Differential enhancer regions in KMT2A::AFF1 cell lines are functional enhancers that drive differential gene expression. (A) Volcano plot of differentially expressed genes between RS4;11 (1883; red) and SEM cells (2468; blue) or no significant change (gray) from 3 biological replicates; false discover rate (FDR) of <0.05. (B) Volcano plot of enhancers with significantly increased accessibility in RS4;11 (1273; red) or SEM cells (1357; blue), or enhancers with unaltered accessibility (gray) from 8 biological replicates, FDR < 0.05. (C) Chromatin immunoprecipitation sequencing (ChIP-seq) tracks at the GNAQ locus for KMT2A, AFF1, H3K27ac, H3K4me1, H3K79me2, and H3K4me3 together with ATAC-seq and Capture-C in SEM cells using the GNAQ promoter as a viewpoint. The SEM-specific GNAQ enhancer (S1) is highlighted in blue. (D) ChIP-seq tracks at the ARID1B locus for KMT2A, AFF1, H3K27ac, H3K4me1, H3K79me2, and H3K4me3 together with ATAC-seq and Capture-C in SEM cells using the ARID1B promoter as a viewpoint. The RS4;11 specific intergenic enhancer (R1) is highlighted in red, and the SEM-specific intragenic enhancer (S2) is highlighted in blue. (E) Reverse transcription-qPCR comparing the expression of enhancer deletion mutants (light shading) with wild type (dark shading) in RS4;11 (red) or SEM cells (blue) when deleting either the intragenic GNAQ enhancer (S1; left) or ARID1B intergenic enhancer (R1; right). Significance of alterations in relative copy number were determined by a 2-sided t test with correction for multiple testing (Benjamini-Hochberg), n = 6 biological replicates. ∗Adjusted P value < .05; ∗∗P < .01. ns, not significant.
Figure 2.

Differential enhancer regions in KMT2A::AFF1 cell lines are functional enhancers that drive differential gene expression. (A) Volcano plot of differentially expressed genes between RS4;11 (1883; red) and SEM cells (2468; blue) or no significant change (gray) from 3 biological replicates; false discover rate (FDR) of <0.05. (B) Volcano plot of enhancers with significantly increased accessibility in RS4;11 (1273; red) or SEM cells (1357; blue), or enhancers with unaltered accessibility (gray) from 8 biological replicates, FDR < 0.05. (C) Chromatin immunoprecipitation sequencing (ChIP-seq) tracks at the GNAQ locus for KMT2A, AFF1, H3K27ac, H3K4me1, H3K79me2, and H3K4me3 together with ATAC-seq and Capture-C in SEM cells using the GNAQ promoter as a viewpoint. The SEM-specific GNAQ enhancer (S1) is highlighted in blue. (D) ChIP-seq tracks at the ARID1B locus for KMT2A, AFF1, H3K27ac, H3K4me1, H3K79me2, and H3K4me3 together with ATAC-seq and Capture-C in SEM cells using the ARID1B promoter as a viewpoint. The RS4;11 specific intergenic enhancer (R1) is highlighted in red, and the SEM-specific intragenic enhancer (S2) is highlighted in blue. (E) Reverse transcription-qPCR comparing the expression of enhancer deletion mutants (light shading) with wild type (dark shading) in RS4;11 (red) or SEM cells (blue) when deleting either the intragenic GNAQ enhancer (S1; left) or ARID1B intergenic enhancer (R1; right). Significance of alterations in relative copy number were determined by a 2-sided t test with correction for multiple testing (Benjamini-Hochberg), n = 6 biological replicates. ∗Adjusted P value < .05; ∗∗P < .01. ns, not significant.

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