DNA methylation-mediated control of Sry gene expression in mouse gonadal development

DNA methylation at CpG sequences is involved in tissue-specific and developmentally regulated gene expression. The Sry (sex-determining region on the Y chromosome) gene encodes a master protein for initiating testis differentiation in mammals, and its expression is restricted to gonadal somatic cells at 10.5-12.5 days post-coitum (dpc) in the mouse. We found that in vitro methylation of the 5'-flanking region of the Sry gene caused suppression of reporter activity, implying that Sry gene expression could be regulated by DNA methylation-mediated gene silencing. Bisulfite restriction mapping and sodium bisulfite sequencing revealed that the 5'-flanking region of the Sry gene was hypermethylated in the 8.5-dpc embryos in which the Sry gene was not expressed. Importantly, this region was specifically hypomethylated in the gonad at 11.5 dpc, while the hypermethylated status was maintained in tissues that do not express the Sry gene. We concluded that expression of the Sry gene is under the control of an epigenetic mechanism mediated by DNA methylation.     

DNA methylation microarray uncovers a permissive methylome for cardiomyocyte differentiation in human mesenchymal stem cells

Differentiation of Wharton's Jelly-Mesenchymal Stem cells (WJ-MSCs) into cardiomyocytes (CMs) in vitro has been reported widely although contradictions remain regarding the maturation of differentiated MSCs into fully functioning CMs. Studies suggest that use of epigenetic modifiers like 5′Azacytidine (5-AC) in MSCs de-methylates DNA and results in expression of cardiac-specific genes (CSGs). However, only partial expression of the CSG set leads to incomplete differentiation of WJ-MSCs to CMs. We used the Agilent 180 K human DNA methylation microarray on WJ-MSCs, 5-AC treated WJ-MSCs and human cardiac tissue (hCT) to analyze differential DNA methylation profiles which were then validated by bisulfite sequencing PCR (BSP). BSP confirmed that only a limited number of CSGs were de-methylated by 5-AC in WJ-MSCs. It also revealed that hCT displays a methylation profile similar to promoter regions of CSG in untreated WJ-MSCs. Thus, the presence of hypo-methylated CSGs indicates that WJ-MSCs are ideal cell types for cardiomyogenic differentiation.     

Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells

DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten–eleven translocation (TET) family proteins converted 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1) promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA.     

Plasticity of DNA methylation in mouse T cell activation and differentiation

Background

Circulating CD4⁺ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4⁺ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results

Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion

We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.     

Generation of Corneal Epithelial Cells from Induced Pluripotent Stem Cells Derived from Human Dermal Fibroblast and Corneal Limbal Epithelium

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6⁺/K12⁺ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.     

Differential methylation of TSP50 and mTSP50 genes in different types of human tissues and mouse spermatic cells

Earlier studies identified human TSP50 as a testis-specific gene that encoded a threonine protease. Most importantly, TSP50 could be a cancer/testis antigen since there was a high frequency of reactivation in breast cancer biopsies. It was also found to be negatively regulated by the p53 gene. To further characterize this gene, we recently examined the DNA methylation patterns of the TSP50 gene promoter in normal human testis, as well as breast tissue and a testicular embryonic carcinoma cell line (HTECCL). Bisulfite genomic sequencing results demonstrated that the promoter exhibited mixed DNA methylation patterns in normal human testis, mainly non-methylation versus slight methylation, which could be attributed to the different stages spermatic cells go through during spermatogenesis. In contrast, it was methylated to a much greater extent in both breast tissue and HTECCL. To find out whether DNA methylation status was related to spermatogenesis stages, we analyzed DNA methylation patterns of the mTSP50 (the mouse ortholog of TSP50) promoter in spermatocytes and spermatozoa isolated from sexually mature mice. The results clearly demonstrated that each group of cells exhibited its preferential DNA methylation pattern that apparently was consistent with the gene expression status observed before. Taken together, our findings suggested that DNA methylation might regulate the TSP50 and mTSP50 gene expressions in different types of tissues and spermatic cells.     

Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses

Differentiation of embryonic stem (ES) cells into embryoid bodies (EBs) provides an in vitro system for the study of early lineage determination during mammalian development. We have previously reported that there are 247 CpG islands that potentially have tissue-dependent and differentially methylated regions (T-DMRs). This provided evidence that the formation of DNA methylation patterns at CpG islands is a crucial epigenetic event underlying mammalian development. Here we present an analysis by the restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme of the genome-wide methylation status of CpG islands of ES cells and EBs and of teratomas produced from ES cells. These results are considered in relation to the methylation status of CpG islands of genomic DNA from normal fetus (10.5 dpc) and adult tissues. We have prepared a DNA methylation panel that consists of 259 T-DMRs and includes novel T-DMRs that are distinctly methylated or unmethylated in the teratomas. The DNA methylation pattern was complex and differed for the ES cells, EBs, and teratomas, providing evidence that differentiation of cells involves both de novo DNA methylation as well as demethylation. Comparison of the numbers of T-DMRs, that were differentially methylated or unmethylated among the cells and tissue types studied, revealed that the teratomas were the most epigenetically different from ES cells. Thus, analysis of the DNA methylation profiles prepared in this study provides new insights into the differentiation of ES cells and development of fetus, EB, teratoma, and somatic tissues.     

Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2^high), differentiated cells (EphB2^negative), Apc^Min/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in Apc^Min/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.     

Genome-wide mapping of DNA methylation in Nile Tilapia

Cytosine DNA methylation is crucial for gene regulation and maintenance of genome stability. However, the detailed nile tilapia methylome remains uncharacterized. In this study, we present the first high-resolution methylome of tilapia gonad generated using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing. In the ovary, 265 and 56 methylation peaks were identified in the genebody and promoter region of 145 genes, respectively. In the testis, 293 and 80 methylation peaks were identified in the genebody and promoter region of 144 genes. Furthermore, 8 and 49 genes showed differentially higher and lower promoter-region methylation rates, respectively, in the ovary relative to those of the testis. Quantitative PCR results revealed that the expression level of fibroblast growth factor 16 (fgf16), sialidase-3-like, fibroblast growth factor 20, aromatase (cyp19a), estrogen receptor, and gonadotropin receptor II precursor were negatively correlated to their methylation levels in the ovary and testis. The methylated levels of cyp19a and fgf16 were validated by bisulfite sequencing PCR technology, and the results were consistent with the MeDIP results. Thus, apart from generating the first methylation map, this study produced a candidate gene repository that provides additional options to explore the relationship between DNA methylation and sex differentiation or maintenance.     

Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development

During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1 ^R132) and IDH2 (IDH2 ^R172) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.     

Differential regulation of DNA methylation in rat testis and its regulation by gonadotropic hormones

Eukaryotic DNA methylation occurs exclusively at the 5'-position of cytosine and has been implicated in the regulation of gene expression. Using high-performance liquid chromatography, the methylation of testis DNA during its development, in different cell populations and during regulation by gonadotropic hormones, were studied. The 5-mC content of testis DNA increased significantly from days 30 to days 150, while in 2-yr-old testis 5-mC content decreased significantly. Among various populations of testicular cells, pachytene spermatocyte DNA contained a significantly high amount of 5-mC when compared to spermatogonia, spermatids and mature sperm DNA. However, the 5-mC content of elongated spermatids was significantly less when compared to the above four fractions. Administration of follicle stimulating hormone to immature rats caused hypomethylation of seminiferous tubular DNA while luteinizing hormone caused similar effects in Leydig cells. These results indicate that in testis, DNA methylation is differentially regulated during development and is controlled by gonadotropic hormones.     

Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation

Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5^mC) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.     

翘嘴鲌胰岛素样生长因子igf3基因的克隆及表达分析

为研究翘嘴鲌(Culter alburnus Basilewsky)胰岛素样生长因子(Insulin-like growth factor 3,igf3)基因在性别调控过程中的作用,基于分子克隆手段RT-PCR和RACE技术相结合方法扩增到翘嘴鲌igf3基因完整cDNA序列,利用qRT-PCR技术检测其在不同组织中的表...