Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia - PubMed (original) (raw)

. 2014 Nov 18;111(46):E4946-53.

doi: 10.1073/pnas.1407079111. Epub 2014 Nov 4.

Hongfang Wang 2, Chongzhi Zang 3, Kelly L Arnett 4, Will Bailis 1, Yugong Ho 5, Birgit Knoechel 6, Claudia Lanauze 1, Lumena Louis 1, Katherine S Forsyth 1, Sujun Chen 7, Yoonjie Chung 1, Jonathan Schug 5, Gerd A Blobel 8, Stephen A Liebhaber 5, Bradley E Bernstein 9, Stephen C Blacklow 10, Xiaole Shirley Liu 3, Jon C Aster 11, Warren S Pear 12

Affiliations

Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia

Yumi Yashiro-Ohtani et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context-specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3' enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia.

Keywords: Brd4; chromatin; enhancer; gene regulation; transcription.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Identification of a site ∼1.27 Mb 3′ of the Myc gene body that binds RBPJ and Notch1 in normal and transformed murine pre-T cells. (A) ChIP-Seq read counts for Notch1, RBPJ, and H3K4me1, H3K4me3, and H3K27me3 marks in a ∼2-Mb region containing Myc. y axis, aligned reads; arrowhead, TSSs. (B) Local ChIP for Notch1 at the 3′ Myc site (NDME) in the murine T-ALL cell line T6E treated with GSI or DMSO for 4 h before harvest. Nonspecific Ig was used as a control. (C and D) Local ChIP analyses of Notch1 occupancy of the binding site 3′ of Myc (NDME) in primary murine T-ALL cells (C) and DN3 thymocytes (D). Previously characterized Notch1 binding sites in the Hes1 promoter and Dtx1 intron2 serve as positive controls; Nanog serves as a negative control. In B_–_D, data were obtained in triplicate in independent experiments; error bars correspond to the SEM. *P < 0.01; **P < 0.001.

Fig. 2.

Fig. 2.

The murine Myc 3′ enhancer contains a SPS that drives NTC dimer-dependent transcription. (A) Schematic of the RBPJ–Notch1–MAML NTC complex on DNA containing an SPS site. The two RBPJ motifs are designated A and B, and the critical R1984 interankyrin repeat domain contact is highlighted. See ref. for details. RBPJ, gold; Notch1 RAM-ANK, purple; MAML1, green. (B) Assembly of dimeric NTCs on the 3′ Myc enhancer element. EMSA was performed with oligonucleotide probes containing the putative SPS and the indicated combinations of recombinant RBPJ, Notch1 RAM-ANKs (RA1), and N-terminal MAML1 (MAML). RA1-R1984A corresponds to the dimerization-defective Notch1 mutant. (C) Schematic of the luciferase reporter gene constructs including the NDME WT and mutant sequences. Note: RBPJ is denoted by its alternative name, CSL. (D) T6E cells were transfected with Renilla luciferase control (pRLTK), the indicated Myc firefly luciferase reporter genes, and either empty vector or vectors driving expression of WT NICD1 or NICD1 with the R1984A mutation. (E) Murine T-ALL T6E cells transfected with pRL-TK and one of the reporter constructs shown in C. Cells were cultured with DMSO or 1 μM compound E for 6 h before harvest. (F) Activity of the murine Myc enhancer reporter construct (_Myc_ProNDME) in various cell lines with and without overexpression of NICD1. In D_–_F, cells were harvested 48 h after transfection. Data were obtained in triplicate in independent experiments; error bars correspond to the SEM. **P < 0.001.

Fig. 3.

Fig. 3.

Interaction of the 3′ Myc enhancer element and the Myc promoter. (A) Schematic of Myc locus and enhancer with superimposed primers used for chromosome conformation capture (3C) assay. (B) The 3C assay in T6E cells. Graphs show the enrichment of PCR products normalized to HindIII-digested and randomly ligated bacterial artificial chromosome (BAC) DNA spanning the region. (C) Short-term Notch inhibition does not affect chromatin looping. T6E cells were treated with DMSO or 1 μM GSI for 6 h. The 3C assay was performed on the Myc promoter and sequences 3′ of the Myc gene body. Positions of primers used in B and C are shown in A. Data were obtained in triplicate in independent experiments; error bars correspond to the SEM.

