Research ArticleNotch Signaling

Genome-wide identification and characterization of Notch transcription complex–binding sequence-paired sites in leukemia cells

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Science Signaling  02 May 2017:
Vol. 10, Issue 477, eaag1598
DOI: 10.1126/scisignal.aag1598
  • Fig. 1 Detection of dimerization of NTCs on DNA by FRET assay.

    (A) Structure of an NTC dimer bound to the Hes1 proximal promoter SPS. The inset shows the key ANK-ANK interface involving the residue R1984A. (B) Cartoon demonstrating the structural basis of the FRET-based NTC dimerization assay. (C) Wavelength scans for labeled NOTCH1 (N1) RAMANK (50:50 mix of RAMANK labeled with Alexa Fluor 488 or Alexa Fluor 546) in various combinations with purified RBPJ, MAML1 (MAML1, residues 13 to 74), and DNA containing the Hes1 SPS. RFU, relative fluorescence units. (D) Quantitation of FRET from data represented in (C). FRET is expressed as 1 − F/Fo, where Fo is the fluorescence intensity in the absence of SPS DNA at the emission maxima for Alexa Fluor 488. Data are means ± SEM of three independent measurements.

  • Fig. 2 Quantitation of NTC dimerization on various DNAs using FRET.

    (A) Example of DNA sequences used in dimerization assays. SPS-1 has paired head-to-head consensus RBPJ motifs separated by a spacer of 16 base pairs. SPS-1–24 is an example of a DNA with a nonconsensus site B sequence, whereas SPS-24 has nonconsensus substitutions in both sites A and B. Nonconsensus base substitutions are shown in lower case. (B) Effect of spacer length on NTC dimerization. Each DNA contains the two consensus RBPJ binding sites shown in SPS-1. (C) NTC dimerization on the indicated palindromic DNA sequences with 16–base pair spacers. (D) NTC dimerization on the indicated nonpalindromic DNA sequences with 16–base pair spacers. In (C) and (D), nonconsensus base substitutions are shown in lower case, and sequence 13 corresponds to a low-affinity RBPJ site found in an SPS located in the promoter of human HES5. Data are means ± SEM of three independent measurements.

  • Fig. 3 Identification of SPS motifs in the genomes of human T-ALL cells.

    (A) PWM logo in the head-to-head orientation used for subsequent calculations. (B) Motif matching scores for secondary RBPJ sites oriented head-to-head (HH), head-to-tail (HT), tail-to-head (TH), and tail-to-tail (TT) calculated for all possible spacer lengths across a 30–base pair (bp) region flanking the highest-scoring RBPJ consensus sequence associated with dynamic and nondynamic NOTCH1 binding sites. Asterisk (*) indicates 16–base pair spacer. (C) Heat map showing K-means clustering of RBPJ sequence motif matching scores generated for sequences flanking dynamic RBPJ binding sites in CUTLL1 cells. Each row represents a single high-confidence dynamic RBPJ site, and each column represents the sequence motif matching score in flanking DNA for positions 1 to 30 base pairs from the primary RBPJ site.

  • Fig. 4 Generation and application of a metric for identifying genomic SPSs.

    (A) Relationship between NTC dimerization measured by FRET and the sums of PWM scores for RBPJ sites A and B. (B) Relationship between NTC dimerization measured by FRET and PBM product scores [PBM(A) × PBM(B)] for RBPJ sites A and B. (C) Bimodal distribution of PBM product scores of dynamic RBPJ/NOTCH1 binding sites in CUTLL1 genomes. On the basis of mixed Gaussian modeling, the red line corresponds to the distribution of PBM product scores corresponding to monomeric RBPJ/NOTCH1 sites, and the green line corresponds to the distribution of PBM product scores for SPSs. Sites with PBM product scores of >0.67 (corresponding to the black line) were designated likely SPSs for purposes of further analysis.

  • Fig. 5 Characteristics of high-confidence SPSs in human T-ALL cell genomes.

    (A) NOTCH1 and (B) RBPJ ChIP-seq signals for predicted SPSs, monomeric RBPJ sites, and dynamic ChIP-seq peaks lacking RBPJ consensus sequences (other sites). (C and D) Location of predicted dynamic RBPJ/NOTCH1 monomeric sites (C) and SPSs (D) relative to transcription start sites (TSS) and RBPJ and NOTCH1 ChIP-seq signal intensity.

  • Fig. 6 Association of predicted SPSs and monomeric NREs with dimer-dependent and dimer-independent Notch target genes in human T-ALL cells.

    (A) Abundance of wild-type NICD1 and R1984A mutant NICD1 in transduced CUTLL1 cells. (B and C) Heat maps of Notch-dependent RNA expression (B) and high-confidence Notch target gene expression (C) in cells treated with vehicle [dimethyl sulfoxide (DMSO)], GSI [1 μM dibenzazepine (DBZ)], or GSI after transduction with wild-type or R1984A mutant NICD1. Expression was determined by sequencing RNA obtained from two independent experiments. (D) Notch dimer–dependent and Notch dimer–independent genes are associated with predicted SPSs and monomeric RBPJ/NOTCH1 sites, respectively. Notch-regulated genes were paired with nearby predicted SPS or monomeric RBPJ/NOTCH1 binding sites as described (17). Genes rescued by R1984A expression in the presence of GSI are preferentially associated with monomeric RBPJ/NOTCH1 binding sites than with SPSs (P < 1 × 10−5, Fisher exact test). (E) Waterfall plot showing the association between the dimer dependency of likely direct Notch target genes and the presence of one or more nearby SPSs (P < 1 × 10−3, Wilcoxon one-sided signed-rank test). The y axis corresponds to the log2 expression ratio of individual Notch target genes in GSI-treated cells transduced with wild-type or R1984A mutant NICD1.

