Journal: bioRxiv

Article Title: Tetramerisation governs SALL transcription factor function in development and disease

doi: 10.1101/2025.10.27.684836

Figure Lengend Snippet: (A) Protein alignment of mouse SALL proteins and their functional domains, including the NuRD-interaction domain (NID), glutamine-rich interaction domain (QID), C2H2 zinc-finger clusters (ZFC1-4) and the nuclear localisation signal (NLS) mapped for SALL4. The domain missing in the SALL4b isoform is bracketed. (B) Cartoon of the experimental design for mapping the QID in SALL4. 3xFlag-tagged SALL4 constructs were used for C-terminal deletions (Figure S1B,C), followed by 3xFlag-GFP-tagged SALL4 construct for N-terminal deletions (Figure S1D). The N-terminal deletions were also fused to the SALL4 NLS to ensure nuclear localisation. The sequence of the QID is shown below, with residue numbers and the minimal domain highlighted. (C) Western blot analysis of co-IPed endogenous SALL1 from transfections of N- and C-terminal deletions as well as Flag-tagged SALL4 full-length into E14/T ESCs. Asterisks indicate IgG heavy chain of the mouse anti-Flag antibody used for IP. Quantified (n=3) in Figure S1E. (D) Diagram of SALL4 full-length (SALL4a) and its functional domains as well as the tested truncation constructs. Ticks indicate that a construct co-IPed endogenous SALL1 and SALL4 while crosses indicate a loss of interaction. The minimal domain construct (205-237) is highlighted in bold. (E) SEC-MALS analysis of recombinant SALL4 QID (192-237) peptide. Green line indicates the UV absorbance at 280nm of the protein elution profile form the SEC while the orange line shows the molecular weight that was measured by the MALS. Grey dashed lines indicate molecular weights expected for monomer (1x), dimer (2x), trimer (3x) and tetramer (4x) assemblies. (F) In silico modelling of the SALL4 QID (205-237) tetramer. All 5 models are aligned to each other and coloured from N-to C-term (blue to red). Residue L221 and its sidechain is highlighted in green.

Article Snippet: 500 μL Protein G Dynabeads (Invitrogen ref. 10003D) were washed once in lysis buffer (50 mM Tris-HCl pH 8, 150 mM KCl, 0.1% (v/v) Triton X-100) and incubated with 50 μg SALL4 antibody (Santa Cruz ref. sc-101147) in 2 mL lysis buffer for one hour at room temperature on a rotating wheel.

Techniques: Functional Assay, Construct, Sequencing, Residue, Western Blot, Transfection, Recombinant, Molecular Weight, In Silico

Journal: bioRxiv

Article Title: Tetramerisation governs SALL transcription factor function in development and disease

doi: 10.1101/2025.10.27.684836

Figure Lengend Snippet: (A) Diagram of SALL4 with its functional domains and the antibody epitope. Mutations designed to disrupt the QID are highlighted in red. The CRISPR strategy used to generate the cell lines is detailed in Figure S3A. (B) Western blot analysis of co-IPed SALL1 from a SALL4 IP with the polyclonal rabbit anti-SALL4 antibody in QIDΔ, QIDmut and control cell lines. (C) Venn diagram showing the overlap of genes that are deregulated in S4KO (clones C1+C2), QIDΔ and QIDmut cell lines compared to WT ESCs (padj>0.05). (D) Plot showing the Pearson correlation values (r) of the 2,938 shared deregulated genes between all mutant lines. One dot plot (with one dot representing the log 2 fold change of one gene vs WT in both genotypes) of the S4KO – QIDmut comparison is shown illustrating that many genes are deregulated in the same way in both genotypes (for more details see Figure S3E,F). (E) Workflow of the spontaneous differentiation experiment using piggyBac cell lines, where Sall4 WT (blue) and QIDΔ and QIDmut (red) cDNAs were integrated into S4KO ESCs. Addition of doxycycline (+Dox) to the culture medium switches transgene expression on, leading to overexpression of SALL4. (F) Representative wells of stem cell colonies stained by alkaline phosphatase staining of all the different piggyBac cell lines after 96 h in N2B27+Dox medium. The quantification on the right shows the mean of the number of AP-positive colonies (AP+) found in each genotype and normalised to WT (positive control) for three biological replicates (individual data points). The error bars indicate the standard deviation (SD). (G) Diagram showing the results of genotyping from pups (P21) following crosses of heterozygous (HET) QIDmut animals. No homozygous (HOM) animals were recovered, indicating complete embryonic lethality. The bar graph below shows quantification and statistical comparison with expected Mendelian distribution using the Fisher’s exact test (p-value< 0.0001, ****). H) Diagram and quantification of WT by HET crosses is shown illustrating the QIDmut heterozygous animals are also born at a sub-Mendelian ratio (Fisher’s exact test, **** = p-value < 0.0001).

