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545 Suppression of Gata3 Binding Drives Selective Abrogation of Notch1-Myc Enhancer Activity By Nucleosome Invasion in Thymocyte Development and Leukemia

Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation: Single Cell Profiling/Actionable Leukemia Targets
Hematology Disease Topics & Pathways:
Diseases, ALL, Leukemia, Animal models, Biological Processes, Technology and Procedures, Study Population, gene editing, Lymphoid Malignancies, NGS, molecular interactions, RNA sequencing
Monday, December 3, 2018: 8:00 AM
Room 9 (San Diego Convention Center)

Laura Belver, PhD1*, Alexander Y Yang1*, Daniel Herranz, PharmD2, Aidan Quinn3*, Francesco G Brundu, PhD4*, Pablo Perez-Duran, PhD5*, Silvia Alvarez, PhD5*, Devya Gurung5*, Pedro P Rocha, PhD6*, Ramya Raviram, PhD7*, Marissa Rashkovan, PhD5*, Raul Rabadan, PhD4,8* and Adolfo A. Ferrando, MD, PhD9

1Institute for Cancer Genetics, Columbia University, New York, NY
2Cancer Institute of NJ, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ
3Columbia University, New York, NY
4Department of Systems Biology, Columbia University, New York, NY
5Institute for Cancer Genetics, Columbia University, New York
6Division of Developmental Biology, National Institutes of Health, Bethesda
7Department of Chemistry and Biochemistry, University of California San Diego, La Jolla
8Department of Biomedical Informatics, Columbia University, New York
9Department of Systems Biology, Columbia University, New York

Long range enhancers play critical roles in the control of gene expression during development and have emerged as key regulators of lineage commitment and oncogenic programs in hematopoiesis and leukemia. The MYC oncogene is dynamically regulated in the hematopoietic system under the control of a network of clustered distal enhancers, which provide modular regulation of MYC expression during lymphoid and myeloid development. In thymocyte development MYC transcription critically depends on the activity of N-Me, a distinct T-cell specific enhancer controlled by NOTCH1 signaling and located 1.4 Mb telomeric to the MYC transcription start site. Yet, the specific mechanisms governing N-Me enhancer activity and lineage specific control of MYC expression remain rudimentarily understood. Analysis of chromatin looping by 4C and chromatin accessibility by ATACseq revealed an unanticipated high density of chromatin contacts between N-Me and additional regulatory elements in the Myc locus and showed a distinct pattern of N-Me chromatin accessibility –opening as progenitors mature into T cell committed CD4 CD8 double negative (DN) 2b cells and returning to a closed configuration in CD4 CD8 double positive (DP) thymocytes–. To explore potential regulators of N-Me activity we performed Mass Spectrometry proteomic profiling of N-Me binding proteins and ChIPseq analyses identifying numerous factors involved in hematopoietic and lymphoid development (ERG, ETS1, GATA3, RUNX1, TCF3 and TCF12) and transcription factor oncogenes with prominent roles in the pathogenesis of T-ALL (HOXA9, MYB, MYC, LMO1, LMO2, TAL1 and TLX1). Moreover, phylogenetic footprinting analyses across vertebrate species identified two ultraconserved elements matching GATA factor binding motifs (GS1 and GS2). To test the functionality of these elements we introduced targeted mutations in the N-Me sequence at these sites using CRISPR/CAS9 directed mutagenesis. Mice homozygous for combined N-Me GS1 and GS2 mutations (GS1+2mut) revealed a marked defect in thymus cellularity with characteristic accumulation of DN and intermediate single positive (ISP) thymocytes and decreased numbers of more mature populations. Mechanistically, immunohistochemical, flow cytometry and single cell RNaseq analyses revealed decreased Myc protein levels in thymocyte poulations of GS1+2 mutant animals. In this context, we hypothesized that GATA3, a prominent N-Me binding transcription factor in our ChIP and proteomic analyses critically implicated in T-cell commitment, could play a major role in N-Me regulation via interaction with the GS1 and GS2 N-Me GATA sites. Consistent with this hypothesis analysis of Gata3 ChIPs from heterozygous GS1+2 mutant mice recovered only the N-Me wild type sequence, formally demonstrating the strict requirement of these sites for N-Me Gata3 binding. Mechanistically, ATACseq analysis revealed a marked reduction in chromatin accessibility and nucleosome invasion in thymocytes from GS1+2 mutant mice in support of a critical pioneering activity for GATA3 in the control of N-Me activity. Finally, given the important role of NOTCH1 induced MYC upregulation in the pathogenesis of T-ALL, we hypothesized that disruption of N-Me activity via targeted mutation of N-Me GATA sites could effectively impair the development of NOTCH1-driven T-ALL in N-Me GS1+2 mutant mice. To test this possibility we infected hematopoietic progenitors from N-Me wild type and N-Me GS1+2 homozygous mice with retroviruses driving the expression of an oncogenic constitutively active form of NOTCH1 (DE-NOTCH1) and transplanted them into sublethally irradiated recipients. In these experiments, mice transplanted with DE-NOTCH1 infected N-Me wild type cells developed overt T-ALL 6 weeks postransplant with 100% penetrance. In contrast, mice transplanted with DE-NOTCH1-expressing N-Me GS1+2 homozygous cells showed complete protection from NOTCH1 induced T-ALL (P <0.001). In all these results identify GATA3 binding to the N-Me enhancer as a critical driver of nucleosome eviction and enhancer activation strictly required for thymocyte development and NOTCH1-induced T-cell transformation.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH