(A) Western blot confirmation of pull-down of MEIS1 from chromatin in CWR22Rv1 LV-MEIS1 and HOXB13ko-LV-MEIS1 cell lines (Top) and heatmap of read density profiles from MEIS1 ChIP-seq for ±2 kb of the peak center (PC) called by MACS2 (Bottom). Graphs are separated vertically into peaks called from LV-MEIS1 samples vs. input DNA (LV-MEIS1 peaks), where both MEIS1 and HOXB13 are expressed, and peaks called from HOXB13ko-LV-MEIS1 samples vs. input DNA (HOXB13ko-LV-MEIS1 peaks), where only MEIS1 is present and HOXB13 has been knocked-out. (B) Spaced Motif (SpaMo) analysis querying MEIS1 as the primary motif demonstrates an inferred secondary motif matching HOXB13 has a conserved spacing from the MEIS1 motif that is most significant at 1 bp away, downstream and on the opposite DNA strand in MEIS1 peaks from LV-MEIS1 ChIP-seq (top). In MEIS1 peaks from HOXB13ko-LV-MEIS1 ChIP-seq, SpaMo analysis identifies an inferred motif matching HOXA9 as the only HOX motif with a significant conservation of spacing with the MEIS1 motif and is most significant at 3 bp away on either DNA strand regardless of up or downstream (bottom). (C) Venn diagram demonstrating the number of genes with differential expression (fold-change >±1.5, FDR < 0.05) in CWR22Rv1-LV-MEIS1 vs. CWR22Rv1-Control cells from RNA-seq of these lines (gray), overlapped with the number of genes that MEIS1 peaks were annotated to using HOMER in either LV-MEIS1 (blue) or HOXB13ko-LV-MEIS1 (orange) ChIP-seq. The 157 genes (red) represent prioritized MEIS effector genes, since they are bound and differentially expressed only when HOXB13 is present. (D) Top 10 significantly enriched pathways from KEGG pathway enrichment analysis using the list of 157 genes identified in (C). (E) Volcano plot of gene expression Log2(fold-change) vs. significance (FDR) in CWR22Rv1 LV-MEIS1 vs. control cells. Dashed lines indicate thresholds for fold-change >2 or<2 and for FDR value <0.01. Highlighted gene symbols indicate genes that: 1) are within the ‘proteoglycans in cancer’ pathway curated by KEGG, 2) are annotated as targets in the MEIS1 ChIP-seq from LV-MEIS1 cells, and 3) have significant differential expression in LV-MEIS1 vs. control RNA-seq (fold-change >±1.5, FDR < 0.05). Gray dots indicate genes with no significant differential expression; green dots indicate genes with fold-change >±2 but FDR > 0.01; blue dots indicate genes with FDR < 0.01 but fold-change <±2; and red dots indicate genes with both FDR < 0.01 and fold-change >±2. (F) Integrated Genome Browser tracks of MEIS1 ChIP in the presence (WT-HOXB13, blue) and absence of HOXB13 (HOXB13-KO, red) at the DCN (2 regions, DCN #1 and DCN #2), LUM, and TFGBR3 loci. (G) ChIP-qPCR of MEIS1 binding using site-specific genomic primers against DCN, LUM, and TGFBR3 loci. MEIS1 binding is significantly diminished when HOXB13 is deleted (HOXB13KO; * indicates p<0.05). (H) Western blot analysis of key genes with significant differential expression in RNA-seq between CWR22Rv1-LV-MEIS1 and control. DCN, TGFBR3, and LUM are direct targets of MEIS1 from ChIP-seq data and ITGB1 is a downstream target of LUM. (I) Gene set enrichment analysis (GSEA) from RNA-seq between CWR22Rv1-LV-MEIS1 and control cells on the oncogenic signatures collection from MSigDB. Enrichment is observed in CWR22Rv-LV-MEIS1 cells for genes known to be suppressed by: active TGFβ signaling (TGFB_UP.V1_DN, NES: 1.43, FDR: 0.039), active EGFR signaling (EGFR_UP.V1_DN, NES: 1.42, FDR: 0.036), active WNT signaling (WNT_UP.V1_DN, NES:1.36, FDR 0.057), and active c-MYC signaling (MYC_UP.V1_DN, NES: 1.43, FDR: 0.045). (J) Heatmaps of expression and unsupervised clustering of RNA-seq from CWR22Rv1 control (purple), LV-MEIS1 (blue), HOXB13ko (green), and HOXB13ko-LV-MEIS1 (orange) cells for all genes in the leading edge of enrichment for each gene set in (G). See also Figure 5—figure supplement 1 and Supplementary files 1–6.