Cell reprogramming into the pluripotent state using graphene based substrates

Graphene has been attracting considerable interest in the field of biomedical engineering because graphene and its derivatives are considered to be ideal platforms for supporting cell growth and differentiation. Here we report that graphene promotes the reprogramming of mouse somatic fibroblasts into induced pluripotent stem cells (iPSCs). We constructed a layer of graphene film on a glass substrate and characterized it as a monolayer using Raman spectroscopy. We found that the graphene substrate significantly improved cellular reprogramming efficiency by inducing mesenchymal-to-epithelial-transition (MET) which is known to affect H3K4me3 levels. Thus, our results reveal that a graphene substrate directly regulates dynamic epigenetic changes associated with reprogramming, providing an efficient tool for epigenetic pluripotent reprogramming.     

Dynamic DNA methylation reprogramming: active demethylation and immediate remethylation in the male pronucleus of bovine zygotes.

DNA methylation reprogramming (DMR) is believed to be a key process by which mammalian zygotes gain nuclear totipotency through erasing epigenetic modifications acquired during gametogenesis. Nonetheless, DMR patterns do not seem to be conserved among mammals. To identify uniform rules underlying mammalian DMRs, we explored DMRs of diverse mammalian zygotes. Of the zygotes studied, of particular interest was the bovine zygote; the paternal DNA methylation first decreased and was then rapidly restored almost to the maternal methylation level even before the two-cell stage. The 5-azadeoxycytidine treatment led to complete demethylation of the male pronucleus. The unusually dramatic changes in DNA methylation levels indicate that the bovine male pronucleus undergoes active demethylation, which is followed by de novo methylation. Our results show that, in bovine, the compound processes of active DNA demethylation and de novo DNA methylation, along with de novo H3-K9 trimethylation also, take place altogether within this very narrow window of pronucleus development.     

Global and gene-specific histone modification profiles of mouse multipotent adult germline stem cells

We previously reported the generation of multipotent adult germline stem cells (maGSCs) from spermatogonial stem cells (SSCs) isolated from adult mouse testis. In a later study, we substantiated the pluripotency of maGSCs by demonstrating their close similarity to pluripotent male embryonic stem cells (ESCs) at the epigenetic level of global and gene-specific DNA methylation. Here, we extended the comparative epigenetic analysis of maGSCs and male ESCs by investigating the second main epigenetic modification in mammals, i.e. global and gene-specific modifications of histones (H3K4 trimethylation, H3K9 acetylation, H3K9 trimethylation and H3K27 trimethylation). Using immunofluorescence staining, flow cytometry and western blot analysis, we show that maGSCs are very similar to male ESCs with regard to global levels and nuclear distribution patterns of these modifications. Chromatin immunoprecipitation real-time PCR analysis of these modifications at the gene-specific level further revealed modification patterns of the pluripotency marker genes Oct4, Sox2 and Nanog in maGSCs that are nearly identical to those of male ESCs. These genes were enriched for activating histone modifications including H3K4me3 and H3K9ac and depleted of repressive histone modifications including H3K27me3 and H3K9me3. In addition, Hoxa11, a key regulator of early embryonic development showed the ESC-typical bivalent chromatin conformation with enrichment of both the activating H3K4me3 and the repressive H3K27me3 modification also in maGSCs. Collectively, our results demonstrate that maGSCs also closely resemble ESCs with regard to their chromatin state and further evidence their pluripotent nature.     

Epigenetic signatures and temporal expression of lineage-specific genes in hESCs during differentiation to hepatocytes in vitro

