Hepatic ontogeny and tissue distribution of mRNAs of epigenetic modifiers in mice using RNA-sequencing

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1–5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1–3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development.     

Zebra fish Dnmt1 and Suv39h1 regulate organ-specific terminal differentiation during development

DNA methylation and histone methylation are two key epigenetic modifications that help govern heterochromatin dynamics. The roles for these chromatin-modifying activities in directing tissue-specific development remain largely unknown. To address this issue, we examined the roles of DNA methyltransferase 1 (Dnmt1) and the H3K9 histone methyltransferase Suv39h1 in zebra fish development. Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the intestine, exocrine pancreas, and retina. Interestingly, not all tissues required Dnmt1, as differentiation of the liver and endocrine pancreas appeared normal. Proper differentiation depended on Dnmt1 catalytic activity, as Dnmt1 morphants could be rescued by active zebra fish or human DNMT1 but not by catalytically inactive derivatives. Dnmt1 morphants exhibited dramatic reductions of both genomic cytosine methylation and genome-wide H3K9 trimethyl levels, leading us to investigate the overlap of in vivo functions of Dnmt1 and Suv39h1. Embryos lacking Suv39h1 had organ-specific terminal differentiation defects that produced largely phenocopies of Dnmt1 morphants but retained wild-type levels of DNA methylation. Remarkably, suv39h1 overexpression rescued markers of terminal differentiation in Dnmt1 morphants. Our results suggest that Dnmt1 activity helps direct histone methylation by Suv39h1 and that, together, Dnmt1 and Suv39h1 help guide the terminal differentiation of particular tissues.     

DNA methyltransferase 3b regulates nerve growth factor-induced differentiation of PC12 cells by recruiting histone deacetylase 2

To elucidate the role of epigenetic reprogramming in cell- or tissue-specific differentiation, we explored the role of DNA methyltransferases (Dnmts) in the nerve growth factor (NGF)-induced differentiation of PC12 (pheochromocytoma) cells into neuronal cells. The mRNA and protein levels of de novo methyltransferase Dnmt3b increased, whereas those of Dnmt3a and Dnmt1 decreased, during NGF-induced neurite outgrowth. Dnmt3b localized in the nucleus, as well as in the growing neurites. When the expression of Dnmt3b was inhibited by antisense or small interfering RNA, PC12 cells continued to proliferate and failed to generate neurites. Cells depleted of Dnmt3b were unable to exit the cell cycle even after 6 days of NGF treatment. Furthermore, this failure in differentiation correlated with significant attenuation in tyrosine phosphorylation of TrkA (a marker for NGF-induced differentiation) and reduced the expression of neuronal markers, Hu antigen, and MAP2. The methyl-CpG content of the PC12 genome or the methylation status of repetitive elements was not significantly altered after differentiation and was not affected by Dnmt3b depletion. This was consistent with the ability of the catalytic-site mutant of Dnmt3b to induce differentiation in Dnmt3b-depleted cells after NGF treatment. The Dnmt3b-mediated differentiation was attributed to its N-terminal domain, which recruits histone deacetylase 2 (Hdac2), as demonstrated by (i) impeding of differentiation by the Hdac inhibitors, (ii) facilitation of the differentiation process by overexpression of the N-terminal domain of Dnmt3b, (iii) higher Hdac activity associated with Dnmt3b after NGF treatment, and (iv) coimmunoprecipitation and cosedimentation of Dnmt3b specifically with Hdac2 in a glycerol density gradient. These data indicate a novel role of Dnmt3b in neuronal differentiation.     

HP1γ links histone methylation marks to meiotic synapsis in mice

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.     

HP1 proteins--what is the essential interaction?

There are three mammalian HP1 genes, Cbx5 (encoding HP1alpha), Cbx1 (encoding HP1beta) and Cbx3 (encoding HP1gamma). Despite their high degree of sequence homology mutational analysis has revealed different phenotypes indicating that they possess different functions. Notably, the Cbx1 mutation is lethal in its homozygous condition. The Cbx1 null phenotype is therefore more severe than the Suv(3)9h1/h2 double-mutant mouse, indicating that the essential function of the Cbx1 gene product, HP1beta, is likely to lie outside its interaction with the heterochromatic H3K9me3 determinant of the "histone code" imposed by the Suv(3)9h1/h2 HMTases. Comparisons of HPI mutants in flies and fungi with corresponding mutations in Suv(3)9 genes show that HP1 mutations are invariably more severe than mutation in Suv(3)9 genes. The implications of these data for HP1 function are discussed.     

Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling

Recent emerging evidences revealed that epigenetic methylation of histone and DNA regulates the lineage commitment of mesenchymal progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 7A (KDM7A) on osteogenic and adipogenic differentiation. Kdm7a expression was up‐regulated in primary marrow stromal cells and established stromal ST2 line after adipogenic and osteogenic treatment. Silencing of endogenous Kdm7a in the cells blocked adipogenic differentiation whereas promoted osteogenic differentiation. Conversely, overexpression of wild‐type Kdm7a in the progenitor cells enhanced adipogenic differentiation whereas inhibited osteogenic differentiation. However, the effect of KDM7A on cell differentiation was largely attenuated when the point mutation was made that abolishes enzymatic activity of KDM7A. Mechanism investigations revealed that silencing of Kdm7a down‐regulated the expression of the CCAAT/enhancer binding protein α (C/EBPα) and secreted frizzled‐related protein 1 (Sfrp1). Chromatin immunoprecipitation (ChIP) assay revealed that KDM7A directly binds to the promoters of C/EBPα and Sfrp1 and removes the histone methylation marks H3K9me2 and H3K27me2. Furthermore, silencing of Kdm7a activated canonical Wnt signalling. Thereafter, activation of canonical Wnt signalling through silencing of Sfrp1 in ST2 attenuated the stimulation of adipogenic differentiation and inhibition of osteogenic differentiation by KDM7A. Our study suggests that KDM7A balances adipogenic and osteogenic differentiation from progenitor cells through epigenetic control of C/EBPα and canonical Wnt signalling and implicates that control of KDM7A action has an epigenetic perspective of curtailing metabolic disorders like osteoporosis.     

HP1 proteins—What is the essential interaction?

There are three mammalian HP1 genes, Cbx5 (encoding HP1α), Cbx1 (encoding HP1β) and Cbx3 (encoding (HP1γ). Despite their high degree of sequence homology mutational analysis has revealed different phenotypes indicating that they possess different functions. Notably, the Cbx1 mutation is lethal in its homozygous condition. The Cbx1 null phenotype is therefore more severe than the Suv(3)9h1/h2 double-mutant mouse, indicating that the essential function of the Cbx1 gene product, HP1β, is likely to lie outside its interaction with the heterochromatic H3K9me3 determinant of the “histone code” imposed by the Suv(3)9h1/h2 HMTases. Comparisons of HP1 mutants in flies and fungi with corresponding mutations in Suv(3)9 genes show that HP1 mutations are invariably more severe than mutation in Suv(3)9 genes. The implications of these data for HP1 function are discussed.     

Thymoquinone reduces intracytoplasmic oxidative stress and improves epigenetic modification in polycystic ovary syndrome mice oocytes,during in‐vitro maturation

Although in‐vitro maturation (IVM) of oocytes has been presented as an alternative treatment to traditional stimulated in‐vitro fertilization, the culture condition can be improved by natural antioxidants. Thus, we investigated the protective effect of Thymoquinone (TQ) during IVM in the polycystic ovary syndrome (PCOS) mice model. The induction of PCOS was made by dehydroepiandrosterone via subcutaneous injection, in prepubertal female B6D2F1‐mice. After 21 days later, germinal vesicle (GV)‐stage‐oocytes were extracted and incubated in IVM media containing 0, 1.0, 10.0, and 100.0 μM of TQ. To assess fertilization and blastulation rates, after 22–24 hr, the treated oocytes were fertilized in‐vitro with epididymal spermatozoa. Some other oocytes were evaluated for maturation, epigenetic, and oxidative stress markers. Similarly, the mRNA expression of epigenetic enzymes genes (Dnmt1 and Hdac1), three maternally derived genes (Mapk, CyclinB, and Cdk1) and apoptosis‐related genes (Bax and Bcl2) were assessed. Our results showed that the maturation, fertilization, and blastulation rates were significantly higher in the 10.0 μM TQ‐treated group compared with the untreated group and likewise with in‐vivo matured oocytes. The Bax expression was reduced in 10.0 μM TQ matured oocytes, but Bcl2, Dnmt1, Hdac1, Cdk1, and Mapk were upregulated in this group compared to other groups. Furthermore, dimethylation of histone‐3 at lysine‐9 (H3K9m2) and DNA methylation were significantly increased whereas H4K12 acetylation (H4K12ac) was decreased in the 10.0 μM TQ‐treated group in comparison with control and in‐vivo matured oocytes. Therefore, our results are suggesting that 10.0 μM TQ may enhance the developmental competence of PCOS oocytes via the modulation of oxidative stress and epigenetic alterations.     

Reduced dosage of the modifiers of epigenetic reprogramming Dnmt1, Dnmt3L,SmcHD1 and Foxo3a has no detectable effect on mouse telomere length in vivo

Studies carried out in cultured cells have implicated modifiers of epigenetic reprogramming in the regulation of telomere length, reporting elongation in cells that were null for DNA methyltransferase DNA methyltransferase 1 (Dnmt1), both de novo DNA methyltransferases, Dnmt3a and Dnmt3b or various histone methyltransferases. To investigate this further, we assayed telomere length in whole embryos or adult tissue from mice carrying mutations in four different modifiers of epigenetic reprogramming: Dnmt1, DNA methyltransferase 3-like, structural maintenance of chromosomes hinge domain containing 1, and forkhead box O3a. Terminal restriction fragment analysis was used to compare telomere length in homozygous mutants, heterozygous mutants and wild-type littermates. Contrary to expectation, we did not detect overall lengthening in the mutants, raising questions about the role of epigenetic processes in telomere length in vivo.     

Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1

De novo DNA methyltransferases, Dnmt3a and 3b, were purified by fractionation of S-100 extract from mouse lymphosarcoma cells through several chromatographic matrices followed by glycerol density gradient centrifugation. Dnmt3a was separated from Dnmt3b and Dnmt1 in the first column, Q-Sepharose whereas Dnmt3b co-purified with Dnmt1 after further fractionation through Mono-S and Mono-Q columns and glycerol density gradient centrifugation. Following purification, the majority of de novo DNA methyltransfearse activity was associated with Dnmt3b/Dnmt1 fractions. By contrast, the fractions containing Dnmt3a alone exhibited markedly reduced activity, which correlated with diminished expression of this isoform in these cells. Histone deacetylase 1(Hdac1) cofractionated with Dnmt3a throughout purification whereas Hdac1 was separated from Dnmt3b/Dnmt1 following chromatography on Mono-Q column. Dnmt3a purified through glycerol gradient centrifugation was also associated with a histone H3 methyltransferase (HMTase) activity whereas purified Dnmt3b/Dnmt1 was devoid of any HMTase activity. The activity of this HMTase was abolished when lysine 9 of N-terminal histone H3 peptide was replaced by leucine whereas mutation of lysine 4 to leucine inhibited this activity only partially. This is the first report on the identification of a few key co-repressors associated with endogenous Dnmt3a and of a complex containing Dnmt3b and a minor form of Dnmt1 following extensive biochemical fractionation.     

Male mice produced by in vitro culture have reduced fertility and transmit organomegaly and glucose intolerance to their male offspring

It has been reported that suboptimal in vitro culture (IVC) of mouse embryos can affect the postnatal expression of epigenetically sensitive alleles, resulting in altered postnatal growth, organ dimensions, health, and behavior in the offspring. Although these detrimental impacts on the offspring are well described, the relative contribution of the IVC-produced fathers is unclear. In this work, we have analyzed if suboptimal IVC (achieved by altering the culture medium by the addition of FCS) can affect male fertility and if organ size and glucose clearance, two of the adverse effects produced by suboptimal IVC conditions, were transmitted to the next two generations. IVC-produced males had lower sperm concentrations (5.8 × 10(6) spermatozoa in IVC vs. 14.5 × 10(6) spermatozoa in control), and these sperm exhibited decreased overall motility (49.6% vs. 72.8% in control) and progressive motility (22.6% vs. 32.2% in control). Fertility tests demonstrated that the percentage of pregnancies was reduced for IVC males (35% for IVC-produced males vs. 86% for in vivo controls). These features were related to a modified gene expression pattern in adult male testes, showing an altered gene expression in genes involved in DNA repair and apoptosis that was confirmed by TUNEL assay. Regarding the IVC related adverse phenotype transmitted to offspring, male glucose intolerance was shown only in F1 and F2 male but not female offspring. The same occurred with male abnormalities in the organ size of the liver, which were transmitted to F1 and F2 males but not to F1 females; moreover, analysis of the F0, F1, and F2 males revealed greater coefficients of variance in body weight and glucose intolerance than the control group. Finally, we analyzed, through gene silencing, the effect of IVC on the mRNA expression at the blastocyst stage for 11 known gene expression modifiers of epigenetic reprogramming. Suboptimal IVC reduced the expression of Kap1, Sox2, Hdac1, Dnmt1, and Dnmt3a, suggesting a molecular epigenetic role for gene expression modifiers in the origin and transmission of these abnormal phenotypes.     

Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development

To ascertain whether p53 deficiency in vivo leads to the deregulation of DNA methylation machinery prior to tumor development, we investigated the expression profile of DNA methyltransferases in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice at 7 weeks of age before tumor development. The expression of DNA methyltransferases was examined in the thymus at 7 weeks of age, since the malignant T-cell lymphoma develops most frequently in p53(-/-) mice around 20 weeks of age. Both mRNA and protein levels of Dnmt1 and Dnmt3b were increased in the thymus and the liver of p53-deficient mice. The expression of Dnmt3a was also increased in the liver but not in the thymus of p53-deficient mice. Dnmt3L expression was reduced in the thymus of p53(+/-) and p53(-/-) mice. The total 5-methylcytosine (5-MeC) in the genomic DNA of p53(+/+), p53(+/-), and p53(-/-) mice was quantitated by dot-blot using antibody against 5-MeC. Global methylation was increased in the thymus and the liver of p53-deficient mice. To correlate the deregulated expression of DNA methyltransferases with the disturbance of the epigenetic integrity, we examined the DNA methylation of the imprinting control region (ICR) at the insulin-like growth factor II (Igf2)/H19 loci in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice. The region containing two CCCTC binding factor (CTCF) binding sites in the 5'-ICR tended to be hypomethylated in the thymus of p53(-/-) mice, but not in the liver. The expression profile of Igf2 and H19 indicated that the thymus-specific changes of Igf2 and H19 expression were coherent to the hypomethylation of the ICR in the thymus. Our results suggest that p53 is required for the maintenance of DNA methylation patterns in vivo.     

Identification of T-cadherin as a novel target of DNA methyltransferase 3B and its role in the suppression of nerve growth factor-mediated neurite outgrowth in PC12 cells

Previously we showed that DNA methyltransferase 3b (Dnmt3b) is required for nerve growth factor (NGF)-induced differentiation of PC12 cells to neuronal phenotype. The present study identified T-cadherin (T-Cad) as one of the targets of Dnmt3b by chromatin immunoprecipitation (ChIP) assay. Combined bisulfite restriction analysis and bisulfite sequencing showed that T-Cad promoter was sparsely methylated in PC12 cells. ChIP-CHOP analysis demonstrated that Dnmt3b is associated with T-Cad promoter irrespective of its methylation status. The mRNA and protein levels of T-Cad were markedly elevated in cells depleted of Dnmt3b by antisense or small interfering RNA. Suppression of T-Cad promoter activity by Dnmt3b was independent of its catalytic activity, which was consistent with the insignificant change in T-Cad promoter methylation status in Dnmt3b-depleted cells. In contrast, deletion of its N-terminal ATRX and PWWP domain abolished its repressor function. Association of histone deacetylase 2 (Hdac2) with T-Cad promoter and restoration of the promoter activity from Dnmt3b-mediated suppression upon treatment with Hdac inhibitor indicated involvement of histone deacetylation in this process. NGF-induced neurite outgrowth was inhibited in a dose dependent manner upon ectopic expression of T-Cad in PC12 cells. Immunofluorescence studies showed that T-Cad was redistributed upon NGF treatment, as evident from its concentration in axon growth cones as opposed to its localization at cell-cell contact region in undifferentiated cells. These results demonstrate a novel role of T-Cad in the NGF-mediated differentiation of PC12 cells to neuronal phenotype.     

Epigenetic regulation of surfactant protein A gene (SP-A) expression in fetal lung reveals a critical role for Suv39h methyltransferases during development and hypoxia

SP-A gene expression is developmentally regulated in fetal lung. Cyclic AMP (cAMP) induction of SP-A expression in human fetal lung type II cells is O(2) dependent and is mediated by increased binding of TTF-1/Nkx2.1 and NF-κB to a critical response element (TBE). This is associated with increased acetylation and decreased methylation of H3K9 at the TBE. Using chromatin immunoprecipitation analysis of fetal lung between 15.5 and 19.0 days of gestation, we observed that the developmental induction of SP-A was associated with increased recruitment of TTF-1, NF-κB, PCAF, and CBP, as well as enhanced acetylation and decreased methylation of histone H3K9 at the TBE. Importantly, expression and TBE binding of the H3K9 methyltransferases, Suv39h1 and Suv39h2, was inversely correlated with the developmental upregulation of SP-A. In human fetal lung epithelial cells, Suv39H1 and Suv39H2 mRNA levels declined with cAMP induction of SP-A. Moreover, hypoxia, which inhibits cAMP stimulation of SP-A, markedly increased Suv39h1 and Suv39h2 binding to the TBE. Finally, short hairpin RNA knockdown of Suv39H1 or Suv39H2 in fetal lung epithelial cells repressed H3K9 methylation and greatly enhanced SP-A expression. Collectively, our findings suggest that Suv39H1 and Suv39H2 are key hypoxia-induced methyltransferases; their decline in fetal lung during late gestation is critical for epigenetic changes resulting in the developmental induction of SP-A.