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Computational Approaches To Elucidate Genome Topology Mechanisms

Thursday, November 3, 2022

12:00 PM-2:00 PM

BIOMED PhD Research Proposal

Computational Approaches To Elucidate Genome Topology Mechanisms That Control Antigen Receptor Gene Diversification

Katharina Hayer, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University

Ahmet Sacan, PhD
Associate Teaching Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University

Craig Bassing, PhD
Children’s Hospital of Philadelphia (CHOP)
Research Institute Investigator
Associate Professor of Pathology and Laboratory Medicine
Perelman School of Medicine
University of Pennsylvania

The 3D folding of genomes controls tissue- and developmental stage-specific gene expression and assembly of antigen receptor (AgR) genes through V(D)J recombination. The importance of this control is highlighted by the estimation that ~10% of disease-linked mutations in humans alter genome topology. Two basic mechanisms fold genomes: i) homotypic interactions among chromatin regions of identical activity, and ii) point-to-point contacts (loops) generated by Cohesin protein mediated loop extrusion between occupied convergent CTCF binding elements (CBEs). However, much remains to be elucidated for how these distinct mechanisms function and cooperate at specific loci. High-throughput chromosome conformation capture (HiC) provides unbiased views of genome folding, but there is no gold standard for computational analysis of HiC and other -omics data to delineate locus specific topologies and link these structures to chromatin activity. The genomic organizations of AgR loci with many V gene segments spanning large sequences far upstream of (D)J gene segments, present challenges for efficient V-to-(D)J recombination and broad utilization of individual V segments across cell populations. Lymphocyte lineage- and developmental stage-specific chromatin activation, RAG endonuclease binding to (D)J segments, and folding of AgR loci serve fundamental roles in promoting long-range V(D)J recombination. Yet, the precise mechanisms that drive AgR locus folding and how these control V(D)J recombination in vivo are mainly unknown due to unique genomic organizations of each locus and comprehensive studies predominantly limited to the IgH locus in immortalized cell lines. The goal of my research is to develop computational pipelines to analyze HiC and chromatin data and apply these on primary mouse cells of distinct lineages or lymphocyte developmental stages and/or harboring CBE or transcriptional element inactivation to elucidate precise mechanisms that fold AgR loci and control V(D)J recombination.
I am starting with the mouse T cell receptor b (Tcrb) locus because it contains fewer V segments and CBEs than the other large mouse AgR loci (Igh, Igk, and Tcra/d) and its rearrangement is controlled by only one enhancer that activates Db-Jb segments. Germline Vb segments are transcriptionally active and recombine only in DN stage thymocytes; resulting TCRb protein signals differentiation of DP thymocytes and silencing of Vb transcription and recombination. Using a pipeline that involves global binning, I have resolved unbiased views of topologies (HiC) and chromatin activities (RNA-Seq, ChIP-Seq) of germline Tcrb loci that are poised for recombination in DN thymocytes, silenced for Vb recombination in DP thymocytes, or not activated for recombination in pro-B cells or a fibroblast cell line. In DN cells, Vb segments contact each other and Db-Jb segments, but not intervening sequences, forming a compacted Tcrb locus structure with homotypic interactions, bi-directional Cohesin/CTCF-mediated looping, or both between Vb and Db-Jbsegments. In DP cells, Vb segments do not interact with each other and contact a transcriptionally upregulated region downstream of DbJb segments in a fashion consistent with uni-directional Cohesin/CTCF-mediated looping from Vb segments through DbJb segments. These altered topological and chromatin features of Tcrb may account for lack of Vb recombination in DP thymocytes. While Tcrb does not fold and lacks transcriptionally active chromatin in fibroblasts as expected, the locus shows some folding and active chromatin in pro-B cells, adopting a configuration similar to DP cells. Together these preliminary data highlight changes in topological and chromatin features that could promote or suppress long-range Vb recombination.
I am developing a pipeline to better integrate the topology and chromatin data by binning HiC data on Tcrbgene segments, transcriptional elements, and CBEs rather than arbitrary global windowing as conventionally used for Hi-C assays and by benchmarking current interaction identification methods to ensure observed interactions are real and not noise. My new computational approach has potential to provide unprecedented resolution of AgR locus topologies and thereby identify key interactions and potential underlying mechanisms that regulate AgR locus folding and shape primary AgR gene repertoires.

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Natalia Broz

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