NOTCH1-RBPJ complexes drive target gene expression through dynamic interactions with superenhancers - PubMed (original) (raw)

NOTCH1-RBPJ complexes drive target gene expression through dynamic interactions with superenhancers

Hongfang Wang et al. Proc Natl Acad Sci U S A. 2014.

Abstract

The main oncogenic driver in T-lymphoblastic leukemia is NOTCH1, which activates genes by forming chromatin-associated Notch transcription 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 sites show dynamic changes in NOTCH1 occupancy when T-lymphoblastic leukemia cells are toggled between the Notch-on and -off states with gamma-secretase inhibiters. Dynamic NOTCH1 sites are functional, being highly associated with Notch target genes, are located mainly in distal enhancers, and frequently overlap with RUNX1 binding. In line with the latter association, we show that expression of IL7R, a gene with key roles in normal T-cell development and in T-lymphoblastic leukemia, is coordinately regulated by Runx factors and dynamic NOTCH1 binding to distal enhancers. Like IL7R, most Notch target genes and associated dynamic NOTCH1-binding sites cooccupy chromatin domains defined by constitutive binding of CCCTC binding factor, which appears to restrict the regulatory potential of dynamic NOTCH1 sites. More remarkably, the majority of dynamic NOTCH1 sites lie in superenhancers, distal elements with exceptionally broad and high levels of H3K27ac. Changes in Notch occupancy produces dynamic alterations in H3K27ac levels across the entire breadth of superenhancers and in the promoters of Notch target genes. These findings link regulation of superenhancer function to NOTCH1, a master regulatory 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.

Keywords: Notch signaling; gene regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

NOTCH1 activation reveals dynamic NOTCH1–RBPJ-binding sites. (A) Scatterplot of NOTCH1 ChIP-seq read counts. Each dot represents a NOTCH1 peak identified in CUTLL1 T-LL cells following GSI washout. ChIP-seq reads within 600 bp of a peak summit were counted in the Notch-on and Notch-off states. Red dots indicate dynamic sites. (B) Classes of NOTCH1-binding sites defined by genomic location and dynamism. Each dot is a NOTCH1 peak plotted according to its distance to the nearest gene’s transcriptional start site (TSS) and its signal-fold change from the Notch-off to the Notch-on states. The red vertical line separates proximal promoters and distal enhancers, and the red horizontal line corresponds to a signal-fold-change threshold with an FDR < 0.05. Inset numbers correspond to peaks found in each quadrant. (C) Heat map of dynamic NOTCH1 sites, ranked by ChIP-Seq signal intensity, and associated transcription factor and histone mark signals across a 1-kb window centered on NOTCH1-binding summits.

Fig. 2.

Fig. 2.

Identification and functional characterization of the IL7R 3′ enhancers. (A) Chromatin landscapes around the IL7R locus in human T-LL cells shows the presence of a pair of 3′ enhancers, E5 and E3, each containing a dynamic NOTCH1–RBPJ site and a RUNX1 site. (B) Diagram showing IL7R enhancer reporter constructs. Regions spanning E5, E3, or both (E53) NOTCH1–RBPJ binding sites were cloned into the pGL3-TATA box plasmid. (C) IL7R enhancer elements are active in CUTLL1 T-LL cells. Here and elsewhere, luciferase assays were carried out in triplicate and normalized to the luciferase activity generated by the empty pGL3-TATA box plasmid. (D) _IL7R_-enhancer activity in CUTLL1 T-LL cells depends on Notch and Runx factors. Notch and Runx factor activity was inhibited by transfection of plasmids encoding DN-MAML and Runt, respectively. (E) Effects of RBPJ and Runx motif mutations on IL7R E53-enhancer reporter gene activity in CUTLL1 cells. The cartoon shows the IL7R E53-enhancer construct and associated RBPJ and Runx motifs. The effects of mutations involving these sites, alone and in combination, are shown in the IL7R E53-enhancer reporter gene assay below. Error bars in C–E represent 1 sd from the mean of data points obtained in triplicate.

Fig. 3.

Fig. 3.

Target gene activation through dynamic NOTCH1-binding sites. (A–C) Dynamic NOTCH1–RBPJ-binding sites are preferentially located near genes that are up-regulated by Notch. The distance from the nearest binding site to the TSS of each gene was recorded (A) and cumulative distributions of 340 up-regulated genes (red), 187 down-regulated genes (blue), and the genomic background (black) were plotted for dynamic NOTCH1 (B) and dynamic RBPJ sites (C). P values were calculated by the Kolmogorov–Smirnov test. (D) Constitutive CTCF-binding sites define domains that restrict NOTCH1 regulation of nearby genes. Genes near dynamic NOTCH1 sites were classified into three categories: A, within 100 kb but in a different CTCF domain; B, within 100 kb and in the same CTCF domain; C, more than 100 kb away and in the same CTCF domain. The ratio between the likelihood of finding an activated gene in each category and the likelihood of finding an activated gene randomly are shown in the bar plot; numbers in each bar correspond to the activated genes in each category. P values were calculated by Fisher exact test. (E) Relationships between regulatory potentials and Notch-dependent changes in gene expression. The schematic shows how regulatory potential is calculated (see Materials and Methods for details). The lower panels show relationships between regulatory potentials of NOTCH1-binding sites and differential expression of genes (see

Supporting Information

for details).

Fig. 4.

Fig. 4.

Dynamic NOTCH1 sites are preferentially located within H3K27ac-marked superenhancers. (A) Chromatin landscapes near the NOTCH1 target gene NRARP. The flanking EXD3 gene is not expressed in T-LL. (B) Composite profiles of H3K27ac flanking dynamic NOTCH1 sites under steady-state (basal), Notch-off, and Notch-on conditions. (C) Enrichment of dynamic NOTCH1 sites in the broadest H3K27ac peaks. Genomic H3K27ac peaks (n = 35,244) were ranked by peak width and grouped into 176 bins, each containing 200 peaks. H3K27ac peaks associated with dynamic or nondynamic NOTCH1 sites were counted in each bin, and the ratio of (i) the likelihood of NOTCH1 binding to H3K27ac peaks and (ii) the average likelihood NOTCH1 binding to all genomic H3K27ac peaks is plotted for each class of NOTCH1 sites. (D) Distribution of H3K27ac peak width for all H3K27ac peaks (gray), all promoter H3K27ac peaks (cyan), H3K27ac peaks associated with nondynamic promoter NOTCH1 (dark blue), nondynamic nonpromoter NOTCH1 (light blue), and dynamic NOTCH1 (red) is shown. (E) Composite profiles of H3K27ac and H3K4me1 on dynamic NOTCH1 associated H3K27ac peaks (see

Supporting Information

for details). (F) Fold-change distribution of H3K27ac peaks associated with dynamic NOTCH1 sites versus all H3K27ac peaks. Fold change is the ratio of H3K27ac level in the Notch-on and Notch-off states. (G) Fold-change distribution of promoter H3K27ac levels on NOTCH1 target genes and all genes. H3K27ac level is measured as the normalized read count within 2 kb of transcriptional start sites.

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