Regulation of RNA Pol II directionality by FACT. Regulation of RNA Pol II directionality by FACT. (A) Scatterplots of log gene body coverage (NET-seq) versus log mRNA expression (RNA-seq) for SSRP1 (n = 4,576) and non-SSRP1 (n = 8,844) target regions in the control state (z-score = 5.3, P < 10−5). (B) Measure of Pol II pause/release. Travelling ratio is defined as NET-seq density of proximal promoter versus gene body area. The log-transformed travelling ratio for each gene class is displayed with boxplots. The Welch two-sided t test was used to calculate significance between control and Ssrp1 KD (*P < 0.05, n.s., not significant). (C) NET-seq density plots (control and Ssrp1 KD group) of up-regulated genes split by FACT-bound status (non-SSRP1 and SSRP1 targets). Solid lines indicate mean values, whereas the shading represents the 95% confidence interval. (D) Cumulative distribution of antisense transcription (NET-seq) in a window 1,000 bp upstream of the TSS. The Welch two-sided t test was used to calculate significance between control and Ssrp1 KD among non-SSRP1 and SSRP1 targets. (E) Boxplots assessing fold change (Ssrp1 KD versus control) in antisense transcription (NET-seq) in a window 1,000 bp upstream of the TSS. The Welch two-sided t test was used to calculate significance between non-SSRP1 and SSRP1 targets. (F) Nucleosome occupancy (MNase-seq), open chromatin (ATAC-seq), and transcriptional activity (NET-seq/RNA-seq) over an SSRP1 (Oct4) and non-SSRP1 (Psmb6) target gene between control and Ssrp1 KD conditions. Nucleosomal loss and increase in antisense transcription at the Oct4 promoter is highlighted in yellow. Constantine Mylonas, and Peter Tessarz LSA 2018;1:e201800085 © 2018 Tessarz and Mylonas