Gene Regulation in Lymphocytes

National Institute On Aging

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Abstract

Enhancers are transcriptional regulatory sequences that modulate gene expression from distant genomic sites. Such sequences are typically marked by DNase1 hypersensitive sites (HS), H3K27ac histone modifications, and co-activators such as p300. Clusters of DNase1 HS having these characteristics are referred to as super-enhancers and implicated in human disease. Remarkably, 80% of human disease-associated single nucleotide polymorphisms (SNPs) are located in or close to enhancer-like genomic sequences. While enhancers are known to bind multiple transcription factors, it is unclear how transcription factor binding confers the many different properties of enhancers. We refer to this combinatorial functionality as the enhancer code. The goals of this project are to gain insight into the enhancer code using IgH and TCR enhancers as paradigms. During FY22 we accomplished the following: We generated many lines of mice that carry mutations in specific transcription factor binding sites in the IgH locus intronic enhancer Em. Our studies revealed unanticipated complexity in the molecular organization of Em. Mutations of both known ETS protein binding sites and two sites that bind E2A (for a total of 4 mutated sites) showed approximately 2-fold reduced enhancer activity as measured by multiple epigenetic and RNA assays. Analysis of compensatory sites that mediate Em activity in the absence of these sites is ongoing using a combination of biochemical and genetic assays. The effects of Em inversion at its normal location were evaluated by chromosome conformation capture techniques and RNA-Seq. Effects on VDJ recombination were assayed by polymerase chase reactions. Inverting the enhancer negatively affected Em function in all assays. We developed a computational model to understand the role of 3-dimensional chromatin structure in establishing a diverse antibody repertoire. Using this model, we investigated the role of regulatory sequences in establishing locus conformation and enabling spatial proximity of gene segments located megabases away to participate in VDJ recombination.