The regulatory role of histone deacetylase inhibitors in Fgf4 expression is dependent on the differentiation state of pluripotent stem cells

The identity of embryonic stem cells (ESCs) is controlled by a set of pluripotency genes, including Oct4, Sox2, Nanog, and Fgf4. How their expression is repressed during differentiation and reactivated during reprogramming is largely unknown. Here, using mouse ESCs as well as F9 and P19 cells (mouse embryonal carcinoma cell lines, P19 being considered further differentiated than F9 cells) as models, we found that HDAC inhibitors elevated Fgf4 expression in P19 cells, but reduced it in F9 cells. We also observed that HDAC inhibitors enhanced the expression of Fgf4 and a subset of pluripotency genes in differentiated ESCs, but reduced their expression in undifferentiated and less differentiated ESCs. Mechanistically, we observed more HDAC1 recruitment and a weaker association of histone 4 lysine 5 acetylation at the Fgf4 enhancer in P19 cells compared to F9 cells. Additionally, we demonstrated the interaction between Sox2 and HDAC1 both in vitro and in vivo, implicating a possible role for Sox2 in the recruitment of HDAC1 to the Fgf4 enhancer. We also found that Nanog bound to the Fgf4 enhancer, and this binding was stronger in F9 cells, indicating the involvement of Nanog in the regulation of Fgf4 expression in undifferentiated and less differentiated pluripotent stem cells. This study uncovers an important role of HDAC1 and histone modifications in the repression of Fgf4 and perhaps other pluripotency genes during ESC differentiation. Our results also suggest that HDAC inhibitors may promote reprogramming partially through activating pluripotency genes at some intermediate stages.     

Identification of genes regulated by nanog which is involved in ES cells pluripotency and early differentiation

Nanog plays an important role in embryonic stem (ES) cells pluripotency and self-renewal, yet the precise mechanism through which Nanog accomplishes this important function remains unclear. To understand comprehensive molecular mechanism by which Nanog mediates, we identified genome-wide molecular changes upon silencing Nanog in ES cells by using microarray technology. In order to downregulate Nanog expression efficiently, four siRNAs were designed on the basis of the conserved Nanog sequence and their effects on the Nanog expression were tested. Among these four siRNAs, Nanog-siRNA-P1 was found to be most effective. Once Nanog was downregulated, ES cells underwent differentiation by showing morphological change and decreased proliferation rate. Microarray analysis was then used to identify the altered gene expression after Nanog was silenced. A series of differentially expressed genes due to reduced expression of Nanog was identified as Nanog-related genes. These genes identified here could provide insights into the roles of Nanog in ES cells self-renewal and early differentiation.     

p300通过homeobox结构域增强Nanog的转录激活活性

胚胎干细胞(ES细胞)能够不断地进行自我更新来维持其多能性,很多转录因子共同调控着ES细胞的自我更新和多能性,Nanog就是其中之一,然而Nanog维持ES细胞多能性的机制并不清楚。p300是真核生物中普遍存在的转录辅助因子,与许多转录因子共同作用调控下游基因的表达。为探索p300是不是能够影响Nanog的转录活性,我们在细胞中共转Nanog(或突变体)及报告基因和p300,结果表明p300能够通过homeobox结构域增强Nanog的转录激活活性。     

Nanog基因的功能与调控研究进展

胚胎干细胞的无限增殖能力和亚全能性决定了它在再生医学、新药开发及发育生物学基础研究中具有巨大的应用前景。探索维持胚胎干细胞亚全能性的因子及其网络的调控功能成为胚胎干细胞生物学研究的热点。已研究发现多个与维持胚胎干细胞亚全能性相关的基因如Oct4, Nanog, Sox2等,其中Nanog是2003年5月末发现的一个基因,它对维持胚胎干细胞亚全能性起关键性作用,能够独立于L1F/Stat3维持ICM和胚胎干细胞的亚全能性。几年来,Nanog的生物学功能及其与 Oct4, Sox2等亚全能性维持基因之间的相互作用关系已有较为深入的研究,并发现多个调控Nanog表达的转录因子,从而进一步明晰Nanog与已知调控胚胎发育的信号通路之间的关系。本文在综述Nanog基因的表达特征和功能的基础上、重点探讨Nanog基因表达调控以及Oct4, Sox2等亚全能性维持基因之间的相互作用关系,并对未来的研究趋势予以展望。     

NPR-A regulates self-renewal and pluripotency of embryonic stem cells

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.     

Gene expression profiling of mouse embryonic stem cell subpopulations

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.     

维持胚胎干细胞多能性的分子机制

胚胎干细胞（ES细胞）来源于早期发育的胚胎,具有分化为任何细胞类型的多能性,因此具有巨大的基础研究及潜在的应用前景.目前认为ES细胞主要通过一些外源性信号分子的作用及某些重要的内源性转录因子的表达共同起作用来达到其维持多能性的目的.外源性信号分子LIF、BMP4以及Wnt等介导的信号传导通路与内源性转录因子Oct4、Nanog、Sox2、FoxD3等共同起作用来抑制那些促进ES细胞分化的基因表达和激活那些有助于维持ES细胞多能性维持的基因表达,进而形成一个相互调控和依存的基因调控网络共同维持ES细胞的多能性.     

FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for na?ve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.