq, RNA-Seq, ATAC-Seq and Hi-C using pro-B cells from young and old mice. We found that Em function was attenuated due to down-regulation of E2A proteins (previously known) and up-regulation of PU.1 (our study). In seeking a mechanism for PU.1 up-regulation we found that the epigenetic regulator Ezh2 was down-regulated in old pro-B cells. As a consequence bivalently-marked genes such as Sfpi1 (that encodes PU.1) and Cebpa and b lost repressive H3K27me3 and were up-regulated at the transcriptional level. Importantly, these genes are important for myeloid lineage differentiation, suggesting that Ezh2 down-regulation with age may contribute to the myeloid bias of hematopoeisis in old mice.