Abstract
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.
TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer