Dynamic Status of REST in the Mouse ESC Pluripotency Network

Citation: Singh SK, Veo BL, Kagalwala MN, Shi W, Liang S, et al. ( Dynamic Status of REST in the Mouse ESC Pluripotency Network Sanjay K. Singh 0 1 Bethany L. Veo 0 1 Mohamedi N. Kagalwala 0 1 Weiwei Shi 0 1 Shoudan Liang 0 1 Sadhan Majumder 0 1 Austin John Cooney, Baylor College of Medicine, United States of America 0 a Current address: Hospital for Sick Children , Toronto, Ontario , Canada b Current address: Laboratory of Genetics, The Salk Institute , La Jolla, California , United States of America 1 1 Department of Genetics, The University of Texas M. D. Anderson Cancer Center , Houston , Texas, United States of America, 2 Department of Bioinformatics, The University of Texas M. D. Anderson Cancer Center , Houston , Texas, United States of America, 3 Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center , Houston , Texas, United States of America, 4 The Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center , Houston , Texas, United States of America, 5 Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston , Houston, Texas , United States of America Background: REST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network. Methods: Employing biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature. Results: We show that Rest+/2 and Rest2/2 AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/2 and Rest2/2 AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a RESTdependent pluripotency when cultured under feeder-free conditions, as well as Rest2/2 AB-1 ESCs, showed no RESTdependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST's role in ESC pluripotency. Conclusions: REST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner. - Funding: This work was partly supported by grants from the NCI (CA97124 and CA81255). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have declared that no competing interests exist. Pluripotent mouse embryonic stem cells (mESCs) derived from the inner cell mass of the mammalian blastocyst are capable of forming all the tissues of the organism and possess the ability to self-renew in an undifferentiated manner. Understanding the mechanisms that control the self-renewal and pluripotency of ESCs has far-reaching implications for the fields of developmental biology, regenerative medicine, and oncology [1,2,3]. The recent successes with reprogramming somatic cells into induced pluripotent stem-like cells have brought intense enthusiasm to this area of research [4,5,6,7,8,9,10,11]. These findings suggest that factors such as Oct4, Nanog, and Sox2 are core components of a large, interconnected network that regulates self-renewal and pluripotency in both mouse and human ESCs [2,12,13,14]. This network is likely regulated by maintenance factors that are triggered by cellular and environmental signals [15]; these factors both protect the self-renewal state from spurious signals and cause differentiation of the cell when required. REST is a transcriptional repressor that was originally discovered to be a repressor of many neuronal differentiation genes [16,17]. Experiments with Rest mutant mice supported RESTs role in neurogenesis [18]. However, later reports indicated that REST can potentially repress about a thousand genes and affect various cellular functions in a context-dependent fashion [19,20,21,22,23]. Although REST expression is higher in ESCs than in most other cell type [24], its function in the selfrenewal network is unclear. REST has been reported to maintain mESC self-renewal and pluripotency by directly suppressing miR21 [25]. In these studies, loss of REST in ESCs accompanied expression of many differentiation markers, including Gata4. In contrast, others found REST to have no role in the maintenance of mESC pluripotency [26,27,28] and loss of REST did not change expression of differentiation markers including Gata4 [27,28]. Yet, another report found that REST was not required to maintain ESC pluripotency and, in contrast, loss of REST actually caused decreased expression of differentiation markers, including Gata4 [29]. However, genome-wide promoter occupancy assays have shown that REST indeed is an integral part of the ESC pluripotency network [30,31,32,33,34], raising the interesting question whether such genome-wide assays truly reflect the biology of ESC pluripotency. Thus, these conflicting results have created a gap in our knowledge of the mESC network, particularly with respect to REST. Using wild-type, Rest+/2 and Rest2/2 ESCs and conditions used by different laboratories, we determined how REST functions in the mESC pluripotency network in coordination with other factors. The work presented here resolves some of the apparent discrepancies in the field. Materials and Methods Mouse ESC lines and culture conditions E14Tg2a.4 were purchased from Bay Genomics (MMRRC item #015890-UCD-CELL) and parental AB1 (N6: Wild-type), N9 (Rest+/2) and N8 (Rest2/2) were obtained as gifts from Amanda Fisher Laboratory [27] (originally obtained in presence of feeder cells and were maintained in presence of feeder layer and LIF; passage numbers in the manuscript represent passage in absence of feeder cells), were cultured without feeder cells in presence of 1000 units ml21 LIF (ESGRO) on gelatin-coated (...truncated)