Article Snippet: 500 μL Protein G Dynabeads (Invitrogen ref. 10003D) were washed once in lysis buffer (50 mM Tris-HCl pH 8, 150 mM KCl, 0.1% (v/v) Triton X-100) and incubated with 50 μg SALL4 antibody (Santa Cruz ref. sc-101147) in 2 mL lysis buffer for one hour at room temperature on a rotating wheel.

Techniques: Functional Assay, CRISPR, Western Blot, Control, Clone Assay, Mutagenesis, Comparison, Expressing, Over Expression, Staining, Positive Control, Standard Deviation

Journal: bioRxiv

Article Title: Tetramerisation governs SALL transcription factor function in development and disease

doi: 10.1101/2025.10.27.684836

Figure Lengend Snippet: (A) Representative nuclei of the different genetically modified ES cell lines after co-immunofluorescence of SALL4 and SALL1. DNA was stained with DAPI. Scale bar = 5 µm. (B) Quantification of all immunofluorescence microscopy experiments, as shown in panel A. All cells were assessed for having SALL4-positive foci or not and results were expressed as ratios over the total cell numbers (n, shown in brackets next to each genotype). Individual data points indicate independent replicate experiment with error bars representing the SD. (C) Heatmaps and average plots of SALL4 CUT&RUN signal over peak regions (17,557 peaks observed in WT cells) in ESC lines from different genotypes. The Reads Per Kilobase per Million mapped reads (RPKM) are all normalised to a E. coli DNA spike-in that was added to each sample. The shaded area in the plots corresponds to the SD of peak signal across all analysed loci. (D) SPR analysis of several SALL4 QID-ZFC4 fusion proteins binding to a DNA probe containing a single ATATTA motif. Mutations in functional domains are highlighted in red and for each construct a K D calculated from a kinetic model is given, unless no binding was detected (N/A).

Article Snippet: 500 μL Protein G Dynabeads (Invitrogen ref. 10003D) were washed once in lysis buffer (50 mM Tris-HCl pH 8, 150 mM KCl, 0.1% (v/v) Triton X-100) and incubated with 50 μg SALL4 antibody (Santa Cruz ref. sc-101147) in 2 mL lysis buffer for one hour at room temperature on a rotating wheel.

Techniques: Genetically Modified, Immunofluorescence, Staining, Microscopy, Binding Assay, Functional Assay, Construct

Journal: bioRxiv

Article Title: Tetramerisation governs SALL transcription factor function in development and disease

doi: 10.1101/2025.10.27.684836

Figure Lengend Snippet: (A) Western blot analysis of co-IPed MTA1 from a SALL4 IPs with the polyclonal rabbit anti-SALL4 antibody in QIDΔ /mut and control cell lines. (B) Quantification of the co-IP of MTA1 with SALL4 WT or QIDΔ /mut protein, as shown in panel A. The data was acquired in three independent replicates (data points), of which the mean is shown. Error bars represent the SD. (C, D, E) Volcano plot showing all proteins detected by mass spectrometry analysis in SALL4 IPs from WT (C), SALL4 QIDΔ (D) and SALL4 QIDmut (E) ESC lines. Relative enrichment (Log2 Fold Change) and statistical significance (-log(p-value)) were calculated in comparison to S4KO negative control samples. SALL proteins are highlighted in green and NuRD co-repressor complex subunits in yellow. (F, G) Stoichiometries of SALL proteins (F) and NuRD co-repressor complex subunits (G) in SALL4 IPs from WT (blue), QIDΔ (red) and QIDmut (salmon) cell lines, as assessed by mass spectrometry analysis. For each protein, label-free absolute quantification (iBAQ values) was obtained and normalised to the corresponding IPed SALL4 levels. Experiments were performed in technical triplicates (individual data points) and error bars show the SD.

