Epigenetic Barrier Research Articles

Abstract 14745: The Histone Reader PHF7 Cooperates With the SWI/SNF Complex at Cardiac Super Enhancers to Promote Direct Cardiac Reprogramming

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through the overexpression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT), and later, Hand2 (GHMT) and Akt1 (AGHMT) were found to further enhance this process. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogram adult fibroblasts. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, increases chromatin accessibility and transcription factor binding at these sites. Further, PHF7 recruits cardiac transcription factors to activate a core regulatory circuit in reprogramming. Importantly, PHF7 is the first epigenetic factor shown to achieve efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic modifiers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

Chromatin accessibility in canine stromal cells and its implications for canine somatic cell reprogramming.

Naturally occurring disease in pet dogs is an untapped and unique resource for stem cell‐based regenerative medicine translational research, given the many similarities and complexity such disease shares with their human counterparts. Canine‐specific regulators of somatic cell reprogramming and pluripotency maintenance are poorly understood. While retroviral delivery of the four Yamanaka factors successfully reprogrammed canine embryonic fibroblasts, adult stromal cells remained resistant to reprogramming in spite of effective viral transduction and transgene expression. We hypothesized that adult stromal cells fail to reprogram due to an epigenetic barrier. Here, we performed assay for transposase‐accessible chromatin using sequencing (ATAC‐seq) on canine stromal and pluripotent stem cells, analyzing 51 samples in total, and establishing the global landscape of chromatin accessibility before and after reprogramming to induced pluripotent stem cells (iPSC). We also studied adult stromal cells that do not yield iPSC colonies to identify potential reprogramming barriers. ATAC‐seq analysis identified distinct cell type clustering patterns and chromatin remodeling during embryonic fibroblast reprogramming. Compared with embryonic fibroblasts, adult stromal cells had a chromatin accessibility landscape that reflects phenotypic differentiation and somatic cell‐fate stability. We ultimately identified 76 candidate genes and several transcription factor binding motifs that may be impeding somatic cell reprogramming to iPSC, and could be targeted for inhibition or activation, in order to improve the process in canines. These results provide a vast resource for better understanding of pluripotency regulators in dogs and provide an unbiased rationale for novel canine‐specific reprogramming approaches.

Analysis of the Epigenetic Signature of Cell Reprogramming by Computational DNA Methylation Profiles

Background: DNA methylation plays an important role in the reprogramming process. Understanding the underlying molecular mechanism of reprogramming is crucial for answering fundamental questions regarding the transition of cell identity. Methods: In this study, based on the genome-wide DNA methylation data from different cell lines, comparative methylation profiles were proposed to identify the epigenetic signature of cell reprogramming. Results: The density profile of CpG methylation showed that pluripotent cells are more polarized than Human Dermal Fibroblasts (HDF) cells. The heterogeneity of iPS has a greater deviation in the DNA hypermethylation pattern. The result of regional distribution showed that the differential CpG sites between pluripotent cells and HDFs tend to accumulate in the gene body and CpG shelf regions, whereas the internal differential methylation CpG sites (DMCs) of three types of pluripotent cells tend to accumulate in the TSS1500 region. Furthermore, a series of endogenous markers of cell reprogramming were identified based on the integrative analysis, including focal adhesion, pluripotency maintenance and transcription regulation. The calcium signaling pathway was detected as one of the signatures between NT cells and iPS cells. Finally, the regional bias of DNA methylation for key pluripotency factors was discussed. Our studies provide new insight into the barrier identification of cell reprogramming. Conclusion: Our studies analyzed some epigenetic markers and barriers of nuclear reprogramming, hoping to provide new insight into understanding the underlying molecular mechanism of reprogramming.