  • Fig. 7 Coregulation of HES5 and HANR1 by enhancer RBPJ/NOTCH1 binding sites.

    (A) HES5/HANR1 coregulation. Upper panels show NOTCH1 and H3K27ac aligned reads from ChIP-seq data for CUTLL1 cells in the Notch-off (GSI) and Notch-on states [GSI washout (WO)]. Dynamic NOTCH1 binding sites designated E1 and E2 have PBM product scores of 0.497 and 0.892, respectively. Lower panels show stranded RNA-seq data obtained from CUTLL1 cells treated with vehicle, GSI (1 μM DBZ), or DBZ after transduction with wild-type (WT) NICD1 or the R1984A NICD1 mutant. HANR1 and HES5 aligned reads from opposite DNA strands are shown in red and blue, respectively. (B and C) Effect of CRISPR/Cas9 editing of E1 and E2 on HES5 and HANR1 expression, as determined by RT-PCR. In (B), HES1 and HANR1 expression was determined in bulk CUTLL1 cells transduced singly or in combination with empty lentiviruses (Cas9GFP and Cas9RFP) or lentiviruses carrying guide RNAs specific for E1 (E1RFP) or E2 (E2GFP). In the left panel in (C), RBPJ motifs are in red, and mutated sequences identified by Sanger sequencing of single PCR products are in blue. Data are means ± SEM of independent determinations done in triplicate. Representative results are shown. N.S., not significant.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/477/eaag1598/DC1

    Fig. S1. Characterization of the dimerization-defective NOTCH1 mutant R1984A in FRET, reporter gene, and local ChIP assays.

    Fig. S2. Sequences used to assess the effect of spacer length on NTC dimerization.

    Fig. S3. Endogenous murine Hes5 promoter and Notch-dependent Myc enhancer SPSs support NTC dimerization in FRET assays.

    Fig. S4. RBPJ binding affinity for DNA correlates with FRET efficiency.

    Fig. S5. Identification of SPSs and Notch dimer–dependent target genes in murine T6E T-ALL cells.

    Fig. S6. HES5 and HANR1 expression depend on NTC dimerization.

    Fig. S7. CRISPR/Cas9 targeting of RBPJ/NOTCH1 binding sites in the E1 and E2 elements 5′ of HES5.

    Fig. S8. Chromatin landscapes near HES5 in T-ALL cell lines.

    Fig. S9. Coregulation of IGF1R and LUNAR1 by an intronic IGF1R SPS.

    Table S1. Dynamic NOTCH1 binding sites in the genome of CUTLL1 T-ALL cells.

    Table S2. Notch-sensitive genes in human CUTLL1 T-ALL cells.

    Table S3. Primers.

    Reference (47)

  • Supplementary Materials for:

    Genome-wide identification and characterization of Notch transcription complex–binding sequence-paired sites in leukemia cells

    Eric Severson, Kelly L. Arnett, Hongfang Wang, Chongzhi Zang, Len Taing, Hudan Liu, Warren S. Pear, X. Shirley Liu, Stephen C. Blacklow,* Jon C. Aster*

    *Corresponding author. Email: stephen_blacklow{at}hms.harvard.edu (S.C.B.); jaster{at}partners.org (J.C.A.)

    This PDF file includes:

    • Fig. S1. Characterization of the dimerization-defective NOTCH1 mutant R1984A in FRET, reporter gene, and local ChIP assays.
    • Fig. S2. Sequences used to assess the effect of spacer length on NTC dimerization.
    • Fig. S3. Endogenous murine Hes5 promoter and Notch-dependent Myc enhancer SPSs support NTC dimerization in FRET assays.
    • Fig. S4. RBPJ binding affinity for DNA correlates with FRET efficiency.
    • Fig. S5. Identification of SPSs and Notch dimer–dependent target genes in murine T6E T-ALL cells.
    • Fig. S6. HES5 and HANR1 expression depend on NTC dimerization.
    • Fig. S7. CRISPR/Cas9 targeting of RBPJ/NOTCH1 binding sites in the E1 and E2 elements 5′ of HES5.
    • Fig. S8. Chromatin landscapes near HES5 in T-ALL cell lines.
    • Fig. S9. Coregulation of IGF1R and LUNAR1 by an intronic IGF1R SPS.
    • Table S3. Primers.
    • Legends for tables S1 and S2
    • Reference (47)

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    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Dynamic NOTCH1 binding sites in the genome of CUTLL1 T-ALL cells.
    • Table S2 (Microsoft Excel format). Notch-sensitive genes in human CUTLL1 T-ALL cells.

    Citation: E. Severson, K. L. Arnett, H. Wang, C. Zang, L. Taing, H. Liu, W. S. Pear, X. S. Liu, S. C. Blacklow, J. C. Aster, Genome-wide identification and characterization of Notch transcription complex–binding sequence-paired sites in leukemia cells. Sci. Signal. 10, eaag1598 (2017).

    © 2017 American Association for the Advancement of Science

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