Gene Regulation in Lymphocytes

National Institute On Aging

Investigators

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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 FY23 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. In continuing studies we found that mutation of 3 E protein bindings sites in Em virtually abolished enhancer activity, despite other binding sites remaining intact. These observations conclusively demonstrate that E protein binding is essential for Em function and raise two key questions. First, are E proteins sufficient for Em function and second, what is the role of other sites if E proteins are not sufficient. To address the first question we designed a mouse strain that substitutes E protein binding sites for the full enhancer. to address the second question we propose to carry out in vivo footprinting experiments determine whether the non-E protein sites are occupied in the triple E deletion. We continued to refine 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. We deleted singly, or in combinations 3 cis-regulatory sequences that configure the 3' end of the IgH locus. We found that the Em directs long-range interactions, reflected in a 'stripe', towards the distal ends of the locus. By substituting Em with an active heterologous promoter we identified a role for anti-sense transcription in promoting stripe formation. We collaborated with Grosschedl and Gittler laboratories at the Max Plank Institute in Freiburg (Germany) to uncover a role for the E1 element of Em. these results have been submitted for publication.