Fig. 4.

Fig. 4.

Notch1 regulates human Myc via a 3′ enhancer element. (A) ChIP-Seq read counts for Notch1, RBPJ, and H3K4me1, H3K4me3, and H3K27me3 marks in a ∼2-Mb region containing human Myc. y axis, aligned sequence tags; arrowhead, TSSs. (B) The 3C assay in the human T-ALL cell line CUTLL1. Graphs show the enrichment of PCR product normalized to HindIII-digested, randomly ligated BAC DNA spanning the region. The arrows beneath the schematic in A denote the position of primers used for 3C analysis. The Myc promoter primer and “probe” were paired with eight primers positioned in flanking sequences to detect ligated HindIII-digestion fragments. (C) The human Myc 3′ enhancer element (NDME) stimulates transcription. (Upper) Schematic of the human Myc NDME and control firefly luciferase reporter genes. A minimal TATA promoter construct was used as a negative control. Note: RBPJ is denoted by its alternative name, CSL. (Lower) CUTLL1 cells were transfected with the indicated reporter genes with and without 1 µM compound E. Luciferase assays were performed 48 h later. (D) KOPT-K1 (Left) and CUTLL1 (Right) cells transduced with either NICD1, NICD1-R1984A, or empty plasmid were cultured in the presence of 1 μM compound E beginning 24 h after transduction. The number of GFP+ transduced cells was determined at various time points and normalized to time 0 (defined as 48 h after transduction). (E) Myc mRNA expression in CUTLL1 cells transduced as in D. Except for the MigR1 empty vector control cells, 1 μM compound E was added to cells 24 h after transduction. Cells were harvested for RT-PCR analysis 48 h later. In B_–_E, data were obtained in triplicate in independent experiments; error bars correspond to the SEM.

Fig. 5.

Fig. 5.

Altered 3′ enhancer activity and chromatin looping in GSI-sensitive and resistant T-ALL cells. (A) Chromatin landscapes in human CUTLL1 cells (i) depleted of NICD1 by treatment with 1 μM compound E for 3 d and (ii) after 4 h of Notch activation by GSI washout. RBPJ, Notch1, and H3K27ac traces are shown in the Notch-on and -off states, whereas Brd4 and Med1 are shown only in the Notch-on state. H3K27ac peaks are seen at the Myc promoter (p) and in 3′ regions (a–l). (B) Chromatin landscapes in GSI-sensitive and -resistant human KOPT-K1 and DND-41 cells. H3K27ac and Brd4 traces are shown for each cell state. The positions of primers and probes used in the 3C analyses in C are shown below the traces. BDME, Brd4-dependent Myc enhancer. (C) The 3C analyses performed with GSI-sensitive (naïve) and GSI-resistant KOPT-K1 cells. Graphs show the enrichment of PCR products normalized to HindIII-digested, randomly ligated BAC DNA spanning the region. The 3C data were obtained in triplicate in independent experiments; error bars correspond to the SEM. **P = 0.008 (comparing GSI-naïve with GSI-resistant samples using Student t test).

References

    1. Aster JC, Blacklow SC, Pear WS. Notch signalling in T-cell lymphoblastic leukaemia/lymphoma and other haematological malignancies. J Pathol. 2011;223(2):262–273. - PMC - PubMed
    1. Hori K, Sen A, Artavanis-Tsakonas S. Notch signaling at a glance. J Cell Sci. 2013;126(Pt 10):2135–2140. - PMC - PubMed
    1. Fryer CJ, Lamar E, Turbachova I, Kintner C, Jones KA. Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev. 2002;16(11):1397–1411. - PMC - PubMed
    1. Tzoneva G, Ferrando AA. Recent advances on NOTCH signaling in T-ALL. Curr Top Microbiol Immunol. 2012;360:163–182. - PubMed
    1. Wang H, et al. NOTCH1-RBPJ complexes drive target gene expression through dynamic interactions with superenhancers. Proc Natl Acad Sci USA. 2014;111(2):705–710. - PMC - PubMed

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