Fig 1.
Broad transcriptional changes in SMC1A-R586W mESCs.
A Lollipop plot of protein sequence mutations observed in SMC1A in the COSMIC cancer genome atlas. B Images of wildtype and two independently derived SMC1A-R586W ES clones growing in complete media with LIF. Scale bar, 500μm. C-D MA plots comparing RNA-seq data from R586W clone (cl) 1 and cl2 to wildtype mESCs. n = 3 biological replicates for all groups. Red, adjusted p<0.1 as determined by DESeq2. E Venn diagram showing overlap of differentially-expressed (DE) genes between two R586W ES clones. F Correlation plot of 5,984 common differentially expressed genes in R586W cl1 and R586W cl2. G Gene ontology analysis using upregulated and downregulated DE genes shared by R586W cl1 and cl2. H Expression of pluripotency genes and I AML tumor suppressors and oncogenes in R586W cl1 and cl2.
Fig 2.
Active histone markings at promoters and enhancers are similar between wildtype and SMC1A-R586W mESCs.
A UCSC Genome Browser coverage tracks showing K4me3 and K27ac distribution at the Pou5f1 (Oct4) locus in mESCs. CTCF data were previously published [68]. B Overlap of K4me3-marked TSSs, C K27ac-marked TSSs, and D K27me3-marked TSSs in wildtype and SMC1A-R586W mESCs. E K4me3 signal at the TSSs of genes downregulated and F upregulated in SMC1A-R586W mESCs. G K27ac signal at the TSSs of genes downregulated and H upregulated in SMC1A-R586W mESCs. I K27me3 signal at the TSSs of genes downregulated and J upregulated in SMC1A-R586W mESCs. K K4me3 signal at TSSs of Super-enhancer associated genes. L K27ac signal at Super-enhancer Domains. M K27me3 signal at Polycomb Domains identified by Dowen et al. (2014). K4me3 and K27me3 data represents merge of two biological replicates for each group. K27ac data shown represents merge of three biological replicates of wildtype and two each of SMC1A-R586W clone 1 and SMC1A-R586W clone 2.
Fig 3.
Cohesin is selectively depleted at promoters and enhancers in SMC1A-R586W mESCs.
A RAD21 enrichment measured by ChIP-seq at the HoxA locus in wildtype and R586W cl1 and cl2. B Heatmap representation of RAD21 and CTCF enrichment, with k-means clustering. Rows are ranked in descending order based on WT RAD21 signal (column 1). C Average signal plots depicting RAD21 enrichment at CTCF sites, a merged list of K27ac peaks (enhancers), and a merged list of K4me3-marked TSSs (promoters) in wildtype and R586W cl1 and cl2. D Overlap of called peaks in wildtype and R586W cl1 and cl2. E Distribution of RAD21 peaks across the given genomic sites in wildtype and R586W mESCs. F Distribution of RAD21 peaks common to both R586W clones but not wildtype mESCs. G Differential analysis using Diffbind of RAD21 signal at peaks common to wildtype and both R586W clones. Red, p-adj<0.1. H Distribution across given genomic sites of peaks with increased or decreased signal in R586W mESCs as measured by Diffbind. For analyses shown, data from 2 biological replicates for each of wildtype, R586W cl1, and R586W cl2 were merged. I Representative western blots showing the levels of indicated proteins in soluble (Sol.) and chromatin-bound (CB) fractions. J Amounts of chromatin-bound protein expressed as a fraction of total (chromatin-bound + soluble) signal for WT, R586W cl1, and R586W cl2. n = 3 biological replicates. All differences not significant as measured by two-way ANOVA. Numerical data are presented in S6 Table.
Fig 4.
Altered chromatin looping in SMC1A-R586W mESCs.
A Hi-C heatmaps at 10kb resolution centered around the HoxA locus in wildtype and R586W mESCs. Yellow lines indicate positions of contact domains called by Arrowhead in each. B Distribution of contact domains by size in WT and SMC1A-R586W mESCs. C Overlap of domain positions in WT and SMC1A-R586W mESCs. D Distribution of loops by size in WT and SMC1A-R586W mESCs. E Left, overlap of loop positions between wildtype and R586W mESCs as measured by Juicer Tools CompareLists routine. Right, overlap of loop positions as measured by Juicer Tools HICCUPsDiff routine F Aggregate peak analysis (APA) in wildtype and R586W mESCs, at wildtype loops (top row) or R586W loops (bottom row). p<0.05 as measured by Kruskal-Wallis test with Dunn’s multiple comparisons correction for wildtype vs R586W when APA is determined at wildtype loop calls (top row), and wildtype vs R586W when APA is determined at R586W loop calls (bottom row). G Distribution of contact distances with K4me3-marked TSSs, H CTCF sites, or I TSS-distal K27ac sites in WT and SMC1A-R586W mESCs. Line is mean of two replicates in each group. Statistical significance tested using Kolmogorov-Smirnoff test. J Binned interactions under 500kb with given genomic feature. Bars represent mean±s.d. for each group. Differences n.s. as measured by two-way ANOVA.
Fig 5.
Physical and functional interactions between NIPBL and Cohesin in SMC1A-R586W mESCs.
A Co-immunoprecipitation experiments were performed in wildtype and SMC1A-R586W mESCs with the indicated antibodies. Western blots shown are representative of 3 independent biological replicates. B Log2-transformed cell counts at the given time points in wildtype and SMC1A-R586W mESCs transfected with the indicated siRNA. n = 4 biological replicates for siControl and siNipbl, and N = 2 biological replicates for siWapl. C Ratio of siNipbl to siControl proliferation in wildtype and SMC1A-R586W mESCs. *, p<0.05 as measured by two-way ANOVA between WT and R586W cl1. #, p<0.05 as measured by two-way ANOVA between WT and R586W cl2. D Transcript levels in siNipbl and siWapl transfected mESCs. n = 4 biological replicates for siNipbl, n = 2 for WT and R586W clone 1 siWapl, and n = 1 for R586W clone 2 siWapl. p<0.05 as measured by two-way ANOVA. E Log2-transformed cell counts at the given time points in wildtype and SMC1A-R586W mESCs transfected with the indicated siRNA. n = 2 biological replicate for siControl and siPds5a+Pds5b. F Ratio of siPds5a+Pds5b to siControl proliferation in wildtype and SMC1A-R586W mESCs. n = 2 biological replicates. All differences not significant as measured by two-way ANOVA. G Transcript levels in siPds5a+Pds5b knockdown mESCs expressed relative to siControl as measured by RT-qPCR. n = 2 biological replicates for all groups. Numerical data are presented in S6 Table.