Figure 3: Usp16 binds to TSS and positively correlates with gene expression and inversely correlates with H2A deubiquitination levels.

(a) Scatter plot of gene expression levels in Usp16^+/+ and Usp16^−/− ESCs as determined by RNA-seq. The fragments per kilobase of exon model per million mapped reads (FPKM) was calculated for each transcript using Cufflinks and transformed to Log2 (FPKM) as relative RNA level. Genes that are significantly deregulated (Log₂ [Fold change]>2) in Usp16^−/− ESCs were shown in the bottom table. (b) Binding of Usp16 (red line) and IgG (black line) to gene-coding regions plus 10 kb upstream of the TSS and downstream of transcription ending site (TES) in Usp16^+/+ ESCs. Parallel experiments in Usp16^−/− ESCs were shown in Supplementary Fig. 3c. (c) ubH2A (orange line, Usp16^−/−; blue line, Usp16^+/+) and H2A (brown line, Usp16^−/−; green line, Usp16^+/+) signals across gene-transcribing regions plus 10 kb upstream of TSS and downstream of TES. (d) Usp16 (first column), Ring1B (second column) and ubH2A (third and fourth columns) distribution at individual genes in Usp16^+/+ and Usp16^−/− ESCs. Top row, top 1,000 expressed genes sorted by relative expression (FPKM). Bottom row, bottom 1,000 expressed genes in gene list re-sorted based on Ring1B signal. Analyses span 10 kb upstream of the TSS, the gene body, and 10 kb downstream of the TES. (e) Binding profiles of ubH2A in bottom 1,000 expressed genes re-sorted by Ring1B binding in Usp16^+/+ and Usp16^−/− ESCs. (f) ubH2A distribution at individual genes in Usp16^+/+ and Usp16^−/− ESCs. Genes are sorted based on ubH2A signals in Usp16^+/+ ESCs. (g) Expression levels of genes in f in Usp16^+/+ and Usp16^−/− ESCs. P-value is calculated by the Student’s t-test. Top whisker and bar represent 98th percentile of genes. Bottom whisker and bar represent 2nd percentile of genes above zero.