Characterization of Transcription Factor-Coactivator Complexes in Acute Lymphoblastic Leukemia
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Authors
Lochhead, Marina
Date
Type
thesis
Language
eng
Keyword
Hematopoiesis , Lymphocyte , Acute Lymphoblastic Leukemia , Protein-Protein Interaction , CBP/p300 , Transcription Factor
Alternative Title
Abstract
Acute lymphoblastic leukemia (ALL) is a cancerous hematological disorder originating in B-lymphoid progenitor cells. In 5% of ALL cases, the oncogenic transcription factor E2A-PBX1 is expressed as a result of the chromosomal translocation 1;19. E2A-PBX1 contains the activation domains of E2A and most of the PBX1 protein, including the DNA-binding homeodomain. These structural features suggest a hypothetical oncogenic model in which E2A-PBX1 binds DNA, recruits transcriptional co-activators by means of the activation domains and induces ALL through deregulation of PBX1 target genes. This transcriptional control is, in part, mediated by the recruitment of the transcriptional coactivator CBP/p300. Indeed, previous studies have shown that E2A-PBX1-mediated oncogenesis requires direct binding of E2A-PBX1 to CBP/p300. However, the transcriptional targets of this complex remain unknown. Additionally, as t(1;19)-mediated ALL is derived from B-lymphoid progenitors, we propose that E2A-PBX1 may be localizing to B-lymphopoietic enhancers bound by the master B-lymphopoietic transcription factors EBF1 and wild-type E2A, perturbing enhancer function and contributing to the development of ALL. The purpose of this thesis was to: 1) identify genomic regions where E2A-PBX1 binds; 2) assess whether other transcription factors are associated with the E2A-PBX1:CBP/p300 complex and further elucidate the structural and biophysical characteristics of those interactions; and 3) develop and characterize an inhibitor to the E2A-PBX1:CBP/p300 complex as a potential therapeutic for ALL. Using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq), we identified over 3700 E2A-PBX1 binding sites in an ALL-derived cell line. We show that E2A-PBX1 recruits CBP/p300 to roughly 50% of its binding sites and localizes with CBP/p300 to consensus sequences bound by EBF1 and wild-type E2A. Using structural and biophysical methods, we characterize interactions between the TAZ2 domain of CBP/p300 and the activation domains of E2A-PBX1 or E2A and EBF1. The structural and ChIP-seq data suggest an additional E2A-PBX1 binding model in which E2A-PBX1 localizes to B-lymphopoietic enhancers by indirectly interacting with EBF1 and wild-type E2A with CBP/p300 as a mediator. Lastly, we developed a disruptor peptide that can bind the KIX domain of CBP/p300 with nanomolar affinity which disrupts E2A-PBX1:CBP/p300 complex formation in ALL cells and downregulates E2A-PBX1 target genes in vivo.
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This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
CC0 1.0 Universal
ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
CC0 1.0 Universal