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. 2020 Jun 11;12(5):397-399.
doi: 10.1093/jmcb/mjz114.

BET inhibition prevents aberrant RUNX1 and ERG transcription in STAG2 mutant leukaemia cells

Affiliations

BET inhibition prevents aberrant RUNX1 and ERG transcription in STAG2 mutant leukaemia cells

Jisha Antony et al. J Mol Cell Biol. .
No abstract available

Keywords: CRISPR-Cas9; ERG; RUNX1; STAG2; chromatin; cohesin; enhancer; inducible; megakaryocyte.

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Figures

Figure 1
Figure 1
STAG2 mutation alters chromatin accessibility and response to cell signaling. (A) Schematic of STAG2 protein showing the position of STAG2 R614* (C>T) mutation. Shown also is the Sanger sequencing plot for CRISPR-Cas9-edited K562 line containing homozygous STAG2 R614* mutation (STAG2-nullA). A silent mutation was introduced at PAM site in STAG2-null cells. (B) Immunoblot analyses of STAG2 protein levels in parental (WT) and STAG2-null cells. Bar graphs show STAG2 protein normalized to γ-tubulin from three biological replicates. Significance was determined by unpaired t-test. L, protein ladder. (C) Images of WT and STAG2-null K562 cells in culture. (D) Gene set enrichment analyses showing upregulation of extracellular matrix (Naba core matrisome) and haematopoietic stem cell genes in STAG2-nullA. Shown are the normalized enrichment score (NES) and FDR q-value. (E) Volcano plot of differential chromatin accessibility in STAG2-nullA compared to WT K562 cells. Significant peaks at adjusted P-value ≤ 0.05 are shown in red (52452 sites showing differential accessibility, 29432 differentially increased and 23020 differentially decreased). Lines indicate log2 fold change cut-off: 2. (F) Enrichment of differentially increased and decreased accessible sites identified in STAG2-nullA at SEs (defined in K562, CD34+ cord blood cells, and CD14+ monocytes). (G) Integrative genome browser view of normalized ATAC-sequencing signals from STAG2-nullA and WT cells at ERG and RUNX1. Significant (P ≤ 0.05) accessible sites at RUNX1-P2 promoter and ERG +85 kb enhancer are boxed. ChromHMM data for K562 (derived from ENCODE) is shown at the top of each plot, and additional tracks are BRD4 binding in K562 following treatment with dimethyl sulfoxide (DMSO) or 6 h of JQ1 (Liu et al., 2017). (H and I) ERG (H) and RUNX1-P2 (I) expression levels examined over a time-course treatment with PMA, JQ1, or a combination of PMA and JQ1. Graphs depict average relative mRNA levels from three biological replicates normalized to two reference genes. Black asterisks denote significant difference between WT and STAG2-null lines following PMA-only treatment. Green asterisks denote significant difference between PMA-only and combination of PMA and JQ1 treatment within each cell type. Significance was determined by two-way Anova. (J) Relative mean fluorescence intensity (MFI) of KIT and CD15 following treatment with control DMSO or JQ1 for 24 h. Relative MFI for each cell type and condition was determined as a ratio of MFI in stained/unstained. Graphs represent the average of three biological replicates. Significance was determined by two-way Anova. Black asterisks denote significant difference between WT and STAG2-null cells for the same condition. Red asterisks denote significant difference between DMSO and JQ1 treatment within each cell type. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

References

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