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R-loops coordinate with SOX2 in regulating reprogramming to pluripotency.

ABSTRACT: R-loops modulate genome stability and regulate gene expression, but the functions and the regulatory mechanisms of R-loops in stem cell biology are still unclear. Here, we profiled R-loops during somatic cell reprogramming and found that dynamic changes in R-loops are essential for reprogramming and occurred before changes in gene expression. Disrupting the homeostasis of R-loops by depleting RNaseH1 or catalytic inactivation of RNaseH1 at D209 (RNaseH1^D209N) blocks reprogramming. Sox2, but not any other factor in the Yamanaka cocktail, overcomes the inhibitory effects of RNaseH1 activity loss on reprogramming. Sox2 interacts with the reprogramming barrier factor Ddx5 and inhibits the resolvase activity of Ddx5 on R-loops and thus facilitates reprogramming. Furthermore, reprogramming efficiency can be modulated by dCas9-mediated RNaseH1/RNaseH1^D209N targeting the specific R-loop regions. Together, these results show that R-loops play important roles in reprogramming and shed light on the regulatory module of Sox2/Ddx5 on R-loops during reprogramming.

SUBMITTER: Li Y

PROVIDER: S-EPMC7360481 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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R-loops coordinate with SOX2 in regulating reprogramming to pluripotency.

Li Yaoyi Y Song Yawei Y Xu Wei W Li Qin Q Wang Xinxiu X Li Kuan K Wang Juehan J Liu Zicong Z Liu Zicong Z Velychko Sergiy S Ye Rong R Xia Qing Q Wang Lei L Guo Rong R Dong Xiaotao X Zheng Zhikai Z Dai Yushuang Y Li Haojie H Yao Mingze M Xue Yuanchao Y Schöler Hans R HR Sun Qianwen Q Yao Hongjie H

Science advances 20200610 24

R-loops modulate genome stability and regulate gene expression, but the functions and the regulatory mechanisms of R-loops in stem cell biology are still unclear. Here, we profiled R-loops during somatic cell reprogramming and found that dynamic changes in R-loops are essential for reprogramming and occurred before changes in gene expression. Disrupting the homeostasis of R-loops by depleting RNaseH1 or catalytic inactivation of RNaseH1 at D209 (RNaseH1<sup>D209N</sup>) blocks reprogramming. Sox ...[more]

PMID: 32704541

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Project description:Several Sox family members, small molecules, and inhibitors of the TGF-β pathway can replace exogenous Sox2 during the generation of induced pluripotent stem cells (iPSCs), suggesting that SOX2 is dispensable for initiation of reprogramming. However, the time point at which the endogenous Sox2 locus is activated in these conditions was not thoroughly evaluated. To identify the Sox2-independent reprogramming steps, we excluded Sox2 from the reprogramming cocktail and used Sox2-deficient mouse embryonic fibroblasts. We show that reprogramming is initiated in the absence of SOX2 as shown by formation of pre-iPSC colonies that have undergone a mesenchymal-to-epithelial transition and that have activated early reprogramming markers. However, these iPSC-like intermediates cannot progress through the final stabilization phase of reprogramming in the absence of SOX2. Overall, our results provide a better understanding of the molecular events underlying the induction of pluripotency and of the role that SOX2 plays in the process. Both pLV-tetO-E1A-2A-Cherry and pLV-tetO-Cherry lentiviral constructs were cloned in house and defective-lentiviral particles were produced as previously described (Han et al. 2013). Next, 5x10 4 ESCs containing a tetracycline-inducible transactivator (irtTA-VP16-GBD, Anastassiadis et al. 2002) plus an Oct4-GFP reporter transgene (Yoshimizu et al. 1999) were transduced twice using 20 µl of replication-defective lentiviral particles plus polybrene (4 µg/ml, Sigma). Cells were washed 24 h after transduction and subsequently induced with doxycycline (1 µg/ml, Sigma) and dexamethasone (10-7 M, Sigma). Samples were harvested at several time-points, 0, 3, 6, 9, 12, 24, and 48 hours after induction, and used for microarray analysis as previously reported (Tapia et al. 2012). Cells transduced with pLV-tetO-Cherry and induced for 48 h were used as control. RNA wasprocessed using the RNeasy micro and mini kits with on-column DNase digestion of QIAGEN as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software. Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab.

Project description:New strategies are needed to predict and overcome metastatic progression and therapy resistance in prostate cancer. One potential clinical target is the stem cell transcription factor SOX2, which has a critical role in prostate development and cancer. We thus investigated the impact of SOX2 expression on patient outcomes and its function within prostate cancer cells. Analyses of SOX2 expression among a case-control cohort of 1028 annotated tumor specimens demonstrated that SOX2 expression confers a more rapid time to metastasis and decreased patient survival after biochemical recurrence. SOX2 ChIP-Seq analyses revealed SOX2-binding sites within prostate cancer cells which differ significantly from canonical embryonic SOX2 gene targets, and prostate-specific SOX2 gene targets are associated with multiple oncogenic pathways. Interestingly, phenotypic and gene expression analyses after CRISPR-mediated deletion of SOX2 in castration-resistant prostate cancer cells, as well as ectopic SOX2 expression in androgen-sensitive prostate cancer cells, demonstrated that SOX2 promotes changes in multiple metabolic pathways and metabolites. SOX2 expression in prostate cancer cell lines confers increased glycolysis and glycolytic capacity, as well as increased basal and maximal oxidative respiration and increased spare respiratory capacity. Further, SOX2 expression was associated with increased quantities of mitochondria, and metabolomic analyses revealed SOX2-associated changes in the metabolism of purines, pyrimidines, amino acids and sugars, and the pentose phosphate pathway. Analyses of SOX2 gene targets with central functions metabolism (CERK, ECHS1, HS6SDT1, LPCAT4, PFKP, SLC16A3, SLC46A1, and TST) document significant expression correlation with SOX2 among RNA-Seq datasets derived from patient tumors and metastases. These data support a key role for SOX2 in metabolic reprogramming of prostate cancer cells and reveal new mechanisms to understand how SOX2 enables metastatic progression, lineage plasticity, and therapy resistance. Further, our data suggest clinical opportunities to exploit SOX2 as a biomarker for staging and imaging, as well as a potential pharmacologic target.

Dataset Information

R-loops coordinate with SOX2 in regulating reprogramming to pluripotency.

Publications

R-loops coordinate with SOX2 in regulating reprogramming to pluripotency.

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