Tet2 Inactivation Enhances the Anti-Tumor Activity of Tumor-Infiltrating Lymphocytes (TILs) to Curtail Melanoma Growth

Inactivation of tumor infiltrating lymphocytes (TILs) is one of the mechanisms mitigating anti-tumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the anti-tumor activity of TILs, with the efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-seq analysis further revealed that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-seq analysis further demonstrated that Tet2 deletion reshapes the chromatin accessibility and favors the binding of transcription factors geared toward CD8+ T cell activation. In summary, our study establishes that Tet2 constitutes one of the epigenetic barriers contributing to dysfunction of TILs, and that Tet2 inactivation could benefit anti-tumor immunity to boost tumor suppression. Disclosures No relevant conflicts of interest to declare.

Small Molecule Epigenetic Modulators in Pure Chemical Cell Fate Conversion.

Although innovative technologies for somatic cell reprogramming and transdifferentiation provide new strategies for the research of translational medicine, including disease modeling, drug screening, artificial organ development, and cell therapy, recipient safety remains a concern due to the use of exogenous transcription factors during induction. To resolve this problem, new induction approaches containing clinically applicable small molecules have been explored. Small molecule epigenetic modulators such as DNA methylation writer inhibitors, histone methylation writer inhibitors, histone acylation reader inhibitors, and histone acetylation eraser inhibitors could overcome epigenetic barriers during cell fate conversion. In the past few years, significant progress has been made in reprogramming and transdifferentiation of somatic cells with small molecule approaches. In the present review, we systematically discuss recent achievements of pure chemical reprogramming and transdifferentiation.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

The iNs and Outs of Direct Reprogramming to Induced Neurons.

Understanding of cell-type specific transcription factors has promoted progress in methods for cellular reprogramming, such as directly reprogramming somatic cells to induced neurons (iN). Methods for direct reprogramming require neuronal-fate determining gene activation via neuron-specific microRNAs, chemical modulation of key neuronal signaling pathways or overexpression via viral vectors, with some reprogramming strategies requiring a combination of these methods to induce the neuronal-cell fate. These methods have been employed in a multitude of cell types, including fibroblasts, hepatocytes, peripheral blood mononuclear, and T cells. The ability to create iN from skin biopsies and blood samples coupled with recent advancements in artificially inducing age- and disease-associated phenotypes are accelerating the development of disease models for late-onset neurodegenerative disorders. Here, we review how activation of the neuronal transcriptome alters the epigenetic landscape of the donor cell to facilitate reprogramming to neurons. We also discuss the advantages of using DNA binding domains such as CRISPR/dCas9 to overcome epigenetic barriers to induce neuronal-cell fate by activating endogenous neuronal cell-fate determining genes.

Characterization of DNA Methylation Patterns and Mining of Epigenetic Markers During Genomic Reprogramming in SCNT Embryos.

Somatic cell nuclear transfer (SCNT), also known as somatic cell cloning, is a commonly used technique to study epigenetic reprogramming. Although SCNT has the advantages of being safe and able to obtain pluripotent cells, early developmental arrest happens in most SCNT embryos. Overcoming epigenetic barriers is currently the primary strategy for improving reprogramming efficiency and improving developmental rate in SCNT embryos. In this study, we analyzed DNA methylation profiles of in vivo fertilized embryos and SCNT embryos with different developmental fates. Overall DNA methylation level was higher in SCNT embryos during global de-methylation process compared to in vivo fertilized embryos. In addition, promoter region, first intron and 3′UTR were found to be the major genomic regions that were hyper-methylated in SCNT embryos. Surprisingly, we found the length of re-methylated region was directly related to the change of methylation level. Furthermore, a number of genes including Dppa2 and Dppa4 which are important for early zygotic genome activation (ZGA) were not properly activated in SCNT embryos. This study comprehensively analyzed genome-wide DNA methylation patterns in SCNT embryos and provided candidate target genes for improving efficiency of genomic reprogramming in SCNT embryos. Since SCNT technology has been widely used in agricultural and pastoral production, protection of endangered animals, and therapeutic cloning, the findings of this study have significant importance for all these fields.

Massively parallel and time-resolved RNA sequencing in single cells with scNT-seq.

Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-Seq), a method for massively parallel analysis of newly-transcribed and pre-existing mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly-transcribed mRNAs with T-to-C substitutions. Using scNT-Seq, we jointly profiled new and old transcriptomes in ~55,000 single cells. These data revealed time-resolved transcription factor activities and cell state trajectories at single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell-embryo-like (2C-like) stem cell states. Finally, integrating scNT-Seq with genetic perturbation identifies DNA methylcytosine dioxygenases as an epigenetic barrier into 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.

In Vitro Conversion of Murine Fibroblasts into Cardiomyocyte-Like Cells.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise as a potential treatment for cardiovascular disease, many of which are associated with tremendous loss of functional cardiomyocytes and simultaneous formation of scar tissue. Burgeoning studies have shown that the introduction of three minimal transcriptional factors, Gata4, Mef2c, and Tbx5 (G/M/T), could convert murine fibroblasts into iCMs that closely resemble endogenous CMs both in vitro and in vivo. Recent studies on iCM cell fate determination have demonstrated that the removal of genetic and epigenetic barriers could facilitate iCM reprogramming. However, varied reprogramming efficiency among research groups hinders its further study and potential applicability. Here, we provide a newly updated and detailed protocol for in vitro generation and evaluation of functional iCMs from mouse embryonic fibroblasts and neonatal cardiac fibroblasts using retroviral polycistronic construct encoding optimal expression of G/M/T factors. We hope that this optimized protocol will lay the foundation for future mechanistic studies of murine iCMs and further improvement of iCM generation.

Abstract 442: Epigenetic Regulation of Ezh2 in Direct Human Cardiac Reprogramming

Cardiovascular disease is still the leading cause of death in the United States. Due to the limited regenerative capacity of adult hearts, the damage caused by heart injury could not be reversed and often progressed into heart failure. In need of cardiovascular disease treatment, many therapies aimed at either cell transplantation or cell regeneration have been proposed. Direct reprogramming of somatic cells into induced cardiomyocytes (iCMs) is considered to be a promising strategy for regenerative medicine. The induction of cardiomyocytes from non-myocytes has been achieved efficiently via ectopic expression of reprogramming factors both in vitro and in vivo with mice models. However, as human cells are more resistant to the reprogramming process, the generation of human iCMs (hiCMs) has been restricted by the factor that using more complex cocktails generated only functionally immature cells with lower efficiency and longer conversion time. The inefficiency of hiCMs production called for the identification and elucidation of underlying species-specific regulatory mechanisms in human, and removal of the additional epigenetic barriers which might be damping the hiCMs reprogramming. Here, we identified a human-specific epigenetic barrier, Enhancer of zesta homolog 2 (EZH2), via an unbiased loss-of-function screening. With the knockdown of EZH2, the hiCM reprogramming efficiency was significantly increased, accompanied with profound repression of collagen and extracellular matrix genes, which are related to the formation of fibrosis. Consistently, Inhibition of EZH2 catalytic activity via small molecules promotes hiCM reprogramming, suggesting that EZH2’s inhibitory effect was mediated by epigenetic regulation of histone modifications. Therefore, our study revealed a previously unrecognized regulatory mechanism of human cardiac reprogramming, which allows us to overcome the fibroblast fate barriers and ease the cardiac cell fate conversion.

Specific patterns of H3K79 methylation influence genetic interaction of oncogenes in AML

AbstractUnderstanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation.

Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

Overcoming Intrinsic H3K27me3 Imprinting Barriers Improves Post-implantation Development after Somatic Cell Nuclear Transfer.

Successful cloning by somatic cell nuclear transfer (SCNT) requires overcoming significant epigenetic barriers. Genomic imprinting is not generally regarded as such a barrier, although H3K27me3-dependent imprinting is differentially distributed in E6.5 epiblast and extraembryonic tissues. Here we report significant enhancement of SCNT efficiency by deriving somatic donor cells carrying simultaneous monoallelic deletion of four H3K27me3-imprinted genes from haploid mouse embryonic stem cells. Quadruple monoallelic deletion of Sfmbt2, Jade1, Gab1, and Smoc1 normalized H3K27me3-imprinted expression patterns and increased fibroblast cloning efficiency to 14% compared with a 0% birth rate from wild-type fibroblasts while preventing the placental and body overgrowth defects frequently observed in cloned animals. Sfmbt2 deletion was the most effective of the four individual gene deletions in improving SCNT. These results show that lack of H3K27me3 imprinting in somatic cells is an epigenetic barrier that impedes post-implantation development of SCNT embryos and can be overcome by monoallelic imprinting gene deletions in donor cells.

