Fig 2: Sall4 is essential for primary tumor formation, but its depletion leads to increased micrometastasis.a Genetics scheme of the Tyr::NrasQ61K; Cdkn2a-/- transgenic mouse model spontaneously developing melanoma, which was crossed with inducible Tyr:CreERT2; Sall4lox/lox; R26R-LSL-GFP mice, allowing ablation of Sall4 from the melanocytic lineage upon tamoxifen administration. b Experimental scheme depicting how at 1 month of age the experimental mice from a undergo Cre-mediated recombination due to 5 consecutive i.p. tamoxifen injections. Hyperplasia gradually develops from birth of the pups. Primary tumors and metastasis were assessed at around 6 months of age. c Photographs of control and heterozygous (Sall4lox/wt; termed Sall4+/- cko) and homozygous (Sall4lox/lox; termed Sall4-/- cko) Sall4 cko animals (left panel). Hematoxylin and eosin staining of back skin from respective control, Sall4+/- cko or Sall4-/- cko animals (right panel), which has been repeated in independent experiments with similar results 7 times. Scale bars 500 µm. d Quantification of primary tumor numbers of control (Sall4+/+ and non-tamoxifen-injected animals), Sall4+/- cko and Sall4-/- cko animals. e Quantification of proliferation rate, assessed by immunohistochemistry (see Supplementary Fig. 3c), in primary tumors of control and Sall4+/- cko animals. f Binocular images of mouse lungs. The endogenous fluorescent GFP signal was imaged under a fluorescent binocular and for visualization inverted and set to black/white (B/W). Dark spots therefore represent inverted GFP+ spots set to B/W. Scale bars 500 µm. g Immunohistochemical stainings of mouse lung sections to verify melanoma identity of GFP+ spots by means of MITF expression. Scale bars top panel 100 µm, second and third panel 25 µm. h Quantification of GFP+ lung metastases of tamoxifen-injected control (Sall4+/+), Sall4+/- cko, and Sall4-/- cko animals. Metastasis score = 0 indicates <5 GFP+ lesions, 1 >5 lesions, 2 >20 lesions, 3 >50 lesions, 4 >100 lesions. In d, e, h, error bars represent mean ± SEM with N indicated in the respective figures. Two-sided t-tests between groups were performed for significance with p values =0.05 = n.s.; <0.05 = *; <0.01 = **, with Sall4-/- cko in d P = 0.0049; Sall4+/- cko in e P = 0.0456; Sall4+/- cko in h P = 0.0011; Sall4-/- cko in h P = 0.0129. Source data for d, e, h are provided as a Source Data file.

Fig 3: RNA sequencing of SALL4 knockdown reveals an invasiveness gene expression signature.a RNA seq row z-score heatmap of differentially expressed (DE) genes after 48 h SALL4 knockdown in the human cell line M010817. Cutoffs were set at Log2 ratio = 0.27 or =-0.27, p value < 0.05 and FDR < 0.05 and resulted in 1004 genes significantly upregulated and 1140 genes significantly downregulated. b Top 12 MetaCoreTM process networks of upregulated genes. Top ten most upregulated genes per process are listed and genes validated in additional cell lines are highlighted in bold. c Top 12 MetaCoreTM process networks of downregulated genes. Top ten most downregulated genes per process are listed and genes validated in additional cell lines are highlighted in bold. d Gene Set Enrichment Analysis (GSEA) of DE genes after SALL4 knockdown (log2 ratio-ranked) as in a with published genesets characterizing invasive versus proliferative melanoma cells40–42. e Log2 expression ratio heatmap of qRT-PCR-based gene expression analysis of specific genes of interest (normalized to PPIA and set relative to siCtrl-treated cells) in human melanoma cell lines (M010817: NRASQ61K; M070302: unknown mutational status; M150548: BRAFV600E; M121224: NRASQ61K and BRAFV600E; MM150536: BRAFV600E) after 48 h treatment with two different siRNAs targeting SALL4. Two-sided t-tests were performed with N = 3 and p values = 0.05 = n.s. Source data for e are provided as a Source Data file.

Fig 4: Sall4 is upregulated in hyperplastic melanocytes.a Experimental scheme of isolated melanocytes for RNA sequencing either from Tyr::CreERT2; R26R-LSL-tdTomato wild-type mice or Tyr::CreERT2; R26R-LSL-tdTomato; Tyr::NrasQ61K; Cdkn2a-/- mice with hyperplastic, melanoma-prone skin. At the age of 4 months, the mice were injected on 5 consecutive days with 80 µg g-1 body weight tamoxifen to induce tdTomato expression in melanocytes. The hair growth cycle in wild-type animals was synchronized by dorsal hair plucking. Melanocytes were isolated from mouse back skin of wild-type and melanoma model mice (3–6 mice per sample) and FACS sorted for cKit (staining) and tdTomato (endogenous). b Row z-score heatmap of normalized counts from RNA sequencing as illustrated in a. Visualized are the top 20 most upregulated genes comparing hyperplastic (Tyr::NrasQ61K; Cdkn2a-/-) to wild-type melanocytes. Genes are ordered top to bottom with genes with highest LogFC values on top. Cutoffs were set at p values < 0.05 and FDR < 0.05. Sall4 (LogFC value = 9.59) represents the only and hence highest upregulated transcription factor within those top 20 upregulated genes. The complete list of all differentially expressed genes can be found under Supplementary Data 1. c Immunohistochemical staining of wild-type melanocytes in murine hair follicles (left panel) and melanoma-prone melanocytes in hyperplastic Tyr::NrasQ61K; Cdkn2a-/- mouse skin (right panel). Arrowheads point towards wild-type melanocytes with undetectable Sall4 expression; arrows point towards hyperplastic and rare wild-type melanocytes with detectable Sall4 protein. Experiment has been repeated independently three times with similar results. Scale bars 25 µm.

Fig 5: SALL4 can regulate invasiveness genes through an epigenetic mechanism.a Read density heatmap of gained and lost H3K27ac ChIP sequencing peaks (±2 kb) upon SALL4 knockdown (left panels) and average read distribution of gained and lost H3K27ac peaks within ±5 kb from peak center (right panels). b Distribution of the gained and lost H3K27ac peaks in different genetic regions. TTS: transcription termination site, TSS: transcription start site, UTR: untranslated region. c–g Representative tracks of invasiveness genes with significantly gained (red bars; highlighted by red, dashed lines) H3K27ac peaks in siSALL4 over siCtrl (no significantly lost H3K27ac peaks were detected for the visualized gene tracks). h Read density heatmap of differential H3K27ac ChIP-seq peaks (within 10 kb) in siSALL4 over siCtrl at direct target genes of SALL4-HDAC2 (CUT&RUN peaks with at least 3 of 4 SALL4/HDAC2 antibodies). i Significantly upregulated genes after SALL4 knockdown (Fig. 4a, b) were overlaid with those genes that have significantly gained H3K27ac marks -15/+10 kb of TSS (a, ocher panel). This resulted in 261 genes with activating chromatin marks that were at the same time upregulated after SALL4 knockdown. j MetaCore™ Process Network enrichment of the 261 genes from i. The top 12 most significant processes are indicated with the specific genes of each process listed in red. k Gene Set Enrichment Analysis (GSEA) of the combined 261 upregulated (i) and 137 downregulated genes (Supplementary Fig. 17a) with differential acetylation after SALL4 knockdown (ranked according to log2 expression ratio of RNA seq results) with the previously published melanoma programs of Verfaillie and colleagues (2015). Source data for b are provided as a Source Data file.