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Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency.

ABSTRACT: Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.

SUBMITTER: Vidal SE

PROVIDER: S-EPMC7724475 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Context-dependent requirement of H3K9 dimethyltransferase activity during reprogramming to pluripotency

Project description:The methylation of histone 3 at lysine 9 (H3K9) is widely regarded as a major roadblock for cellular reprogramming and interference with associated methyltransferases such as EHMT1 and EHMT2 (also known as GLP and G9A) facilitates the derivation of induced pluripotent stem cells (iPSCs). In addition, activation of demethylases by ascorbic acid (AA) has become a common approach to facilitate the extensive epigenetic remodeling required for iPSC formation. The functional interaction between the H3K9 methylation machinery and AA-stimulated enzymes, and consequences of interfering with these pathways, remain insufficiently explored. Here, we show that reduction of EHMT1/2 activity paradoxically counteracts iPSC formation in an optimized reprogramming system in the presence of AA. At the cellular and molecular level, EHMT1/2 activity is required for efficient downregulation of somatic genes and transition into an epithelial state. Genetic interference with the demethylase KDM3B partially rescues the adverse effect of EHMT1/2 inhibition on iPSC formation. Transient inhibition of EHMT1/2 during reprogramming yields iPSCs that fail to efficiently give rise to viable mice, suggesting persistent molecular defects in these cells. Together, our observations document a more nuanced function of H3K9 methyltransferases during iPSC formation and suggest that the balancing of AA-stimulated enzymes by EHMT1/2 is required for efficient and error-free reprogramming to pluripotency.

2020-09-28 | GSE130490 | GEO

Context-dependent requirement of H3K9 dimethyltransferase activity during reprogramming to pluripotency

Project description:Context-dependent requirement of H3K9 dimethyltransferase activity during reprogramming to pluripotency

| PRJNA540469 | ENA

Context-Dependent Functions of NANOG Phosphorylation in Pluripotency and Reprogramming.

Project description:The core pluripotency transcription factor NANOG is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Although NANOG is phosphorylated at multiple residues, the role of NANOG phosphorylation in ESC self-renewal is incompletely understood, and no information exists regarding its functions during reprogramming. Here we report our findings that NANOG phosphorylation is beneficial, although nonessential, for ESC self-renewal, and that loss of phosphorylation enhances NANOG activity in reprogramming. Mutation of serine 65 in NANOG to alanine (S65A) alone has the most significant impact on increasing NANOG reprogramming capacity. Mechanistically, we find that pluripotency regulators (ESRRB, OCT4, SALL4, DAX1, and TET1) are transcriptionally primed and preferentially associated with NANOG S65A at the protein level due to presumed structural alterations in the N-terminal domain of NANOG. These results demonstrate that a single phosphorylation site serves as a critical interface for controlling context-dependent NANOG functions in pluripotency and reprogramming.

| S-EPMC5425684 | biostudies-literature

Context-Dependent Impact of RAS Oncogene Expression on Cellular Reprogramming to Pluripotency.

Project description:Induction of pluripotency in somatic cells with defined genetic factors has been successfully used to investigate the mechanisms of disease initiation and progression. Cellular reprogramming and oncogenic transformation share common features; both involve undergoing a dramatic change in cell identity, and immortalization is a key step for cancer progression that enhances reprogramming. However, there are very few examples of complete successful reprogramming of tumor cells. Here we address the effect of expressing an active oncogene, RAS, on the process of reprogramming and found that, while combined expression with reprogramming factors enhanced dedifferentiation, expression within the context of neoplastic transformation impaired reprogramming. RAS induces expression changes that promote loss of cell identity and acquisition of stemness in a paracrine manner and these changes result in reprogramming when combined with reprogramming factors. When cells carry cooperating oncogenic defects, RAS drives cells into an incompatible cellular fate of malignancy.

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Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events.

Project description:Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to histones H3 and H4, thereby creating rare histone biotinylation marks in the epigenome. These marks co-localize with K9-methylated histone H3 (H3K9me), an abundant gene repression mark. The abundance of H3K9me marks in transcriptionally competent loci decreases when HCS is knocked down and when cells are depleted of biotin. Here we tested the hypothesis that the creation of H3K9me marks is at least partially explained by physical interactions between HCS and histone-lysine N-methyltransferases. Using a novel in silico protocol, we predicted that HCS-interacting proteins contain a GGGG(K/R)G(I/M)R motif. This motif, with minor variations, is present in the histone-lysine N-methyltransferase EHMT1. Physical interactions between HCS and the N-terminal, ankyrin and SET domains in EHMT1 were confirmed using yeast-two-hybrid assays, limited proteolysis assays and co-immunoprecipitation. The interactions were stronger between HCS and the N-terminus in EHMT1 compared with the ankyrin and SET domains, consistent with the localization of the HCS-binding motif in the EHMT1 N-terminus. HCS has the catalytic activity to biotinylate K161 within the binding motif in EHMT1. Mutation of K161 weakened the physical interaction between EHMT1 and HCS, but it is unknown whether this effect was caused by loss of biotinylation or loss of the motif. Importantly, HCS knockdown decreased the abundance of H3K9me marks in repeats, suggesting that HCS plays a role in creating histone methylation marks in these loci. We conclude that physical interactions between HCS and EHMT1 mediate epigenomic synergies between biotinylation and methylation events.

| S-EPMC3633699 | biostudies-literature

Augmented Production of Platelets From Cord Blood With Euchromatic Histone Lysine Methyltransferase Inhibition.

