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. 2022 Dec 30;141(14):1737–1754. doi: 10.1182/blood.2022016528

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Figure 4. — H9SB together repress the transcription of myeloid differentiation genes. (A) Heat maps of HOXA9 or SAFB signal (relative to the input) from CUT&RUN experiments in MOLM13 cells. The y-axis represents individual regions centered at SAFB-bound genomic peaks (± 5 kilobases). Regions were sorted based on the increasing distance to TSS. The relationship between coloring and signal intensity is shown at the (bottom). (B) Schematic representation of overlapping regions occupied by HOXA9 and SAFB. (C) A bar graph showing the genomic distribution of H9SB cobound regions. (D) Venn diagram shows an overlap between total differentially regulated genes in HOXA9- and SAFB-CRISPR knockout cells. Data analyzed using 3 independent replicates at P < .001; for upregulated or downregulated genes, the threshold was set to 1 ± 0.2. In the hypergeometric test, P < .00001 for all overlaps. (E) RNA sequencing (RNA-seq) volcano plot showing genes downregulated (left, blue) and genes upregulated (right, red) in HOXA9-CRISPR compared with nontargeting control MOLM13 cells (n = 3 in both conditions). (F) Volcano plot representing RNA-seq in SAFB-CRISPR MOLM13 cells, as described in panel E. (G) The Venn diagram shows the overlap between genes commonly dysregulated on HOXA9 and SAFB perturbation in MOLM13 cells (green) and genes that are linked to H9SB co-occupied genomic regions (orange). List of these genes provided in supplemental Table 4. Hypergeometric test P = 2.438e-13. (H) Heat map showing expression log₂ fold change in (HOXA9-Cr vs NT) or (SAFB-Cr vs NT) MOLM13 cells of those 1505 target genes. (I) Venn diagram shows the overlap between genes commonly regulated by H9SB in MOLM13 cells (blue) and genes that are regulated by HOXA9-MEIS1 in MOLM13 cells (orange). The overlap showed 99 genes shared by HOXA9/SAFB/MEIS1. A list of these genes is provided in supplemental Table 4. Hypergeometric test P < 3.782e-50. (J) Heat map showing expression log₂ fold change in (MEIS1-Cr vs NT) MOLM13 cells of those 99 target genes (HOXA9/MEIS1/SAFB). (K) Heat map showing expression log₂ fold change in (MEIS1-Cr vs NT) MOLM13 cells of those 151 target genes (HOXA9/MEIS1). (H,J,K) The relationship between the coloring and expression values is shown in the bar (right). The top 10 genes from up- or downregulated gene lists are shown in the plot. DEG, differentially expressed genes.

Figure 4. — H9SB together repress the transcription of myeloid differentiation genes. (A) Heat maps of HOXA9 or SAFB signal (relative to the input) from CUT&RUN experiments in MOLM13 cells. The y-axis represents individual regions centered at SAFB-bound genomic peaks (± 5 kilobases). Regions were sorted based on the increasing distance to TSS. The relationship between coloring and signal intensity is shown at the (bottom). (B) Schematic representation of overlapping regions occupied by HOXA9 and SAFB. (C) A bar graph showing the genomic distribution of H9SB cobound regions. (D) Venn diagram shows an overlap between total differentially regulated genes in HOXA9- and SAFB-CRISPR knockout cells. Data analyzed using 3 independent replicates at P < .001; for upregulated or downregulated genes, the threshold was set to 1 ± 0.2. In the hypergeometric test, P < .00001 for all overlaps. (E) RNA sequencing (RNA-seq) volcano plot showing genes downregulated (left, blue) and genes upregulated (right, red) in HOXA9-CRISPR compared with nontargeting control MOLM13 cells (n = 3 in both conditions). (F) Volcano plot representing RNA-seq in SAFB-CRISPR MOLM13 cells, as described in panel E. (G) The Venn diagram shows the overlap between genes commonly dysregulated on HOXA9 and SAFB perturbation in MOLM13 cells (green) and genes that are linked to H9SB co-occupied genomic regions (orange). List of these genes provided in supplemental Table 4. Hypergeometric test P = 2.438e-13. (H) Heat map showing expression log₂ fold change in (HOXA9-Cr vs NT) or (SAFB-Cr vs NT) MOLM13 cells of those 1505 target genes. (I) Venn diagram shows the overlap between genes commonly regulated by H9SB in MOLM13 cells (blue) and genes that are regulated by HOXA9-MEIS1 in MOLM13 cells (orange). The overlap showed 99 genes shared by HOXA9/SAFB/MEIS1. A list of these genes is provided in supplemental Table 4. Hypergeometric test P < 3.782e-50. (J) Heat map showing expression log₂ fold change in (MEIS1-Cr vs NT) MOLM13 cells of those 99 target genes (HOXA9/MEIS1/SAFB). (K) Heat map showing expression log₂ fold change in (MEIS1-Cr vs NT) MOLM13 cells of those 151 target genes (HOXA9/MEIS1). (H,J,K) The relationship between the coloring and expression values is shown in the bar (right). The top 10 genes from up- or downregulated gene lists are shown in the plot. DEG, differentially expressed genes.