PTEN modulates gene transcription by redistributing genome-wide RNA polymerase II occupancy

Abstract

Control of gene expression is one of the most complex yet continuous physiological processes impacting cellular homeostasis. RNA polymerase II (Pol II) transcription is tightly regulated at promoter-proximal regions by intricate dynamic processes including Pol II pausing, release into elongation and premature termination. Pol II pausing is a phenomenon where Pol II complex pauses within 30-60 nucleotides after initiating the transcription. Negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) contribute in the establishment of Pol II pausing, and positive transcription elongation factor b releases (P-TEFb) paused complex after phosphorylating DSIF that leads to dissociation of NELF. Pol II pausing is observed in most expressed genes across the metazoan. The precise role of Pol II pausing is not well understood; however, it's required for integration of signals for gene regulation. In the present study, we investigated the role of phosphatase and tensin homolog (PTEN) in genome-wide transcriptional regulation using PTEN overexpression and PTEN knock-down models. Here we identify that PTEN alters the expression of hundreds of genes, and its restoration establishes genome-wide Pol II promoter-proximal pausing in PTEN null cells. Furthermore, PTEN re-distributes Pol II occupancy across the genome and possibly impacts Pol II pause duration, release and elongation rate in order to enable precise gene regulation at the genome-wide scale. Our observations demonstrate an imperative role of PTEN in global transcriptional regulation that will provide a new direction to understand PTEN-associated pathologies and its management.

Figure 1

PTEN modulates global gene expression. (A) Overexpression of PTEN in BT-549 cells. (B) Volcano plot showing differential gene expression in BT-549 cells after overexpression of PTEN followed by RNA-seq analysis. A total of 2146 genes were differentially expressed (FDR-corrected P-value <0.05) including 400 and 299 genes 2-fold or more overexpressed and underexpressed, respectively. (C) IPA analysis of top canonical pathways in BT-549 cells with and without PTEN overexpression. (D) Immunoblot showing knock-down of PTEN in MCF7 cells. (E) Volcano plot showing differential gene expression (1242 genes, FDR-corrected P-value <0.05) in MCF7 cells after knock-down of PTEN using shRNA followed by RNA-seq analysis. With ≥2-fold change cut-off, 167 genes are relatively underexpressed and 87 genes are overexpressed. (F) IPA analysis of top canonical pathways of DEGs in MCF7 cells followed by PTEN knock-down.

Figure 2

PTEN expression regulates genome-wide RNA Pol II pausing. (A) Metagene plot showing Pol II signals at TSS (average of 16 290 genes) in BT-549 cells after re-expression of PTEN followed by Pol II ChIP-seq. (B) Pol II occupancy at representative genes are shown using UCSC browser. (C) A small group of genes (n = 136) showing decrease in Pol II pausing after PTEN overexpression. (D) Decrease in Pol II occupancy after PTEN re-expression in BT-549 cells at representative genes are shown using UCSC browser. (E) PTEN expression after 48 h of tetracycline treatment (left), and Metagene plot showing Pol II signals at TSS in BT-549-PTEN +/− tetracycline treatment followed by Pol II ChIP-seq (right). (F) Metagene plot showing Pol II signals at TSS (average of 16 290 genes) in MCF7 cells after knock-down of PTEN using shRNA followed by Pol II ChIP-seq. (G) Pol II occupancy at representative genes are shown using UCSC browser.

Figure 3

PTEN regulates RNA Pol II pause release. (A) PRR was calculated by taking the ratio of gene body (+300 bp to +2 kb from TSS) counts to promoter (−50 bp to +300 bp form TSS) counts. PRR is represented by cumulative frequency plots showing an overall decrease in PRR after re-expression of PTEN in BT-549 PTEN-null cells. (B) All the genes (n = 10.141) in panel ‘A’ were classified in to three groups based on their PRR. Genes are categorized under groups representing decreased (left, 1.5-fold reduction in PRR after PTEN re-expression in BT-549 cells), unchanged (center, between 0.67 and 1.5-fold change in PRR) and increased (right, 1.5-fold increase) PRR after PTEN expression in BT-549 cells. (C) Top GO categories for genes with an increase in PRR after PTEN re-expression in BT-549 cells. (D) Cumulative frequency plot representing an overall increase in PRR after knock-down of PTEN in MCF7 cells (n = 14 300 genes). (E) Box plots represent changes in PRR in three groups of genes. Genes are grouped based on their PRR: increased PRR group (left), 1.5-fold increase in PRR after PTEN knock-down in MCF7 cells; unchanged PRR group (center), between 0.67 and 1.5-fold change in PRR; and decreased PRR group (right), 1.5-fold reduction in PRR after PTEN knock-down in MCF7 cells. (F) Top GO categories for genes with a decrease in PRR after knock-down of PTEN in MCF7 cells. (F) BT-549 cells were treated with 0.5 mM Trp for 1 and 2 min and Pol II ChIP signals (% input) were used to generate a decay curve. Representative genes showing increase in Pol II pause duration after re-expression of PTEN in BT-549 cells.

Figure 4

PTEN redistributes genome-wide Pol II occupancy. (A) Metagene plot showing Pol II signals on gene bodies in BT-549 cells after overexpression of PTEN followed by Pol II ChIP-seq. (B) Heatmap showing Pol II signals on gene bodies in various cell types (Breast epithelium, SRR2301044; T47D, SRR7965856; MCF7, SRR1290687; MDA-MB-231, SRR5919406; iPSCs, SRR5935361; and Dermal fibroblasts, SRR4340777). (C) Metagene plot showing Pol II signals on gene bodies in MCF7 cells after knock-down of PTEN followed by Pol II ChIP-seq. (D) Pol II elongation was compared after 5 and 10 min of Trp treatment (0.5 μM) followed by ChIP-qPCR for Pol II signal at the terminal exon of TBL2 gene. (E) Pol II elongation was compared after 5 and 10 min of Trp treatment followed by ChIP-qPCR for Pol II signal at the terminal exon of CTSB gene. (F) Pol II elongation was compared after 5 and 10 min of Trp treatment followed by ChIP-qPCR for Pol II signal at the terminal exon of RND3 gene. Dotted vertical red line indicates the primer position used in ChIP-qPCR.