Article Snippet: 500 μL Protein G Dynabeads (Invitrogen ref. 10003D) were washed once in lysis buffer (50 mM Tris-HCl pH 8, 150 mM KCl, 0.1% (v/v) Triton X-100) and incubated with 50 μg SALL4 antibody (Santa Cruz ref. sc-101147) in 2 mL lysis buffer for one hour at room temperature on a rotating wheel.

Techniques: Western Blot, Control, Co-Immunoprecipitation Assay, Mass Spectrometry, Comparison, Negative Control, Quantitative Proteomics

Journal: bioRxiv

Article Title: Tetramerisation governs SALL transcription factor function in development and disease

doi: 10.1101/2025.10.27.684836

Figure Lengend Snippet: (A) Diagram of SALL1 with its functional domains as well as the location of TBS-causing non-sense and frameshift mutations in red (Table S1) and neutral mutations found in the general population (gnomAD ) in blue. Of note, missense mutations are not shown in this diagram. The modelled 826C>T mutation (S1N) is highlighted in green and with a star. Lines indicate the first mutated residue with missense tails not being shown. Below the disease-causing variants, a grey brackets indicates the location of the previously defined hotspot region . (B) Depiction of SALL4-mScarlet3 fusion protein expressed by genetically engineered ESCs and the human SALL1 constructs that were transiently transfected with C-terminal GFP 3x-Flag tags. The mutation introduced within SALL1 QID (I241P, homologous to mouse SALL4 L221P) disrupts homo- and heteromultimerisation with SALL proteins (see Figure S5C). (C) Representative pictures of live cells expressing endogenously SALL4-mScarlet3 cells and transfected with GFP-tagged constructs: GFP, S1N or S1Nmut All samples were analysed with the same brightness/contrast indicating different transfection efficiencies. In the SALL4 mScarlet3 channel, white arrows highlight transfected cells. (D) Zoom on single nuclei, either transfected with S1N or S1Nmut. The cartoon in the middle depicts how the automated segmentation and quantification programme measured area-normalised SALL4 signal in foci and nucleoplasm. Relative enrichment was quantified as a fluorescence ratio, as shown on the right. (E) Quantification of all live-cell imaging data from (n=5) replicate experiments. Only transfected cells were analysed with each data point representing SALL4 fluorescence ratio (see panel D) in a single cell and the black line representing the median. Statistical analysis was performed using an ordinary one-way ANOVA test with a p-value lower than 0.05 being considered statistically significant (**** p< 0.0001). (F) Venn diagram showing the overlap of genes that are deregulated WT cells transfected with S1N, S1Nmut or GFP. The 7,000 genes that are only deregulated in S1N (not GFP or S1Nmut) are then overlapped with genes deregulated in S4KO (clones C1+C2). All deregulated genes are obtained from comparisons to WT ESCs (padj>0.05). (G) Plot showing the Pearson correlation values (r) of the 4,317 shared deregulated genes between all cell lines. Additionally, the dot plot (with one dot representing the log 2 fold change of one gene vs WT in both genotypes) of the S1N – S4KO comparison is shown illustrating that many genes are deregulated in the same way (for more details see Figure S5F).

Article Snippet: 500 μL Protein G Dynabeads (Invitrogen ref. 10003D) were washed once in lysis buffer (50 mM Tris-HCl pH 8, 150 mM KCl, 0.1% (v/v) Triton X-100) and incubated with 50 μg SALL4 antibody (Santa Cruz ref. sc-101147) in 2 mL lysis buffer for one hour at room temperature on a rotating wheel.

Techniques: Functional Assay, Mutagenesis, Residue, Construct, Transfection, Expressing, Fluorescence, Live Cell Imaging, Clone Assay, Comparison

Journal: bioRxiv

Article Title: Tetramerisation governs SALL transcription factor function in development and disease

doi: 10.1101/2025.10.27.684836

Figure Lengend Snippet:

Article Snippet: 500 μL Protein G Dynabeads (Invitrogen ref. 10003D) were washed once in lysis buffer (50 mM Tris-HCl pH 8, 150 mM KCl, 0.1% (v/v) Triton X-100) and incubated with 50 μg SALL4 antibody (Santa Cruz ref. sc-101147) in 2 mL lysis buffer for one hour at room temperature on a rotating wheel.

Techniques: Functional Assay, Sequencing, Variant Assay