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 FY20 we accomplished the following: - Continued with analyses of IgH alleles mutated at specific transcription factor binding motifs. In the previous Annual report we had noted that ChIP-seq analysis of transcription factor binding revealed that mE1 mutation abolished YY1 binding to Em, whereas mE2/5 mutation had no effect on E47 binding. Absence of YY1 binding was in accordance with our experimental design, whereas continued E47 binding was completely unanticipated. We first investigated whether the introduced mutations affected E47 binding in vitro and found that both mutations abolished protein binding by EMSA. The stark distinction between the biochemical result and the in vivo results could have interesting implications that we are now addressing experimentally. We carried out in vitro DNA binding and competitions with a series of overlapping fragments that covered a genomic region extending from 1kb 5' of the enhancer to 1kb 3' of the enhancer. In this extended search we found that the mE4 element bound E47 in vitro, though with lower affinity than the combined E2/5 motifs. this led to the hypothesis that the unchanged recruitment of E47 to mE2/5-mutated alleles may be directed by interaction between mE4 and E47. To test this we initiated generation of mE4-mutated mice and mE2/4/5 triply-mutated mice. - we generated mE2/5/A/B quadruply mutated IgH alleles to test the synergistic roles of ETS and bHLH proteins in mediating enhancer function. Preliminary analyses indicated accentuated effects of the quad mutation on B cell development, epigenetic state and sterile transcription in the IgH locus. Further studies are ongoing. - in collaborative studies we showed that mE1-mutated IgH alleles lacked H4K16ac modifications, indicating that YY1 binding recruited the MOF complex to the locus. - we generated mice in which the Em enhancer was flipped in orientation at the normal site to rigorously test the textbook dogma that enhancer function is orientation-independent. we generated mice with a transcriptional stop sequence upstream of Em to test the effects of anti-sense transcripts initiating at Em. Unfortunately, the engineered mutation did not effectively block anti-sense transcription, thus precluding its use in further studies.