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The Role Of A Locus Control Region Upstream Of Bcl6 In Germinal Center Formation

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Abstract

Germinal Center B-cells (GCB) arise upon antigen stimulation and undergo somatic hypermutation (SHM) in order to produce high-affinity antibodies. BCL6, a transcriptional repressor, sustains GCB survival despite the genotoxic stress associated with SHM by repressing apoptosis and cell cycle arrest genes. Studies from our lab show intra-chromosomal interactions between a putative locus control region (LCR) and the BCL6 promoter in human GCBs. In both human and mice, this LCR is characterized by histone modifications associated with active enhancers, DNase hypersensitive regions, and increased binding of transcription factors and chromatin modifiers. This evidence suggested that the LCR is ‘switched on’ during the transition from naïve B-cells to GCBs and led us to hypothesize that the LCR plays a role in the transcriptional re-programming characterizing this stage of B-cell differentiation. We utilized CRISPR-Cas9 technology, to knock out the LCR in mice, which resulted in complete abrogation of GC formation. Additionally, we found that this defect is GCB intrinsic, and the loss of the LCR does not affect other tissues that require BCL6. We also discovered that a single copy of the LCR, in cis with Bcl6, is sufficient for GCB formation. However, when the LCR is in trans with Bcl6 it results in the loss of GC formation. Furthermore, to study the effect of this LCR on gene regulation we tried to utilize an ex-vivo culture system that mimics aspects of B-cell differentiation into GC B-cells. These B-cell follicle cultures or GC ‘organoids’ were extensively utilized to grow and differentiate murine B-cells. During the course of this investigation, we optimized many aspects of the culture conditions. However, the ex-vivo system did not phenocopy the in-vivo observations made in LCR KO mice. Our current efforts are focused on identifying the necessary functional motifs within the LCR by utilizing lymphoma cell lines. This work adds to our current understanding of how chromatin architectural modulation may be controlled by critical loci around master transcription factors, thereby influencing widespread transcriptional regulation required for differentiation. Additionally, an understanding of this particular LCR upstream of Bcl6 has the potential to guide investigations into the manner in which chromosomal translocations—like the 3q27 translocations frequently observed in diffuse large B-cell lymphomas (DLBCL)—play a role in lymphomagenesis and the maintenance of such pathologies.

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2018

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BCL6; Chromatin Architecture; CRISPR; Enhancer; Locus Control Region; Super Enhancer

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Immunology & Microbial Pathogenesis

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Doctor of Philosophy

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Government Document

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Attribution-NonCommercial-NoDerivatives 4.0 International

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dissertation or thesis

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