Lingyi Chen

A genomic code for nucleosome positioning

Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting with most DNA binding proteins. Nucleosomes have higher affinity for particular DNA sequences, reflecting the ability of the sequence to bend sharply, as required by the nucleosome structure. However, it is not known whether these sequence preferences have a significant influence on nucleosome position in vivo, and thus regulate the access of other proteins to DNA. Here we isolated nucleosome-bound sequences at high resolution from yeast and used these sequences in a new computational approach to construct and validate experimentally a nucleosome–DNA interaction model, and to predict the genome-wide organization of nucleosomes. Our results demonstrate that genomes encode an intrinsic nucleosome organization and that this intrinsic organization can explain ∼50% of the in vivo nucleosome positions. This nucleosome positioning code may facilitate specific chromosome functions including transcription factor binding, transcription initiation, and even remodelling of the nucleosomes themselves.

Molecular basis of pluripotency

Embryonic stem (ES) cells are pluripotent and capable of self-renewal, thus holding promise for regenerative medicine. Recent studies have begun to provide insights into the molecular mechanisms underlying pluripotency and self-renewal. In this article, we discuss the roles of transcriptional regulation, epigenetic regulation and miRNAs in the maintenance of pluripotency and the differentiation of ES cells.

A genomewide study identifies the Wnt signaling pathway as a major target of p53 in murine embryonic stem cells

Both p53 and the Wnt signaling pathway play important roles in regulating the differentiation of mouse embryonic stem cells (mESCs). However, it is not known whether they directly and/or functionally crosstalk in mESCs. Here we report a surprising antidifferentiation function of p53 in mESCs through directly regulating the Wnt signaling pathway. A chromatin-immunoprecipitation-based microarray (ChIP-chip) and gene expression microarray assays reveal that the Wnt signaling pathway is significantly (P value, 0.000048) overrepresented in p53-regulated genes in mESCs. The expression of five Wnt ligand genes is robustly induced by various genotoxic and nongenotoxic insults in a p53-dependent manner. Moreover, the induction of these Wnt genes is greatly attenuated in mouse embryonic fibroblast (MEF) cells and ESC-derived neural stem/progenitor cells, suggesting that the induction is mESC specific. It is established that the activation of the Wnt signaling pathway inhibits the differentiation of mESCs. Consistent with this notion, we detected an antidifferentiation activity from the conditioned medium (CM) collected from UV (UV)-treated mESCs. This antidifferentiation activity can be lowered by either the addition of Wnt antagonists into the CM or the reduction of p53 levels in UV-treated mESCs. Therefore, reminiscent of its dual functions on death and survival in somatic cells, p53 appears to regulate both prodifferentiation and antidifferentiation programs in mESCs. Our findings uncover a direct and functional connection between p53 and the Wnt signaling pathway, and expand the catalog of p53 regulated genes in mESCs.

Mechanism of Transcriptional Silencing in Yeast

Transcriptional silencing is a phenomenon in which the transcription of a gene by RNA polymerase II or III is repressed or not, dependent only on the genes chromosomal location. Two prevailing models exist for silencing: (1) steric hindrance in silenced chromatin inhibits the binding of upstream activator proteins or polymerase or (2) silencing primarily blocks steps downstream of transcription preinitiation complex formation. Here, we test these models quantitatively for the case of SIR2-dependent silencing in budding yeast, using foreign and endogenous reporter proteins, at transgenic and endogenous loci. Our results contradict both models and show instead that transcriptional silencing at several URA3 transgenes, and at the naturally silenced endogenous HMRa and HMLalpha mating type genes, acts downstream of gene activator protein binding to strongly reduce the occupancy of TFIIB, RNA polymerase II, and TFIIE at the silenced promoters.

Cross-regulation of the Nanog and Cdx2 promoters

The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass (ICM) and trophectoderm (TE), is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx2, while the pluripotency factor, Nanog, is essential to specify the epiblast. We have analyzed the promoters of Nanog and Cdx2, and have found that these two transcription factors are likewise regulated reciprocally. Using an embryonic stem cell line with conditional TE differentiation, we show that Nanog overexpression suppresses the upregulation of TE markers, while Nanog knockdown upregulates the expression of TE markers. We further show that Nanog and Cdx2 bind to and repress each others promoters. However, whereas Nanog knockout results in detectable Cdx2 expression in the ICM, we observe no overt disruption of blastocyst development, indicating that Nanog plays a subservient role to Oct4 in segregation of the ICM and TE.

Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc−/− and Terc+/−) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.

Molecular basis of the first cell fate determination in mouse embryogenesis.

Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the trophectoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, microRNAs, and signal transduction.

Rif1 Maintains Telomere Length Homeostasis of ESCs by Mediating Heterochromatin Silencing

Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.

Current progress and prospects of induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells are derived from somatic cells by ectopic expression of few transcription factors. Like embryonic stem (ES) cells, iPS cells are able to self-renew indefinitely and to differentiate into all types of cells in the body. iPS cells hold great promise for regenerative medicine, because iPS cells circumvent not only immunological rejection but also ethical issues. Since the first report on the derivation of iPS cells in 2006, many laboratories all over the world started research on iPS cells and have made significant progress. This paper reviews recent progress in iPS cell research, including the methods to generate iPS cells, the molecular mechanism of reprogramming in the formation of iPS cells, and the potential applications of iPS cells in cell replacement therapy. Current problems that need to be addressed and the prospects for iPS research are also discussed.

Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells.

Significance Signaling pathways regulate the self-renewal and differentiation of embryonic stem cells (ESCs). Suppression of Mek/Erk signaling by pharmacological inhibitors promotes self-renewal and pluripotency maintenance of mouse ESCs, supporting the prevailing view that Erk signaling is dispensable for ESC self-renewal. However, using inducible Erk knockout ESCs, we demonstrate that Erk signaling is critical for ESC self-renewal. ESCs cannot be maintained for more than four passages after Erk depletion, associated with misregulated expression of pluripotency genes, reduced proliferation rate, G1 cell-cycle arrest, increased apoptosis, rapid shortening of telomeres, and impaired genomic stability. We further demonstrate an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC self-renewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs.