For KD rescue assays, cells where infected with pLKO-pim-based lentiviruses with two shRNAs and one vacant control, and selected with 1?g/mL puromycin for a few days For miR-200 rescues, viruses were prepared and cells?infected as described in the Supplemental Experimental Procedures. ChIP-qPCR Assays ChIP experiments were performed as in (Lee et?al., 2006) with a few modifications described in Supplemental Experimental Procedures. Author Contributions F.F., M.F., and J.W. essential for proper expression of by a dual regulatory mechanism: it facilitates NANOG binding to the promoter and fine-tunes its expression; most importantly, it downregulates the repressor ZEB1 directly via transcriptional repression and indirectly via post-transcriptional activation of the miRNAs. Our study thus uncovers a previously unappreciated role for the pluripotency regulator NAC1 in promoting efficient somatic cell reprogramming. was surprisingly dispensable for early embryo development (Yap et?al., 2013). Not unexpectedly, thereafter we were able to derive knockout (KO) mouse embryonic stem cells (mESCs), which Punicalin undergo normal self-renewal and maintain pluripotency (our unpublished data). In this study, we dissected the functional contribution of NAC1 in establishing pluripotency during somatic cell reprogramming. We identified a critical role for?NAC1 in transcriptionally and post-transcriptionally modulating and expression during the generation of iPSCs. Punicalin In the absence of NAC1 functions, reprogramming is usually diverted to an anomalous state that can be fully rescued with the re-expression of E-CADHERIN, but not NANOG or ESRRB. Our data thus uncover a previously unappreciated reprogramming factor that plays an indispensable role, beyond the mesenchymal-to-epithelial transition (MET), in controlling expression and establishing the pluripotency of iPSCs. Results NAC1 Depletion Impairs Somatic Cell Reprogramming Several pluripotency factors, including NANOG, TET1, and TET2, are essential for somatic cell reprogramming, while dispensable for stem cell maintenance once pluripotency is established (Golipour et?al., 2012). Although NAC1 functions in the maintenance of pluripotency in ESCs were mostly superfluous (our unpublished data), we decided to explore whether NAC1 could play a role in the establishment of pluripotency during somatic cell reprogramming. To test the effects of NAC1 on reprogramming, we knocked down its expression in mouse embryonic fibroblasts (MEFs) harboring an distal enhancer-driven GFP reporter that is only expressed in fully pluripotent iPSCs (Yeom et?al., 1996). Subsequently, we transduced the four Yamanaka factors, as depicted in Physique?S1A. knockdown (KD) was efficient (Physique?S1D, top) and minimally altered MEF proliferation (Determine?S1B). However, it drastically affected the total number and morphology of alkaline phosphatase (AP) positively stained iPS colonies, as well as the intensity of the staining (Figures 1AC1C). When scoring for GFP-positive colonies, we found that NAC1 downregulation not only diminished total GFP-positive populations (Physique?S1C), but also compromised the morphology of iPS colonies, compared with scramble small hairpin RNA (shRNA) control (shSCR) (Physique?1D). Data from three impartial reprogramming experiments revealed that the majority of the iPS colonies upon KD were GFP unfavorable (Physique?1E). Open in a separate window Physique?1 Is Required for Somatic Cell Reprogramming (A) Images of AP-stained wells for MEF-derived iPSCs upon control and KD. (B) Images of AP-stained iPS colonies upon control and KD. (C) Quantification of control and KD iPS colonies scored based on intensity of AP staining. (D) Images in Punicalin bright field and GFP fluorescence for iPS colonies upon control and KD MEF reprogramming. (E) Quantification of control and KD iPS colonies scored for GFP expression. (F) Representative pictures of wells of AP-stained iPS derived from WT (+/+), het (+/?), and null (?/?) MEFs. (G) Quantification of WT, het, and null iPS colonies based on AP staining. (H) Images of representative WT, het, and null iPS colonies in bright field (top panel) and after AP staining (bottom panel). (I) Pictures of duplicated wells for WT, het, and null iPS colonies stained with AP upon incubation in serum/LIF or 2i/LIF medium. (J) Average qPCR gene expression profiling for three WT, three het, and nine null clonal iPSC lines. Indicated are selected pluripotency markers, late reprogramming markers, and MET/cell-adhesion genes. stands for KO mouse was not embryonic lethal, we were able to derive wild-type (WT), heterozygous (het), and null MEFs (Physique?S1D, bottom). We then employed these fibroblasts in our reprogramming assays. As shown in Figures 1F and 1G, there was minimal difference in total number of iPS colonies upon AP staining among Rabbit Polyclonal to RREB1 WT, het, and null cells. However, null colonies stained less efficiently for AP, due to their pre-iPS-like morphology (Figures 1G and 1H) compared with WT and het cells. We also crossed our mice with the mutant MEFs Punicalin harboring the GFP reporter (Physique?S1E, top). Consistent with KD experiments, (Physique?S1E, bottom). To assess whether WT iPSCs survived in the 2i/LIF medium. In contrast, null cells showed significantly lower.