Figure 4.
Che-1 modulates active chromatin and global transcription in MM cells. (A) Line traces of representative gel lanes of micrococcal nuclease (MNase) assay (bottom) and corresponding WB analysis with the indicated antibodies of total cell extracts (top) from Kms27 cells transfected with siRNA control (siControl) or siRNA Che-1 (siChe-1). (B) ATAC-seq and H3K27ac signal in siControl (blue) and siChe-1 (yellow) samples at c-Myc, AATF, SCN1A, and WDR48 loci. Signals of each replicate in the 2 categories are overlaid and fitted with transparency. Signal enrichment scale is given on the y-axis of each site. (C) Enrichment density of ATAC-seq significant peaks identified in siControl (blue) and siChe-1 (yellow) samples. Enrichment density was calculated on the relative size of each peak. (D) Top: Venn diagram showing shared and unique peaks in the 2 categories siControl (blue) and siChe-1 (yellow) samples. Middle: Motifs of the top 10 most enriched transcription factors for each category. Q value <10−3. Bottom: Enrichment density of ATAC-seq significant peaks of each category identified in siControl (blue) and siChe-1 (yellow) samples. Enrichment density was calculated on the relative size of each peak. (E) Disease ontology enrichment analysis of the only siControl (blue) significant peaks with the relative P value associated with each entry. Test statistic for binomial distribution. (F) Pathway enrichment analysis of the shared peaks between siControl (blue) and siChe-1 (yellow) samples with the relative P value associated with each entry. Test statistic for binomial distribution. (G) Heat map representation of normalized fold change in RNA spike-in normalized gene expression in Kms27 cells after siChe-1 transfection (left). Spike-in normalized transcriptional output of Kms27 cells transfected as in A (right). (H) Top: Bar chart of Che-1 ChIP-seq signals to the closest gene. Bar chart is representing the absolute distances of Che-1 signals (N = 6000) to the closest gene. x-axis: Absolute distance expressed in kilobase (kb) to the closest transcription start site (TSS). y-axis: Percentage of peak association to the closest genes. Bottom: Motif analysis of Che-1 ChIP signals. Left: Top 10 most significant motifs obtained from the 6000 Che-1 ChIP binding sites (Q value  <10−41). Right: Significant motifs of Che-1 binding signals at >5 kb to the closest TSS (Q value <0.05). (I) Left: ATAC and H3K27ac signal intensity of siControl (blue) and siChe (yellow) at 6000 Che-1 ChIP binding sites. Signal intensity from 1 (weak) to 8 (strongest). Right: Normalized Che-1 ChIP (red), H3K27ac ChIP and ATAC-seq signal at IRF4, CASC11, c-Myc, FGFR3, LTM1, and NSD2 genes. See also supplemental Figure 4. MSigDB, Molecular Signature Database.

Che-1 modulates active chromatin and global transcription in MM cells. (A) Line traces of representative gel lanes of micrococcal nuclease (MNase) assay (bottom) and corresponding WB analysis with the indicated antibodies of total cell extracts (top) from Kms27 cells transfected with siRNA control (siControl) or siRNA Che-1 (siChe-1). (B) ATAC-seq and H3K27ac signal in siControl (blue) and siChe-1 (yellow) samples at c-Myc, AATF, SCN1A, and WDR48 loci. Signals of each replicate in the 2 categories are overlaid and fitted with transparency. Signal enrichment scale is given on the y-axis of each site. (C) Enrichment density of ATAC-seq significant peaks identified in siControl (blue) and siChe-1 (yellow) samples. Enrichment density was calculated on the relative size of each peak. (D) Top: Venn diagram showing shared and unique peaks in the 2 categories siControl (blue) and siChe-1 (yellow) samples. Middle: Motifs of the top 10 most enriched transcription factors for each category. Q value <10−3. Bottom: Enrichment density of ATAC-seq significant peaks of each category identified in siControl (blue) and siChe-1 (yellow) samples. Enrichment density was calculated on the relative size of each peak. (E) Disease ontology enrichment analysis of the only siControl (blue) significant peaks with the relative P value associated with each entry. Test statistic for binomial distribution. (F) Pathway enrichment analysis of the shared peaks between siControl (blue) and siChe-1 (yellow) samples with the relative P value associated with each entry. Test statistic for binomial distribution. (G) Heat map representation of normalized fold change in RNA spike-in normalized gene expression in Kms27 cells after siChe-1 transfection (left). Spike-in normalized transcriptional output of Kms27 cells transfected as in A (right). (H) Top: Bar chart of Che-1 ChIP-seq signals to the closest gene. Bar chart is representing the absolute distances of Che-1 signals (N = 6000) to the closest gene. x-axis: Absolute distance expressed in kilobase (kb) to the closest transcription start site (TSS). y-axis: Percentage of peak association to the closest genes. Bottom: Motif analysis of Che-1 ChIP signals. Left: Top 10 most significant motifs obtained from the 6000 Che-1 ChIP binding sites (Q value  <10−41). Right: Significant motifs of Che-1 binding signals at >5 kb to the closest TSS (Q value <0.05). (I) Left: ATAC and H3K27ac signal intensity of siControl (blue) and siChe (yellow) at 6000 Che-1 ChIP binding sites. Signal intensity from 1 (weak) to 8 (strongest). Right: Normalized Che-1 ChIP (red), H3K27ac ChIP and ATAC-seq signal at IRF4, CASC11, c-Myc, FGFR3, LTM1, and NSD2 genes. See also supplemental Figure 4. MSigDB, Molecular Signature Database.

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