Embryonic stem cells (ESCs) maintain unique epigenetic states to maintain their pluripotency. Differentiation of ESCs into specialized cell types requires changes in these epigenetic states. However, the dynamics of epigenetic marks found in hESCs during differentiation are poorly understood. Here, we report the variation in the dynamics of epigenetic modifications associated with the expression of lineage-specific genes during differentiation of hESCs to hepatocytes in vitro. The promoter regions of pluripotency marker genes characterized by permissive histone marks such as trimethylation of H3 at lysine 4 (H3K4me3) and acetylation of H3 at lysine 9 (H3K9ac) in hESCs were instead enriched with repressive histone marks such as dimethylation of H3 at lysine 9 (H3K9me2), trimethylation of H3 at lysine 9 (H3K9me3) and trimethylation of H3 at lysine 27 (H3K27me3) during differentiation to hepatocytes. Interestingly, expression of definitive endoderm marker genes containing bivalent and non-bivalent domains may be modulated by a marked reduction in H3K27me3 and a significant enhancement of permissive marks such as H3K4me3 and H3K9ac during hESC differentiation. Expression of hepatocyte marker genes regulated by histone modifications was similar to that of pluripotency marker genes. Our findings provide insight into the epigenetic mechanisms regulating expression of developmental genes. Of particular interest, they may be differentially regulated either in a bivalent or non-bivalent domain manner during hESC differentiation.     

Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.     

组蛋白赖氨酸甲基化在表观遗传调控中的作用

组蛋白赖氨酸的甲基化在真核基因表观遗传调控中起着关键作用。迄今已知,在组蛋白H3中有5个赖氨酸(K4、K9、K27、K36、K79)和组蛋白H4中的1个赖氨酸(K20)可被特异的组蛋白赖氨酸甲基转移酶甲基化。这不同位点的甲基化效应是不同的,H3-K9、H3-K27、H4-K20甲基化具有抑制效应;H3-K4、H3-K36、H3-K79甲基化具有激活效应,而且组蛋白甲基化与其它组蛋白共价修饰之间以及DNA甲基化之间存在对话。     

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.     

Phosphorylation of serine‐10 of histone H3 shields modified lysine‐9 selectively during mitosis

Post‐translational modifications of histones play important roles in regulating chromatin dynamics and epigenetic inheritance during mitosis. The epigenetic significance and stability of histone H3‐lysine 9 (H3K9) modifications have been well studied in interphase cells, whereas not as much in mitotic cells. Here, we inspected mitosis‐coupled alterations in the global modifications of H3K9. Signals for H3K9 mono‐, di‐methylation and acetylation became invisible as cells entered mitosis in contrast to the pattern observed for H3‐serine 10 phosphorylation (H3S10ph). Treatment with the aurora‐B inhibitor ZM447439 or expression of the dominant negative mutant Aur‐B^K106R resulted in prometaphase chromosomes that lacked signals for H3S10ph but were positive for H3K9 modifications. Trimethylation was the sole K9 modification that remained consistently detectable throughout the cell cycle. This phenomenon was specific for H3K9‐S10, as this pattern was not observed at H3K27‐S28. Methylated H3K27 remained detectable throughout the cell cycle, despite phosphorylation of the adjacent H3S28. Contrastingly, our dot‐blot experiment using synthetic peptides showed that phosphorylation of serine residue basically kept adjacent lysine from antibody access. Together, these results suggest that phosphorylation of serine residue occurs in a selective manner, being influenced by the types of modifications and the nature of neighboring lysine residues.     

Chromosome-wide regulation of euchromatin-specific 5mC to 5hmC conversion in mouse ES cells and female human somatic cells

DNA cytosine methylation (5mC) is indispensable for a number of cellular processes, including retrotransposon silencing, genomic imprinting, and X chromosome inactivation in mammalian development. Recent studies have focused on 5-hydroxymethylcytosine (5hmC), a new epigenetic mark or intermediate in the DNA demethylation pathway. However, 5hmC itself has no role in pluripotency maintenance in mouse embryonic stem cells (ESCs) lacking Dnmt1, 3a, and 3b. Here, we demonstrated that 5hmC accumulated on euchromatic chromosomal bands that were marked with di- and tri-methylated histone H3 at lysine 4 (H3K4me2/3) in mouse ESCs. By contrast, heterochromatin enriched with H3K9me3, including mouse chromosomal G-bands, pericentric repeats, human satellite 2 and 3, and inactive X chromosomes, was not enriched with 5hmC. Therefore, enzymes that hydroxylate the methyl group of 5mC belonging to the Tet family might be excluded from inactive chromatin, which may restrict 5mC to 5hmC conversion in euchromatin to prevent nonselective de novo DNA methylation.     

Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1/KDM1A

Endogenous retroviruses (ERVs) constitute a substantial portion of mammalian genomes, and their retrotransposition activity helped to drive genetic variation, yet their expression is tightly regulated to prevent unchecked amplification. We generated a series of mouse mutants and embryonic stem (ES) cell lines carrying "deletable" and "rescuable" alleles of the lysine-specific demethylase LSD1/KDM1A. In the absence of KDM1A, the murine endogenous retrovirus MuERV-L/MERVL becomes overexpressed and embryonic development arrests at gastrulation. A number of cellular genes normally restricted to the zygotic genome activation (ZGA) period also become up-regulated in Kdm1a mutants. Strikingly, many of these cellular genes are flanked by MERVL sequences or have cryptic LTRs as promoters that are targets of KDM1A repression. Using genome-wide epigenetic profiling of Kdm1a mutant ES cells, we demonstrate that this subset of ZGA genes and MERVL elements displays increased methylation of histone H3K4, increased acetylation of H3K27, and decreased methylation of H3K9. As a consequence, Kdm1a mutant ES cells exhibit an unusual propensity to generate extraembryonic tissues. Our findings suggest that ancient retroviral insertions were used to co-opt regulatory sequences targeted by KDM1A for epigenetic silencing of cell fate genes during early mammalian embryonic development.     

In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification‐specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac‐Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac‐Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac‐Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.     

Global mapping of H3K4me3 and H3K27me3 reveals chromatin state-based regulation of human monocyte-derived dendritic cells in different environments

Depending on the environment, dendritic cells (DCs) may become active or tolerogenic, but little is known about whether heritable epigenetic modifications are involved in these processes. Here, we have found that epigenetic histone modifications can regulate the differentiation of human monocyte-derived DCs (moDCs) into either activated or tolerized DCs. The inhibition or silencing of methyltransferases or methylation-associated factors affects the expression of multiple genes. Genome mapping of transforming growth factor (TGF-β)- or lipopolysaccharide (LPS)-associated H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) demonstrated the presence of histone modification of gene expression in human TGF-β- or LPS-conditioned moDCs. Although the upregulated or downregulated genes were not always associated with H3K4me3 and/or H3K27me3 modifications in TGF-β-conditioned (tolerized) or LPS-conditioned (activated) moDCs, some of these genes may be regulated by the increased and/or decreased H3K4me3 or H3K27me3 levels or by the alteration of these epigenetic marks, especially in TGF-β-conditioned moDCs. Thus, our results suggested that the differentiation and function of moDCs in tumor and inflammation environments are associated with the modification of the H3K4me3 and K3K27me3 epigenetic marks.     

Global histone modification of histone H3 in colorectal cancer and its precursor lesions

Chromatin remodeling through histone modification is an important mechanism of epigenetic gene dysregulation in human cancers. However, little is known about global alteration of histone status during tumorigenesis and cancer progression. Histone H3 status was examined in benign and malignant colorectal tumors by immunohistochemistry and Western blotting. For immunohistochemical evaluation, 4 anti-histone H3 antibodies, specific to dimethylation at lysine 4 (H3K4me2), acetylation at lysine 9 (H3K9ac), dimethylation at lysine 9 (H3K9me2), and trimethylation at lysine 27 (H3K27me3), were used. On immunohistochemistry, H3K4me2, H3K9ac, and H3K27me3 showed no significant changes between normal and colorectal tumors. On the other hand, the global level of H3K9me2 was distinctly higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells. In addition, it was significantly higher in adenocarcinoma than in adenoma. Correspondingly, Western blotting confirmed that H3K9me2 expression was significantly higher in adenocarcinomas than in normal colorectal mucosa. No alteration of H3K9me2 was observed with tumor differentiation and with the histological subtypes of colorectal cancers. These results suggest that aberration of the global H3K9me2 level is an important epigenetic event in colorectal tumorigenesis and carcinogenesis involved with gene regulation in neoplastic cells through chromatin remodeling.     

Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis.