Identification of NOTCH1-Controlled Transcriptional Programs In Human T-Cell Development

Abstract 2495

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive malignancy characterized by constitutive activation on the NOTCH signaling pathway. The NOTCH1 oncogene encodes a ligand activated transcriptional activator that regulates gene expression in association with the RBPJK DNA binding protein. NOTCH1 signaling is strictly required for thymocyte development and activating mutations resulting in aberrant NOTCH1 signaling are present in over 60% of T-ALLs. Over the last years gene expression profiling studies have identified NOTCH1 as a critical transcriptional regulator of cell growth and metabolism in T-ALL. However, the specific mechanisms that mediate the differential expression of NOTCH1 target genes in T-cell development and T-ALL remains to be elucidated. Here we used ChIP-seq technology to define the complete repertoire of NOTCH1 targets in T-ALL. Furthermore, we performed an integrative analysis of these results in the context of gene expression signatures induced NOTCH1 signaling in T-ALL and in the context of thymocyte development. NOTCH1 ChIP-seq analysis was performed by SOLiD3 ultra deep sequencing of chromatin immunoprecipitates generated with two different anti-NOTCH1 antibodies in HPB-ALL cells, a NOTCH1-mutated T-ALL leukemic cell line with high levels of NOTCH1 signaling. Analysis of the distribution of NOTCH1 binding sites relative to annotated genes (3,078 genes associated to NOTCH1 peaks; MACS ChIP-seq with P< 1e–9) showed that 58% of high confidence NOTCH1 peaks are intragenic, 28% are located in promoter regions and 14% are intergenic. Moreover, detailed analysis of these results showed that NOTCH1 binding is preferentially located in the proximal promoter of target genes and revealed a highly significant enrichment of NOTCH1 peaks located between −1Kb and +1kb from the transcription start site. Motif analysis of DNA sequences occupied by NOTCH1 revealed a high level of enrichment of “CTCCCA” NOTCH/RBPJK DNA binding sites (P<0.0001). Further characterization of NOTCH1/RBPJK DNA binding sequences showed high enrichment of an “ACTACANN” motif adjacent to canonical NOTCH/RBPJK sites (MDSCAN score=5.43) and a marked overrepresentation of paired NOTCH/RBPJK sites. Functional annotation using Gene Ontology analysis indicated a significant enrichment of NOTCH1 targets corresponding to transcriptional regulation and protein biosynthesis (P <0.001). NOTCH1 ChIPseq target genes were characteristically downregulated in T-ALL cells upon inhibition of NOTCH1 signaling with a gamma secretase inhibitor. Moreover, functional annotation of top NOTCH1 regulated targets confirmed the dominance of genes involved in protein biosynthesis and cellular anabolic processes. Next, we analyzed the differential expression of NOTCH1 ChIP-seq targets in different stages of T-cell development. This analysis revealed five distinct clusters of NOTCH1 target genes, which were associated with distinct functional categories such as metabolism, cell cycle and signal transduction (P <0.00001). These results are consistent with a combinatorial model of gene regulation where different transcription factors coordinately interact with NOTCH1 signaling to define distinct patterns of gene expression during T-cell development. In order to identify transcription factors involved in the differential regulation of NOTCH1 target genes we analyzed the presence of transcription factor motifs paired at specific distances with RBPJK sites. This analysis revealed a highly significant enrichment of STAT3, AP1, NKX2.5 and YY-1 binding motifs (P <0.01) in association with NOTCH/RBPJK binding sites. Overall these results unravel a complex combinatorial program involved in the regulation of NOTCH1 target genes and point to a coordinated role for JAK/STAT, MAPK/AP1 and NOTCH1 signaling in T-cell development and T-ALL.