NOTCH1-RBPJ complexes drive target gene expressionthrough dynamic interactions with superenhancers

Abstract

The main oncogenic driver in T-lymphoblastic leukemia is NOTCH1,which activates genes by forming chromatin-associated Notchtranscription complexes. Gamma-secretase-inhibitor treatment prevents NOTCH1 nuclear localization, but most genes with NOTCH1-binding sites are insensitive to gamma-secretase inhibitors. Here,we demonstrate that fewer than 10% of NOTCH1-binding sitesshow dynamic changes in NOTCH1 occupancy when T-lympho-blastic leukemia cells are toggled between the Notch-on and -offstates with gamma-secretase inhibiters. Dynamic NOTCH1 sites arefunctional, being highly associated with Notch target genes, arelocated mainly in distal enhancers, and frequently overlap withRUNX1 binding. In line with the latter association, we show thatexpression of IL7R, a gene with key roles in normal T-cell development and in T-lymphoblastic leukemia, is coordinately regulatedby Runx factors and dynamic NOTCH1 binding to distal enhancers.Like IL7R, most Notch target genes and associated dynamicNOTCH1-binding sites cooccupy chromatin domains defined byconstitutive binding of CCCTC binding factor, which appears torestrict the regulatory potential of dynamic NOTCH1 sites. Moreremarkably, the majority of dynamic NOTCH1 sites lie in super-enhancers, distal elements with exceptionally broad and high levels of H3K27ac. Changes in Notch occupancy produces dynamicalterations in H3K27ac levels across the entire breadth of super-enhancers and in the promoters of Notch target genes. These findings link regulation of superenhancer function to NOTCH1, a masterregulatory factor and potent oncoprotein in the context of immature T cells, and delineate a generally applicable roadmap for identifying functional Notch sites in cellular genomes.