Significance Studies focused on understanding how transcription factors control gene expression have shown that transcription-factor binding sites generally greatly exceed the number of regulated genes, making it challenging to identify functional binding sites. Using Notch pathway inhibitors, we identified a subset of Notch-binding sites in leukemia cell genomes that are dynamic, changing in occupancy relatively rapidly when Notch signaling is perturbed. Dynamic Notch sites are highly associated with genes that are directly regulated by Notch and mainly lie in large regulatory switches termed superenhancers, which control genes with key roles in development and cancer. This work links Notch signaling to superenhancers and suggests that assessment of transcription factor–genome dynamics can help to identify functionally important regulatory sites.

The identification of activating NOTCH1 mutations in T-cell acute lymphoblastic leukemia (T-ALL) led to clinical testing of γ-secretase inhibitors (GSI) that prevent NOTCH1 activation1–3. However, responses have been transient4,5, suggesting that resistance limits clinical efficacy. Here we modeled T-ALL resistance, identifying GSI-tolerant ‘persister’ cells that expand in the absence of NOTCH signaling. Rare persisters are already present in naïve T-ALL populations, and the reversibility of the phenotype suggests an epigenetic mechanism. Relative to GSI-sensitive cells, persisters activate distinct signaling and transcriptional programs, and exhibit chromatin compaction. A knockdown screen identified chromatin regulators essential for persister viability, including BRD4. BRD4 binds enhancers near critical T-ALL genes, including MYC and BCL2. The BRD4 inhibitor JQ1 down-regulates these targets and induces growth arrest and apoptosis in persisters, at doses well tolerated by GSI-sensitive cells. Consistently, the GSI-JQ1 combination was found to be effective against primary human leukemias in vivo. Our findings establish a role for epigenetic heterogeneity in leukemia resistance that may be addressed by incorporating epigenetic modulators in combination therapy.

Epstein-Barr virus nuclear antigen 2 (EBNA2) regulation of transcription through the cell transcription factor RBPJ is essential for resting B-lymphocyte (RBL) conversion to immortal lymphoblast cell lines (LCLs). ChIP-seq of EBNA2 and RBPJ sites in LCL DNA found EBNA2 at 5,151 and RBPJ at 10,529 sites. EBNA2 sites were enriched for RBPJ (78%), early B-cell factor (EBF, 39%), RUNX (43%), ETS (39%), NFκB (22%), and PU.1 (22%) motifs. These motif associations were confirmed by LCL RBPJ ChIP-seq finding 72% RBPJ occupancy and Encyclopedia Of DNA Elements LCL ChIP-seq finding EBF, NFκB RELA, and PU.1 at 54%, 31%, and 17% of EBNA2 sites. EBNA2 and RBPJ were predominantly at intergene and intron sites and only 14% at promoter sites. K-means clustering of EBNA2 site transcription factors identified RELA-ETS, EBF-RUNX, EBF, ETS, RBPJ, and repressive RUNX clusters, which ranked from highest to lowest in H3K4me1 signals and nucleosome depletion, indicative of active chromatin. Surprisingly, although quantitatively less, the same genome sites in RBLs exhibited similar high-level H3K4me1 signals and nucleosome depletion. The EBV genome also had an LMP1 promoter EBF site, which proved critical for EBNA2 activation. LCL HiC data mapped intergenic EBNA2 sites to EBNA2 upregulated genes. FISH and chromatin conformation capture linked EBNA2/RBPJ enhancers 428 kb 5′ of MYC to MYC. These data indicate that EBNA2 evolved to target RBL H3K4me1 modified, nucleosome-depleted, nonpromoter sites to drive B-lymphocyte proliferation in primary human infection. The primed RBL program likely supports antigen-induced proliferation.pstein Barr virus (EBV) infection is highly prevalent in all human populations. EBV is also an important cause of endemic Burkitt Lymphoma (1), Hodgkin Lymphoma (2), and lymphoproliferative disorders in immune-suppressed (3) and HIV-infected people (4). In early primary human infection, EBV infects peripheral resting B lymphocytes (RBLs) and expresses six nuclear (Epstein-Barr virus nuclear antigen, EBNA) and two integral membrane (LMP) proteins. EBNAs and LMPs convert RBLs into proliferating lymphoblastoid cells (LCLs), which are malignant in the absence of effective T-cell responses. Because EBNAs and LMPs include >4,000 amino acids, T-cell immune responses normally eliminate most EBV-infected cells. Lymphocytes with down-regulated EBV protein expression persist in tonsils and lymph nodes and are reservoirs for reactivated EBV infection (4). EBV conversion of RBLs into LCLs in vitro (5) is a relevant and experimentally useful model for EBV proliferative effects in B-lymphocytes.In converting RBLs to LCLs, EBNA2 and EBNALP are expressed first. EBNA2 up-regulates EBV and cell gene expression, including EBV LMP1 and cell MYC, CD23, and CD21 (6-9). EBNA2 associates with DNA through RBPJ, which also mediates Notch binding to DNA (10, 11). The B-lymphocyte and macrophage lineage ETS protein, PU.1, is also important in EBNA2 activation of the EBV LMP1 promoter (12, 13). The EBNA2 C-terminal acidic domain recruits basal ...

Rapid climate change jeopardizes tree populations by shifting current climate zones. To avoid extinction, tree populations must tolerate, adapt, or migrate. Here we investigate geographic patterns of genetic variation in valley oak, Quercus lobata Née, to assess how underlying genetic structure of populations might influence this species' ability to survive climate change. First, to understand how genetic lineages shape spatial genetic patterns, we examine historical patterns of colonization. Second, we examine the correlation between multivariate nuclear genetic variation and climatic variation. Third, to illustrate how geographic genetic variation could interact with regional patterns of 21st Century climate change, we produce region-specific bioclimatic distributions of valley oak using Maximum Entropy (MAXENT) models based on downscaled historical (1971-2000) and future (2070-2100) climate grids. Future climatologies are based on a moderate-high (A2) carbon emission scenario and two different global climate models. Chloroplast markers indicate historical range-wide connectivity via colonization, especially in the north. Multivariate nuclear genotypes show a strong association with climate variation that provides opportunity for local adaptation to the conditions within their climatic envelope. Comparison of regional current and projected patterns of climate suitability indicates that valley oaks grow in distinctly different climate conditions in different parts of their range. Our models predict widely different regional outcomes from local displacement of a few kilometres to hundreds of kilometres. We conclude that the relative importance of migration, adaptation, and tolerance are likely to vary widely for populations among regions, and that late 21st Century conditions could lead to regional extinctions.

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.