SiRNA inhibition and not chemical inhibition of Suv39h1/2 enhances pre-implantation embryonic development of bovine somatic cell nuclear transfer embryos.

The efficiency of somatic cell nuclear transfer (SCNT) is low due to the strong resistance of somatic donor cells to epigenetic reprogramming. Many epigenetic drugs targeting DNA methylation and histone acetylation have been used in attempts to improve the in vitro and in vivo development of SCNT embryos. H3K9me3 has been shown to be an important reprogramming barrier for generating induced pluripotent stem cells (iPSCs) and SCNT embryos in mice and humans. In this study, we examined the effects of selective siRNA and chemical inhibition of H3K9me3 in somatic donor cells on the in vitro development of bovine SCNT embryos. Chaetocin, an inhibitor of SUV39H1/H2, was supplemented during the culture of donor cells. In addition, the siRNA knockdown of SUV39H1/H2 was performed in the donor cells. The effects of chaetocin and siSUV39H1/H2 on H3K9me3 and H3K9ac were quantified using flow cytometry. Furthermore, we assessed chaetocin treatment and SUV39H1/H2 knockdown on the blastocyst formation rate. Both chaetocin and siSUV39H1/H2 significantly reduced and elevated the relative intensity level of H3K9me3 and H3K9ac in treated fibroblast cells, respectively. siSUV39H1/H2 transfection, but not chaetocin treatment, improved the in vitro development of SCNT embryos. Moreover, siSUV39H1/H2 altered the expression profile of the selected genes in the derived blastocysts, similar to those derived from in vitro fertilization (IVF). In conclusion, our results demonstrated H3K9me3 as an epigenetic barrier in the reprogramming process mediated by SCNT in bovine species, a finding which supports the role of H3K9me3 as a reprogramming barrier in mammalian species. Our findings provide a promising approach for improving the efficiency of mammalian cloning for agricultural and biomedical purposes.

Epigenetic Features of Human Perinatal Stem Cells Redefine Their Stemness Potential.

Human perinatal stem cells (SCs) can be isolated from fetal annexes without ethical or safety limitations. They are generally considered multipotent; nevertheless, their biological characteristics are still not fully understood. The aim of this study was to investigate the pluripotency potential of human perinatal SCs as compared to human induced pluripotent stem cells (hiPSCs). Despite the low expression of the pluripotent factors NANOG, OCT4, SOX2, and C-KIT in perinatal SC, we observed minor differences in the promoters DNA-methylation profile of these genes with respect to hiPSCs; we also demonstrated that in perinatal SCs miR-145-5p had an inverse trend in comparison to these stemness markers, suggesting that NANOG, OCT4, and SOX2 were regulated at the post-transcriptional level. The reduced expression of stemness markers was also associated with shorter telomere lengths and shift of the oxidative metabolism between hiPSCs and fetal annex-derived cells. Our findings indicate the differentiation ability of perinatal SCs might not be restricted to the mesenchymal lineage due to an epigenetic barrier, but other regulatory mechanisms such as telomere shortening or metabolic changes might impair their differentiation potential and challenge their clinical application.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

AP-1 Contributes to Chromatin Accessibility to Promote Sarcomere Disassembly and Cardiomyocyte Protrusion During Zebrafish Heart Regeneration.

