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 FY25 we accomplished the following: 1) Generated several additional mouse strains by CRISPR/Cas9 methodolgy to probe enhancer function. Amongst the most interesting observations are: a) the Igh enhancer appears to be composed of two independently functional parts. Each part contains binding sites for E2A and ETS proteins. Additionally, the 3' half has 3 binding sites for Runx proteins. b) Addition of three more E2A binding sites just 5' of the enhancer phenocopies the enhancer deletion with regard to B cell development. Based on molecular characterization of Igh alleles our working hypothesis is that the new binding sites block anti-sense transcription initiated at the enhancer, which has a detrimental effect on VDJ recombination. Predictions of this hypothesis are being tested. c) the Em enhancer is marked by a DNase 1 hypersensitive site in hematopoietic stem cells, but is transcriptionally inert and lacks activation-related epigenetic histone modifications. We carried ATAC-seq in HSC with Em mutated Igh alleles to identify the role of transcription factors that confer chromatin accessibility without transcription in HSC. 2) We completed the first phase of a biophysical model to understand molecular mechanisms that generate diverse Igh repertoires during B cell development. A manuscript has been submitted for publication. 3) We published a comprehensive review on the structures and functions of antigen receptor gene enhancers with a focus on mechanisms that direct their tissue- and developmental timing specific activation.