Genome-wide binding patterns for Prmt5. (A) Genome Browser tracks showing Prmt5 ChIP-seq at day 0, day 1, and day 2 of 3T3-L1 differentiation. Significant ChIP peaks determined by MACS2 are indicated below the genome browser tracks in gray for the Abhd2 promoter and (B) the Pdgfra promoter. (C) Gene annotation of all significant Prmt5 peaks at day 0. (D) Top biological processes identified from Gene Ontology analysis of significant day 0 Prmt5 peaks. (E) The HOMER motif discovery algorithm identified Runx1 and Nrf1 as the top motifs present within Prmt5 peaks. (F) Prmt5 ChIP-Seq tag density plots over the gene body ± 2 kb displayed as heat maps, order determined by k-means clustering. Expression data displayed as log2TPM (transcripts per million mapped reads) for day 0, day 1, day 2, and day 7 of 3T3-L1 differentiation.

Association between histone modifications and Prmt5 binding. (A) Average tag density plots for Prmt5, H3K27ac, H3K4me3, and H3K27me3 ChIP-Seq, in relation to Prmt5 peak centers. (B) Tag density plots for day 0 and day 2 ChIP-Seq for Prmt5, H3K27ac, H3K4me3, and H3K27me3, plotted +/- 2kb of Prmt5 peak centers. (C) Genome Browser tracks showing Prmt5 ChIP-seq at day 0, day 1, and day 2 of 3T3-L1 differentiation, along with the corresponding tracks for H3K27ac, H3K4me3, H3K27me3 ChIP-Seq at day 0 and day 2 of 3T3-L1 differentiation. Significant ChIP peaks determined by MACS2 are indicated below the genome browser tracks. (D) bedtools intersect was used to calculate significant peak overlap between Prmt5 ChIP-Seq and histone modification ChIP-Seqs at day 0 and day 2 of differentiation.

Prmt5-dependent gene expression changes. (A) Genome Browser tracks showing Prmt5 ChIP-seq at day 0, day 1, and day 2 of 3T3-L1 differentiation. Significant ChIP peaks determined by MACS2 are immediately below the genome browser tracks in gray. (B) Real- time RT-qPCR analysis of Prmt5-bound genes in 3T3-L1 cells transfected with scrambled or Prmt5-targeting siRNA. Cells were collected at time points between D0 and D4 of differentiation. Data represent averages from 3-6 replicates and are presented as means +/- standard deviations (SD). The level of expression in scrambled day 0 samples were set to a value of 1 for genes Ptn, Thbs2, and Pdgfra, and other values are relative to that sample value. *, P<0.05; **, P<0.001; ***, P< 0.0001 (versus scrambled by Student’s t test). (C) Volcano plots displaying differentially expressed genes between scrambled and Prmt5 siRNA transfected 3T3-L1 cells harvested at day 0 of differentiation. The y-axis data correspond to the mean log10 expression levels (padjusted-values). The red dots represent significantly upregulated genes (padj < 0.05, log2foldchange > 0.5) and blue dots represent significantly downregulated genes (padj < 0.05, log2foldchange < −0.5). The black dots represent genes whose differential expression levels did not reach statistical significance. Flanking the volcano plot are Gene Ontology Biological processes charts for the top upregulated and downregulated genes. (D) Differential expression of Prmt5-bound Prmt5- dependent genes ranked by log2foldchange displayed on the right, with corresponding day 0 Prmt5 ChIP-Seq tag density plots over the gene body ± 2 kb displayed to the left.

Co-localization of Prmt5 with loop anchors and regulators of genome structure. (A) Genome Browser tracks for showing Prmt5 ChIP-seq at day 0 of 3T3-L1 differentiation, along with the corresponding tracks from H3K27ac, H3K4me3, Med1, Smc1, and Ctcf ChIP- Seqs with D0 Promoter Capture Hi-C displayed above. Significant ChIP peaks determined by MACS2 are indicated below the genome browser tracks. (B) Average tag density plots for Prmt5, Ctcf, Med1, and Smc1 ChIP-Seq, in relation to Prmt5 peak centers. (C) Tag density plots for day 0 and day 2 ChIP-Seq for Prmt5, Med1, Smc1, and Ctcf, plotted +/- 2kb of Prmt5 peak centers. (D) bedtools intersect was used to calculate significant peak overlap between Prmt5 ChIP-Seq and Med1, Smc1, and Ctcf ChIP-Seqs at day 0 and day 2 of differentiation. (E) Average tag density plots showing Smc1, Med1 and Prmt5 binding at DNA loop anchors +/-2kb, in relation to Ctcf binding.

Effect of Prmt5 knockdown on TADs and TAD boundaries. (A) Compartment profiles (the first principal components) of scrambled siRNA and Prmt5 siRNA data for Chr 1. The A-type (open) compartments are shown in green, and the B-type (closed) compartments are shown in red. Below this, contact heatmaps are displayed. (B) Insulation plot profiles at 10-kb intervals are displayed genomic loci around genes Cdk14 and Myc for scrambled siRNA and Prmt5 siRNA transfected samples. Below that, a genome browser track for the same locus is displayed showing Prmt5 and Ctcf ChIP-seq peaks at day 0 of 3T3-L1 differentiation. High-confidence D0 3T3-L1 looping event interactions are depicted as purple lines below this plot. (C) Insulation scores were plotted for all Prmt5-bound TAD boundaries for day 0 scrambled siRNA and Prmt5 siRNA transfected samples.