Xiaochen Kou

Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming.

DNA methylation and demethylation have been proposed to play an important role in somatic cell reprogramming. Here, we demonstrate that the DNA hydroxylase Tet1 facilitates pluripotent stem cell induction by promoting Oct4 demethylation and reactivation. Moreover, Tet1 (T) can replace Oct4 and initiate somatic cell reprogramming in conjunction with Sox2 (S), Klf4 (K), and c-Myc (M). We established an efficient TSKM secondary reprogramming system and used it to characterize the dynamic profiles of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and gene expression during reprogramming. Our analysis revealed that both 5mC and 5hmC modifications increased at an intermediate stage of the process, correlating with a transition in the transcriptional profile. We also found that 5hmC enrichment is involved in the demethylation and reactivation of genes and regulatory regions that are important for pluripotency. Our data indicate that changes in DNA methylation and hydroxymethylation play important roles in genome-wide epigenetic remodeling during reprogramming.

Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos.

Histone modifications have critical roles in regulating the expression of developmental genes during embryo development in mammals. However, genome-wide analyses of histone modifications in pre-implantation embryos have been impeded by the scarcity of the required materials. Here, by using a small-scale chromatin immunoprecipitation followed by sequencing (ChIP–seq) method, we map the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3), which are associated with gene activation and repression, respectively, in mouse pre-implantation embryos. We find that the re-establishment of H3K4me3, especially on promoter regions, occurs much more rapidly than that of H3K27me3 following fertilization, which is consistent with the major wave of zygotic genome activation at the two-cell stage. Furthermore, H3K4me3 and H3K27me3 possess distinct features of sequence preference and dynamics in pre-implantation embryos. Although H3K4me3 modifications occur consistently at transcription start sites, the breadth of the H3K4me3 domain is a highly dynamic feature. Notably, the broad H3K4me3 domain (wider than 5 kb) is associated with higher transcription activity and cell identity not only in pre-implantation development but also in the process of deriving embryonic stem cells from the inner cell mass and trophoblast stem cells from the trophectoderm. Compared to embryonic stem cells, we found that the bivalency (that is, co-occurrence of H3K4me3 and H3K27me3) in early embryos is relatively infrequent and unstable. Taken together, our results provide a genome-wide map of H3K4me3 and H3K27me3 modifications in pre-implantation embryos, facilitating further exploration of the mechanism for epigenetic regulation in early embryos.

Allelic reprogramming of the histone modification H3K4me3 in early mammalian development

Histone modifications are fundamental epigenetic regulators that control many crucial cellular processes. However, whether these marks can be passed on from mammalian gametes to the next generation is a long-standing question that remains unanswered. Here, by developing a highly sensitive approach, STAR ChIP–seq, we provide a panoramic view of the landscape of H3K4me3, a histone hallmark for transcription initiation, from developing gametes to post-implantation embryos. We find that upon fertilization, extensive reprogramming occurs on the paternal genome, as H3K4me3 peaks are depleted in zygotes but are readily observed after major zygotic genome activation at the late two-cell stage. On the maternal genome, we unexpectedly find a non-canonical form of H3K4me3 (ncH3K4me3) in full-grown and mature oocytes, which exists as broad peaks at promoters and a large number of distal loci. Such broad H3K4me3 peaks are in contrast to the typical sharp H3K4me3 peaks restricted to CpG-rich regions of promoters. Notably, ncH3K4me3 in oocytes overlaps almost exclusively with partially methylated DNA domains. It is then inherited in pre-implantation embryos, before being erased in the late two-cell embryos, when canonical H3K4me3 starts to be established. The removal of ncH3K4me3 requires zygotic transcription but is independent of DNA replication-mediated passive dilution. Finally, downregulation of H3K4me3 in full-grown oocytes by overexpression of the H3K4me3 demethylase KDM5B is associated with defects in genome silencing. Taken together, these data unveil inheritance and highly dynamic reprogramming of the epigenome in early mammalian development.

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of “4N-ON” and the corresponding “4N-OFF” iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.

Enhanced Telomere Rejuvenation in Pluripotent Cells Reprogrammed via Nuclear Transfer Relative to Induced Pluripotent Stem Cells

Although somatic cell nuclear transfer (SCNT) and induction of pluripotency (to form iPSCs) are both recognized reprogramming methods, there has been relatively little comparative analysis of the resulting pluripotent cells. Here, we examine the capacity of these two reprogramming approaches to rejuvenate telomeres using late-generation telomerase-deficient (Terc(-/-)) mice that exhibit telomere dysfunction and premature aging. We found that embryonic stem cells established from Terc(-/-) SCNT embryos (Terc(-/-) ntESCs) have greater differentiation potential and self-renewal capacity than Terc(-/-) iPSCs. Remarkably, SCNT results in extensive telomere lengthening in cloned embryos and improved telomere capping function in the established Terc(-/-) ntESCs. In addition, mitochondrial function is severely impaired in Terc(-/-) iPSCs and their differentiated derivatives but significantly improved in Terc(-/-) ntESCs. Thus, our results suggest that SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming. Understanding the basis of this differential could help optimize reprogramming strategies.

Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing

Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos.

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self‐renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with β‐thalassemia into transgene‐free naïve iPSCs with molecular signatures of ground‐state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross‐species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene‐correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient‐specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy.

Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming.

The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies.

Unique features of mutations revealed by sequentially reprogrammed induced pluripotent stem cells

Although viable mice can be generated from induced pluripotent stem cells (iPSCs), the impact of accumulated mutations on the developmental potential of the resulting iPSCs remains to be determined. Here, we demonstrate that all-iPSC mice generated through tetraploid blastocysts complementation can tolerate the accumulation of somatic mutations for up to six generations using a Tet-on inducible reprogramming system. But, the viability of the all-iPS mice decreased with increasing generations. A whole-genome sequencing survey revealed that thousands of single-nucleotide variations (SNVs), including 44 non-synonymous ones, accumulated throughout the sequential reprogramming process. Subsequent analysis provides evidence that these accumulated SNVs account for the gradual reduction in viability of the resultant all-iPSC mice. Unexpectedly, our present reprogramming system revealed that pluripotent stem cells are heterogeneous in terms of possessing a set of copy-number alterations (CNAs). These CNAs are unique for pluripotent cells and subsequently disappear in the differentiating progenies.

Xist Repression Shows Time‐Dependent Effects on the Reprogramming of Female Somatic Cells to Induced Pluripotent Stem Cells

Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X‐inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer studies. In this study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the downregulation of Xist by an isopropyl β‐d‐1‐thiogalactopyranoside (IPTG)‐inducible short hairpin RNA (shRNA) system can greatly impair the mesenchymal‐to‐epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre‐iPSCs to iPSCs in the late phase. Furthermore, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was released from the inactivated X chromosome (Xi). This enables the X chromosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist repression enables the transition from pre‐iPSCs to iPSCs. Stem Cells 2014;32:2642–2656