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. 2014 Dec 12;346(6215):1373-7.
doi: 10.1126/science.1259037. Epub 2014 Nov 13.

Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element

Marc R Mansour  1 , Brian J Abraham  2 , Lars Anders  2 , Alla Berezovskaya  3 , Alejandro Gutierrez  4 , Adam D Durbin  3 , Julia Etchin  3 , Lee Lawton  2 , Stephen E Sallan  4 , Lewis B Silverman  4 , Mignon L Loh  5 , Stephen P Hunger  6 , Takaomi Sanda  7 , Richard A Young  8 , A Thomas Look  9
Affiliations

Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element

Marc R Mansour et al. Science. .

Abstract

In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, that recruit much of the cell's transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits its H3K27 acetylase-binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, which suggests a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.

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Figures

Figure 1
Figure 1. Mutations at an intergenic site are associated with the TAL1 super-enhancer in T-ALL cells
(A) Normalized ChIP-seq tracks for H3K27ac at the STIL-TAL1 locus in two human purified normal hematopoietic stem cell samples (CD34), the RPMI-8402 T-ALL cell line that over-expresses TAL1 as a result of TAL1d, DND-41 T-ALL cells that do not express TAL1, human fetal thymic tissue, and MOLT-3 and Jurkat cells that have mutations at a non-coding site 7.5 kb from the TAL1 transcriptional start site (red arrow). ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. (B) Sequence alignments of the −7.5 kb site showing wild-type (WT) sequences in black and inserted sequences in red for Jurkat and MOLT-3 T-ALL cell lines and eight pediatric T-ALL patients. (C) TAL1 mRNA expression as determined by quantitative PCR and expressed as percentage of GAPDH. Error bars are ±SEM from two independent experiments performed in triplicate.
Figure 2
Figure 2. Mutations of the TAL1 enhancer activate through recruitment of MYB
(A) All TAL1 enhancer mutations introduce de novo MYB binding sites as determined by UniPROBE (19). The MYB primary binding motif is shown above the mutation-derived MYB motifs, with inserted nucleotides shown in red. (B) A 400 bp fragment of the −7.5 kb TAL1 enhancer containing either the wild-type sequence or each of the mutant alleles was cloned upstream of luciferase and a minimal promoter. Constructs were nucleofected into Jurkat cells, together with either control siRNA, or two independent siRNAs targeting MYB. Firefly luciferase activity was measured at 24 hrs, normalized to renilla luciferase to control for cell number and transfection efficiency, and expressed as a ratio relative to activity of the wild-type STIL-TAL enhancer construct. Error bars are ±SEM from two independent experiments performed in triplicate. Corresponding immunoblots for MYB and tubulin are shown below.
Figure 3
Figure 3. MYB binds the mutant TAL1 enhancer site and is a member of the TAL1 complex
(A) ChIP-seq tracks at the STIL-TAL1 locus from Jurkat and MOLT-3 T-ALL cells for GATA3, HEB, RUNX1, TAL1, CBP, MYB (ab45150 antibody), MYB (05-175 antibody), RNA polymerase II (Pol II), and Mediator 1 (MED1). The mutation site at −7.5 kb is depicted with a red arrow. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. (B) Heatmaps showing genome-wide co-occupancy of MYB binding sites (+/− 1 kb) with those from TAL1, RUNX1, GATA3, and CBP sites as determined by ChIP-seq. For each region (y axis), the sequence density centered at 0 indicates overlapping bound regions. (C) Co-immunoprecipitation (Co-IP) and reciprocal Co-IP experiments performed from Jurkat lysates for MYB and TAL1. WCL, whole cell lysate; IgG, isotype control IgG antibody.
Figure 4
Figure 4. Targeted deletion of the TAL1 enhancer mutation collapses the TAL1 super-enhancer
(A) Targeted deletion of 177–193 bp of the mutant but not wild-type allele in Jurkat cells abrogates expression of endogenous TAL1, as determined by qRT-PCR. Data are mean ± SD of two independent experiments performed in triplicate. Agarose gel of products from PCR amplification across the MuTE site for CRISPR/Cas9 Jurkat clones. All clones, including parental cells, express MSCV-TAL1. Hyperladder IV on right. Genotype for each clone is shown below: +, allele present; Δ, deleted allele. (B) ChIP-seq tracks for H3K27ac and MYB at the STIL-TAL1 locus from selected CRISPR/Cas9 clones. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. (C) Sequence alignments of Jurkat clones targeted by CRISPR/Cas9 gsRNA#3 (target sequence is highlighted in grey, PAM sequence in yellow), which targets the 12 bp insertion in Jurkat cells (red font), but not the wild-type allele. Endogenous TAL1 expression as determined by qRT-PCR for respective clones are shown. Data are mean ± SD of two independent experiments performed in triplicate.
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Comment in

  • Cancer. Cancer by super-enhancer.
    Vähärautio A, Taipale J. Vähärautio A, et al. Science. 2014 Dec 12;346(6215):1291-2. doi: 10.1126/science.aaa3247. Science. 2014. PMID: 25504702 No abstract available.

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