Project description:Cord blood hematopoietic stem/progenitor cells (CB-HSPCs) have emerged as a promising supply for functional platelets to potentially alleviate the increasing demand for platelet transfusions, but the clinical application has been limited by the undefined molecular mechanism and insufficient platelet production. Here, we performed single-cell profiling of more than 16 160 cells to construct a dynamic molecular landscape of human megakaryopoiesis from CB-HSPCs, enabling us to uncover, for the first time, cellular heterogeneity and unique features of neonatal megakaryocytes (MKs) and to also offer unique resources for the scientific community. By using this model, we defined the genetic programs underlying the differentiation process from megakaryocyte-erythroid progenitors (MEPs) to MKs via megakaryocyte progenitors (MKPs) and identified inhibitors of euchromatic histone lysine methyltransferase (EHMT), which, when applied at the early stage of differentiation, significantly increase the final platelet production. At the mechanistic level, we found that EHMT inhibitors act to selectively induce the expansion of MEPs and MKPs. Together, we uncover new mechanistic insights into human megakaryopoiesis and provide a novel chemical strategy for future large-scale generation and clinical applications of platelets.

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Histone deacetylase inhibitors in cell pluripotency, differentiation, and reprogramming.

Project description:Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies.

| S-EPMC3328162 | biostudies-other

Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1?.

Project description:Activity-dependent transcription is critical for the regulation of long-term synaptic plasticity and plastic rewiring in the brain. Here, we report that the transcription of neurexin1? (nrxn1?), a presynaptic adhesion molecule for synaptic formation, is regulated by transient neuronal activation. We showed that 10?minutes of firing at 50?Hz in neurons repressed the expression of nrxn1? for 24?hours in a primary cortical neuron culture through a transcriptional repression mechanism. By performing a screening assay using a synthetic zinc finger protein (ZFP) to pull down the proteins enriched near the nrxn1? promoter region in vivo, we identified that Ash1L, a histone methyltransferase, is enriched in the nrxn1? promoter. Neuronal activity triggered binding of Ash1L to the promoter and enriched the histone marker H3K36me2 at the nrxn1? promoter region. Knockout of Ash1L in mice completely abolished the activity-dependent repression of nrxn1?. Taken together, our results reveal that a novel process of activity-dependent transcriptional repression exists in neurons and that Ash1L mediates the long-term repression of nrxn1?, thus implicating an important role for epigenetic modification in brain functioning.

| S-EPMC4882582 | biostudies-literature

Histone H1 variants are differentially expressed and incorporated into chromatin during differentiation and reprogramming to pluripotency.

Project description:There are seven linker histone variants in human somatic cells (H1.0 to H1.5 and H1X), and their prevalence varies as a function of cell type and differentiation stage, suggesting that the different variants may have distinct roles. We have revisited this notion by using new methodologies to study pluripotency and differentiation, including the in vitro differentiation of human embryonic stem (ES) and teratocarcinoma cells and the reprogramming of keratinocytes to induced pluripotent stem cells. Our results show that pluripotent cells (PCs) have decreased levels of H1.0 and increased levels of H1.1, H1.3, and H1.5 compared with differentiated cells. PCs have a more diverse repertoire of H1 variants, whereas in differentiated cells, H1.0 expression represents ?80% of the H1 transcripts. In agreement with their prevalent expression in ES cells, the regulatory regions of H1.3 and H1.5 genes were found to be occupied by pluripotency factors. Moreover, the H1.0 gene promoter contains bivalent domains (H3K4me2 and H3K27me3) in PCs, suggesting that this variant is likely to have an important role during differentiation. Indeed, the knockdown of H1.0 in human ES did not affect self-renewal but impaired differentiation. Accordingly, H1.0 was recruited to the regulatory regions of differentiation and pluripotency genes during differentiation, confirming that this histone variant plays a critical role in the regulation of these genes. Thus, histone H1 variant expression is controlled by a variety of mechanisms that produce distinct but consistent H1 repertoires in pluripotent and differentiated cells that appear critical to maintain the functionality of such cells.

| S-EPMC3195578 | biostudies-literature

Reprogramming towards pluripotency requires AID-dependent DNA demethylation.

Project description:Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

| S-EPMC2906123 | biostudies-literature

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