H9SB forms a repressive complex on chromatin with NuRD and HP1γ. (A) Volcano plot displaying the label-free quantitative MS result of HOXA9 and SAFB immunoprecipitation by RIME in MOLM13 cells. The plot shows the log₂ ratio of averaged peptide MS intensities between HOXA9-IP vs IgG (left) or SAFB IP vs IgG (right) samples (x-axis), plotted against the negative log₁₀P values (y-axis) calculated across the triplicate data sets. Student t test, n = 3 technical replicates. The dashed black line marks 1.5 log FC. Chromatin-associated proteins enriched in HOXA9 or SAFB pulldowns are marked as red dots. The complete list of SAFB-HOXA9 commonly enriched proteins is given in supplemental Table 1. (B) Bar graph displays the enrichment (label-free quantification [LFQ] values) of NuRD complex members (MTA2 and GATAD2A) or heterochromatin protein HP1γ in HOXA9 or SAFB or IgG immunoprecipitated samples. The data shown here are intensities from LFQ values obtained via mass spectrometric analyses of all replicates for IgG (n = 3), SAFB (n = 3), and HOXA9 (n = 2) pull downs, average ± SD. The program does not plot for zero values. (C) Western blots showing HOXA9 interaction with SAFB, MTA2, and GATAD2A via coimmunoprecipitation of endogenous HOXA9 pulldown in MOLM13 cells. (D) Heat maps of HOXA9, SAFB, MTA2, GATAD2A, and HP1γ signal (relative to Input) on H9SB co-occupied genomic regions measured by the CUT&RUN method in MOLM13 cells. The y-axis represents individual regions centered at H9SB-bound genomic regions (± 10 kilobases). Regions were sorted based on the increasing distance to TSS. The relationship between coloring and signal intensity is shown in the bar (bottom of the plot). (E) Exemplar loci demonstrating co-occurrence of NuRD, HP1γ, and repressive histone modifications with H9SB that correlated with derepression of the associated genes upon H9SB perturbation are shown in the genome browser track on selected loci S100A8 in the Hg38 genome, obtained from CUT&RUN sequencing in MOLM13 cells. The upper 2 tracks show the transcripts signal obtained from RNA-seq in MOLM13 cells after HOXA9 (blue) or SAFB (pink) perturbation. Transcripts signal for HOXA9- or SAFB-CRISPR samples are shown relative to the nontargeting control (gray). (F) Venn diagram showing the overlap of high-confident NuRD (GATAD2A + MTA2) and HP1γ peaks with H9SB-cobound genomic regions in MOLM13 cells. The numbers represent the genomic regions. The differentiation-associated target genes of the H9SB-repressive complex that were also upregulated upon H9SB perturbation are highlighted; the top box shows gene targets of HOXA9/SAFB/NuRD; the lower box shows gene targets of HOXA9/SAFB/NuRD/HP1γ. (G) Heat maps show genomic coenrichment of HOXA9 and SAFB in primary AML cells (n = 5). NuRD and HP1γ co-occupancy as determined by the intersection of NuRD (MTA2 and GATAD2A) and Hp1γ peaks with H9SB cobound peaks obtained in the same AML cells by CUT&RUN. Because of limited material availability, only selected antibodies were used for samples 4 and 5 for CUT&RUN experiments. The relationship between coloring and signal intensity is shown in the bar at the side of the plot. (H) The genome browser track shows the peak colocalization of HOXA9, SAFB, and NuRD complex (MTA2 and GATAD2A); HP1γ on selected loci S100A8 (left) and CEBPD/SPIDR (right) in the Hg38 genome, obtained from CUT&RUN sequencing in primary AML cells. (I) The average signal of MTA2 (left) and HP1γ (right) (intensity on the y-axis as normalized read count) centered at H9SB-cobound regions determined using CUT&RUN in MOLM13 cells with or without SAFB knockdown using shRNA. (J) The average signal of H3K27Ac (left) and BRD4 (right) (intensity on the y-axis as normalized read count) centered at H9SB-cobound regions determined using CUT&RUN in MOLM13 cells with or without SAFB knockdown using shRNA. (K) The genome browser track shows the reduction in the enrichment of NuRD complex (MTA2 and GATAD2A) and HP1γ after SFAB knockdown in MOLM13 cells. Lower tracks show the gained enrichment of BRD4 and H3K27Ac after SFAB knockdown in MOLM13 cells. Selected loci CEBPD/SPIDR in the Hg38 genome, the signal obtained from CUT&RUN sequencing.