Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape.
Ontology highlight
ABSTRACT: In 2006, the "wall came down" that limited the experimental conversion of differentiated cells into the pluripotent state. In a landmark report, Shinya Yamanaka's group described that a handful of transcription factors (Oct4, Sox2, Klf4 and c-Myc) can convert a differentiated cell back to pluripotency over the course of a few weeks, thus reprograming them into induced pluripotent stem (iPS) cells. The birth of iPS cells started off a rush among researchers to increase the efficiency of the reprogramming process, to reveal the underlying mechanistic events, and allowed the generation of patient- and disease-specific human iPS cells, which have the potential to be converted into relevant specialized cell types for replacement therapies and disease modeling. This review addresses the steps involved in resetting the epigenetic landscape during reprogramming. Apparently, defined events occur during the course of the reprogramming process. Immediately, upon expression of the reprogramming factors, some cells start to divide faster and quickly begin to lose their differentiated cell characteristics with robust downregulation of somatic genes. Only a subset of cells continue to upregulate the embryonic expression program, and finally, pluripotency genes are upregulated establishing an embryonic stem cell-like transcriptome and epigenome with pluripotent capabilities. Understanding reprogramming to pluripotency will inform mechanistic studies of lineage switching, in which differentiated cells from one lineage can be directly reprogrammed into another without going through a pluripotent intermediate.
Project description:Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.
Project description:Recent attention has focussed on conversion of human pluripotent stem cells to a more naïve transcriptional and epigenetic status. Here we provide a simple method for resetting without transgenes via transient exposure to histone deacetylase inhibition. This protocol is effective across multiple embryo-derived and induced pluripotent stem cells. Reset cells can retain a normal karyotype over multiple passages. Specific markers and global transcriptome profile, including transposable element signature, diverge markedly from conventional human pluripotent stem cells and are consistent with a naïve-like identity. Global DNA methylation is reduced to a level similar to that reported for the inner cell mass, although gain of methylation is also apparent at certain loci. Imprinted methylation is mostly lost, however. In female cells, X chromosome reactivation is evidenced by bi-allelic transcription detected by RNA-FISH. XIST expression is also regained but is predominantly monoallelic. Reset cells are competent for tri-lineage differentiation, both via embryoid body formation and in adherent culture, and can also undergo germline specification. This reliable conversion system and associated transcriptome and methylome datasets will facilitate routine resetting for side by side evaluation with primed pluripotent stem cells and comprehensive interrogation and refinement of candidate naïve pluripotent stem cells.
Project description:As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.
Project description:Enforced ectopic expression of a cocktail of pluripotency-associated genes such as Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). The remarkable proliferation ability of iPSCs and their aptitude to redifferentiate into any cell lineage makes these cells a promising tool for generating a variety of human tissue in vitro. Yet, pluripotency induction is an inefficient process, as cells undergoing reprogramming need to overcome developmentally imposed epigenetic barriers. Recent work has shed new light on the molecular mechanisms that drive the reprogramming of somatic cells to iPSCs. Here, we present current knowledge on the transcriptional and epigenetic regulation of pluripotency induction and discuss how variability in epigenetic states impacts iPSCs' inherent biological properties.
Project description:Although extended pluripotent stem cells (EPSCs) have the potential to form both embryonic and extraembryonic lineages, how their transcriptional regulatory mechanism differs from that of embryonic stem cells (ESCs) remains unclear. Here, we discovered that YY1 binds to specific open chromatin regions in EPSCs. Yy1 depletion in EPSCs leads to a gene expression pattern more similar to that of ESCs than control EPSCs. Moreover, Yy1 depletion triggers a series of epigenetic crosstalk activities, including changes in DNA methylation, histone modifications and high-order chromatin structures. Yy1 depletion in EPSCs disrupts the enhancer-promoter (EP) interactions of EPSC-specific genes, including Dnmt3l. Yy1 loss results in DNA hypomethylation and dramatically reduces the enrichment of H3K4me3 and H3K27ac on the promoters of EPSC-specific genes by upregulating the expression of Kdm5c and Hdac6 through facilitating the formation of CCCTC-binding factor (CTCF)-mediated EP interactions surrounding their loci. Furthermore, single-cell RNA sequencing (scRNA-seq) experiments revealed that YY1 is required for the derivation of extraembryonic endoderm (XEN)-like cells from EPSCs in vitro. Together, this study reveals that YY1 functions as a key regulator of multidimensional epigenetic crosstalk associated with extended pluripotency.
Project description:Mechanisms specifying cancer cell states and response to therapy are incompletely understood. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas, the most common tumors of the peripheral nervous system. We find schwannomas are comprised of 2 molecular groups that are distinguished by activation of neural crest or nerve injury pathways that specify tumor cell states and the architecture of the tumor immune microenvironment. Moreover, we find radiotherapy is sufficient for interconversion of neural crest schwannomas to immune-enriched schwannomas through epigenetic and metabolic reprogramming. To define mechanisms underlying schwannoma groups, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of tumor evolution and establish a paradigm of epigenetic and metabolic reprograming of cancer cells that shapes the immune microenvironment in response to radiotherapy.
Project description:Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination.
Project description:Ideally, disease modeling using patient-derived induced pluripotent stem cells (iPSCs) enables analysis of disease initiation and progression. This requires any pathological features of the patient cells used for reprogramming to be eliminated during iPSC generation. Hutchinson-Gilford progeria syndrome (HGPS) is a segmental premature aging disorder caused by the accumulation of the truncated form of Lamin A known as Progerin within the nuclear lamina. Cellular hallmarks of HGPS include nuclear blebbing, loss of peripheral heterochromatin, defective epigenetic inheritance, altered gene expression, and senescence. To model HGPS using iPSCs, detailed genome-wide and structural analysis of the epigenetic landscape is required to assess the initiation and progression of the disease. We generated a library of iPSC lines from fibroblasts of patients with HGPS and controls, including one family trio. HGPS patient-derived iPSCs are nearly indistinguishable from controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles, and can differentiate into affected cell lineages recapitulating disease progression, despite the nuclear aberrations, altered gene expression, and epigenetic landscape inherent to the donor fibroblasts. These analyses demonstrate the power of iPSC reprogramming to reset the epigenetic landscape to a revitalized pluripotent state in the face of widespread epigenetic defects, validating their use to model the initiation and progression of disease in affected cell lineages.
Project description:Shoot regeneration can be achieved in vitro through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root meristem-gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant-cell pluripotency. These factors might be useful in reprogramming lineage-specified plant cells to pluripotency.
Project description:Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC), which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hESCs, we find that iPSCs tend to form large-scale (100 kb-1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly, these 5hmC aberrant hotspots largely coincide (~80%) with aberrant iPSC-ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC-hESC differences.