Fig 2: Union peak set comprehensiveness and accuracy. (A) Fingerprint plot for aligned sequence files. Negligible separation between the SALL4 and control curves indicates poor enrichment in the SALL4 samples, an experiment typically considered as having failed. (B) Upset plot describing the distribution of peaks observed by each peak caller. The left histogram represents the total number of called peaks per caller. The top histograms represent the size of the sub-sets in question. The connected circles represent highlighted overlap. (C) Venn diagram showing the overlapping number of peaks between the SALL4 union ChIP-seq dataset and the Cut&Run dataset, both of which were done on SNU-398 cells. (D) The motif sequence used for the directed motif search in the SALL4 ChIP-seq union set, which was found in 55.2% of the union set. (E) The STREME de novo motif search for the SALL4 union peak set identified the AT-rich binding motif as the second result. (F) The HOMER de novo motif search for the SALL4 union peak set identified the AT-rich binding motif as the third result. (G) The STREME identified motif (shown in D) was found centrally enriched in the union peak set as compared to background sequences; an accepted indicator of a valid motif result. (H) Venn diagram depicting the degree of overlap in the number of genes between the SALL4 ChIP-seq union peak set, the SALL4 Cut&Run peak set and the SALL4 KD DEG list.

Fig 3: RNA-seq Data Revealed Direct SALL4 Target Genes(A) Volcano plot showing genes that are down- or upregulated significantly after SALL4 KD with log2 fold change (FC) represented on the x axis; red circles denote differentially expressed genes with false discovery rate (FDR) of <0.05.(B) Number of differentially expressed genes after SALL4 KD (2,695) as well as those with annotated SALL4 peaks nearby (430) (Tables S3 and S4).(C) Bar graph representing number of up- and downregulated genes after SALL4 KD (Table S3) and their Gene Ontology (GO) molecular pathway analysis focusing on GO 0140110.(D) Volcano plot from (A) with genes encoding KDM proteins labeled; green circles denote genes containing SALL4 CUT&RUN peaks; the size of the circles corresponds to log2FC in expression.(E) Quantitative real-time PCR analysis of two SALL4 direct targets 40 h after SALL4 KD, summarized from either 3 or 4 independent experiments (primer sequences found in Table S5); SCR, scrambled shRNA control.(F) EMSA showing that SALL4 binds KDM3A promoter region containing the WT AT-rich motif but not the mutated sequence (Key Resources Table)

Fig 4: SALL4 CUT&RUN Showing Its Binding Genome wide in Liver Cancer Cells(A) Representative genomic tracks of three SALL4 CUT&RUN replicates (rep) and their isotype rabbit IgG control experiments; scale is 0–25.(B) The genomic distribution of SALL4 CUT&RUN peaks.(C) The top five HOMER motifs from de novo analysis of three CUT&RUN reps with their respective p values, resulting in the two composite motifs shown on right.(D) Bar graph showing the percentage of peaks containing Motif 2 in de novo (black bars) and direct (gray bars) analyses from SALL4 CUT&RUN reps.

Fig 5: Knockdown of SALL4 leads to increased COL1A and COLXII deposition. (a, b) Immunofluorescence analysis of Collagen XII 21 days post injury after mismatch morpholino or Sall4 morpholino treatment. (c) Quantification of mean fluorescence intensity of collagen XII at 21 days post injury after morpholino treatment. (d) Relative expression of COL12A1 to 18S at 14 days post injury after morpholino treatment. (e, f) Immunofluorescence analysis of Collagen I 21 days post injury, SALL4 depletion also leads to increases Collagen I protein and mRNA levels (g, h). (c–h) significance determined by an unpaired t-test. Error bars are s.d. unless noted. **=P<0.01, ***P<0.001. For 21 days post injury, Replicas=2 with 10 animals total per condition. Scale bar = 50 µm.