Article

Genome-wide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells

Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2011; 108(36):14908-13. DOI: 10.1073/pnas.1109023108
Source: PubMed

ABSTRACT

Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is oncogenic in murine and human T-cell progenitors. Using ChIP-Seq, we find that in human and murine T-lymphoblastic leukemia (TLL) genomes Notch1 binds preferentially to promoters, to RBPJ binding sites, and near imputed ZNF143, ETS, and RUNX sites. ChIP-Seq confirmed that ZNF143 binds to ∼40% of Notch1 sites. Notch1/ZNF143 sites are characterized by high Notch1 and ZNF143 signals, frequent cobinding of RBPJ (generally through sites embedded within ZNF143 motifs), strong promoter bias, and relatively low mean levels of activating chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in cells. K-means clustering of Notch1 binding sites and associated motifs identified conserved Notch1-RUNX, Notch1-ETS, Notch1-RBPJ, Notch1-ZNF143, and Notch1-ZNF143-ETS clusters with different genomic distributions and levels of chromatin marks. Although Notch1 binds mainly to gene promoters, ∼75% of direct target genes lack promoter binding and are presumably regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R, and the GIMAP cluster. Human and murine TLL genomes also have many sites that bind only RBPJ. Murine RBPJ-only sites are highly enriched for imputed REST (a DNA-binding transcriptional repressor) sites, whereas human RPBJ-only sites lack REST motifs and are more highly enriched for imputed CREB sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in TLL cells, as well as unique classes of divergent RBPJ-only sites that also likely regulate transcription.

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    • "To compare results for H4ac regions that were differentially enriched in the knockdown or the control, and random expectations for each, we summarized the spatial relationships as a bar chart showing the Y-coordinate of each empirical distribution function at a center-to-center distance of 1 kb (Fig. 7 A). We used block bootstrap v0.8.1 (Encode Statistics) to estimate p-values for the concordance between locations of 2,112 genome-wide strong enriched regions from Wang et al. (2011) and locations of genome-wide 500-bp regions that edgeR called as differentially enriched in H4ac in the RBPJ knockdown (BH-corrected P < 0.05) and in the control (BH-corrected P < 0.01). For each pairwise comparison, we set R = 0.05 and reported the mean p-value from thirty 10,000-iteration runs. "
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    ABSTRACT: Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.
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    • "It has been observed that some sites in the genome are occupied only when Notch signaling is active (''dynamic sites'') (Bray and Bernard, 2010; Castel et al., 2013; Housden et al., 2013; Krejcí et al., 2009; Wang et al., 2014, 2011). These dynamic sites show increased recoverability by RBP ChIP when NICD is present, leading to the proposal that the NICD/RBP complex acts as a pioneer factor to modify chromatin at these sites (Castel et al., 2013). "
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    • "Interestingly, the proximity of RBP-J binding sites of mice and human differ for the enrichment of either the REST repressor or CREB binding site, respectively. Furthermore, the authors revealed that only in about 25% of the target genes Notch binds within the promoter leaving $75% of the genes that are probably regulated via enhancer activity, instead (Wang et al., 2011). In a continuation of this work, the authors showed recently that only $10% of the Notch1 binding sites are modulated in Notch1 occupancy upon GSI treatment, which they define as dynamic Notch-RBP-J sites (Wang et al., 2014). "
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