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Date of Award

Spring 5-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Non-Hodgkin Lymphoma (NHL) is a cancer of B- and T-cells that affects nearly 75,000 people in the US every year, leading to 20,000 deaths according to the American Cancer Society. While NHL has been heavily genetically profiled, no single identified driver mutations sufficient to initiate lymphomagenesis have been identified. A large number of mutations have been found across all sub-types with an enrichment in genes involved in epigenetic regulation of gene expression. As a means to understanding how the epigenome and regulation of gene expression are altered in B-NHL, we processed over 100 B-NHL tumors from the clinic, in addition to normal B-cells from patient peripheral blood or healthy donors. We assessed: 1) active chromatin by ChIP-seq of histone modifications (H3K4ME3, H3K27AC, H3K4ME1), 2) chromatin accessibility by FAIRE-seq and 3) gene expression through microarrays and rRNA-depleted RNA-seq. These data lead to the identification of two previously undefined sub-types of Follicular Lymphoma (FL), which we published in Immunity in 2015. Our ongoing work with sequence-specific chromatin modifiers (SSCMs) makes these areas of aberrant chromatin activity viable targets for epigenetic therapeutics in future. In addition to identifying hundreds of altered regulatory elements in NHL, we also discovered hundreds of novel and previously-annotated long non-coding RNAs (lncRNAs), which represented some of the most differentially-expressed transcripts between cancerous and normal B cells. Despite an influx of contemporary publications concerning the myriad functionalities and criticality of many lncRNAs, we were unable to find a database or analytical tool capable of providing us with putative mechanisms of action for our lncRNAs of interest. To expedite the selection of lncRNAs for further analysis, we created an algorithm we named Predicting LncRNA Activity by Integrating Data-driven Omics and Heuristics (PLAIDOH). PLAIDOH requires a single user-derived input file with expression of lncRNAs and coding gene transcripts across 3 or more samples. By incorporating statistical correlation of lncRNAs and their cognate coding genes, in addition to multiple publicly-available genomic and interactomic datasets PLAIDOH calculates three separate scores designed to rank lncRNAs by 1) their cis-regulatory potential 2) enhancer-like qualities 3) sub-cellular location and RNA-binding protein profile. PLAIDOH correctly identified many of the previously-identified lncRNAs involved in transcriptional regulation of neighboring genes in addition and provided much-needed insight into the roles of highly-expressed NHL lncRNAs, allowing us to follow-up several transcripts with experiments performed in NHL cell lines. One of those lncRNAs of interest was RP11-96018L.1(LncRNA-PLCG2). RP11-96018L.1is a cytoplasmic lncRNA, which is specifically and highly expressed in B cells. Knock out or Knock down of RP11-96018L.1leads to a defect in BCR-mediated calcium signaling, but not through modulation of transcription or post-translational interaction with the most proximal gene, PLCG2. Pull down experiments have identified PLD1 as a cytoplasmic interactor of RP11-96018L.1and experiments are underway to identify the mechanism of action by which RP11-96018L.1affects the down-stream calcium signaling after BCR stimulation. Taken in its entirety, my thesis work is a holistic, ‘omics-based interrogation of gene regulation and lncRNA activity in B cells and NHL, including the production of a novel bioinformatics tool (PLAIDOH), which will contribute to the mounting number of analyses focused on lncRNA activity. Our continued efforts to identify the role of RP11-96018L.1in B cell signaling and lymphomagenesis will result in the functional annotation of a previously unidentified component of the BCR signaling cascade.

Language

English (en)

Chair and Committee

Jacqueline Payton

Committee Members

Grant Challen, John Welch, Douglas Chalker, Christopher Maher,

Comments

Permanent URL: https://doi.org/10.7936/ajr8-4032

Available for download on Monday, May 15, 2119

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