The adult human heart is an organ with low regenerative potential. Heart failure following acute myocardial infarction is a leading cause of death due to the inability of cardiomyocytes to proliferate and replenish lost cardiac muscle. While the zebrafish has emerged as a powerful model to study endogenous cardiac regeneration, the molecular mechanisms by which cardiomyocytes respond to damage by disassembling sarcomeres, proliferating, and repopulating the injured area remain unclear. Furthermore, we are far from understanding the regulation of the chromatin landscape and epigenetic barriers that must be overcome for cardiac regeneration to occur. To identify transcription factor regulators of the chromatin landscape, which promote cardiomyocyte regeneration in zebrafish, and investigate their function. Using the Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq), we first find that the regenerating cardiomyocyte chromatin accessibility landscape undergoes extensive changes following cryoinjury, and that activator protein-1 (AP-1) binding sites are the most highly enriched motifs in regions that gain accessibility during cardiac regeneration. Furthermore, using bioinformatic and gene expression analyses, we find that the AP-1 response in regenerating adult zebrafish cardiomyocytes is largely different from the response in adult mammalian cardiomyocytes. Using a cardiomyocyte-specific dominant negative approach, we show that blocking AP-1 function leads to defects in cardiomyocyte proliferation as well as decreased chromatin accessibility at the fbxl22 and ilk loci, which regulate sarcomere disassembly and cardiomyocyte protrusion into the injured area, respectively. We further show that overexpression of the AP-1 family members Junb and Fosl1 can promote changes in mammalian cardiomyocyte behavior in vitro. AP-1 transcription factors play an essential role in the cardiomyocyte response to injury by regulating chromatin accessibility changes, thereby allowing the activation of gene expression programs that promote cardiomyocyte dedifferentiation, proliferation, and protrusion into the injured area.

DNA demethylation is a driver for chick retina regeneration

ABSTRACT Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectomy, via retinal pigment epithelium (RPE) reprogramming in the presence of FGF2. In this study, we systematically analysed the reprogramming competent chick RPE prior to injury, and during different stages of reprogramming. In addition to changes in the expression of genes associated with epigenetic modifications during RPE reprogramming, we observed dynamic changes in histone marks associated with bivalent chromatin (H3K27me3/H3K4me3) and intermediates of the process of DNA demethylation including 5hmC and 5caC. Comprehensive analysis of the methylome by whole-genome bisulphite sequencing (WGBS) confirmed extensive rearrangements of DNA methylation patterns including differentially methylated regions (DMRs) found at promoters of genes associated with chromatin organization and fibroblast growth factor production. We also identified Tet methylcytosine dioxygenase 3 (TET3) as an important factor for DNA demethylation and retina regeneration, capable of reprogramming RPE in the absence of exogenous FGF2. In conclusion, we demonstrate that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation. In the presence of FGF2, these dynamic modifications are further sustained in the commitment to form a new retina. Our findings reveal active DNA demethylation as an important process that may be applied to remove the epigenetic barriers in order to regenerate retina in mammals. Abbreviations bp: Base pair; DMR: Differentially methylated region; DMC: Differentially methylated cytosines; GFP: Green fluorescent protein; PCR: Polymerase chain reaction. TET: Ten-eleven translocation; RPE: retinal pigment epithelium.

Wnt-3a Induces Epigenetic Remodeling in Human Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) from adult teeth show the expression of a very complete repertoire of stem pluripotency core factors and a high plasticity for cell reprogramming. Canonical Wnt and Notch signaling pathways regulate stemness and the expression of pluripotency core factors in DPSCs, and even very short-term (48 h) activations of the Wnt pathway induce a profound remodeling of DPSCs at the physiologic and metabolic levels. In this work, DPSC cultures were exposed to treatments modulating Notch and Wnt signaling, and also induced to differentiate to osteo/adipocytes. DNA methylation, histone acetylation, histone methylation, and core factor expression levels where assessed by mass spectroscopy, Western blot, and qPCR. A short-term activation of Wnt signaling by WNT-3A induced a genomic DNA demethylation, and increased histone acetylation and histone methylation in DPSCs. The efficiency of cell reprogramming methods relies on the ability to surpass the epigenetic barrier, which determines cell lineage specificity. This study brings important information about the regulation of the epigenetic barrier by Wnt signaling in DPSCs, which could contribute to the development of safer and less aggressive reprogramming methodologies with a view to cell therapy.