The early life social environment and DNA methylation

Although epidemiological data provides evidence that there is an interaction between genetics (nature) and the social and physical environments (nurture) in human development; the main open question remains the mechanism. The pattern of distribution of methyl groups in DNA is different from cell-type to cell type and is conferring cell specific identity on DNA during cellular differentiation and organogenesis. This is an innate and highly programmed process. However, recent data suggests that DNA methylation is not only involved in cellular differentiation but that it is also involved in modulation of genome function in response to signals from the physical, biological and social environments. We propose that modulation of DNA methylation in response to environmental cues early in life serves as a mechanism of life-long genome "adaptation" that molecularly embeds the early experiences of a child ("nurture") in the genome ("nature"). There is an emerging line of data supporting this hypothesis in rodents, non-human primates and humans that will be reviewed here. However, several critical questions remain including the identification of mechanisms that transmit the signals from the social environment to the DNA methylation/demethylation enzymes.     

鼠肝细胞癌变中DNA甲基化作用的研究

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.     

The early life social environment and DNA methylation: DNA methylation mediating the long-term impact of social environments early in life

Although epidemiological data provides evidence that there is an interaction between genetics (nature) and the social and physical environments (nurture) in human development; the main open question remains the mechanism. The pattern of distribution of methyl groups in DNA is different from cell-type to cell type and is conferring cell specific identity on DNA during cellular differentiation and organogenesis. This is an innate and highly programmed process. However, recent data suggests that DNA methylation is not only involved in cellular differentiation but that it is also involved in modulation of genome function in response to signals from the physical, biological and social environments. We propose that modulation of DNA methylation in response to environmental cues early in life serves as a mechanism of life-long genome “adaptation” that molecularly embeds the early experiences of a child (“nurture”) in the genome (“nature”). There is an emerging line of data supporting this hypothesis in rodents, non-human primates and humans that will be reviewed here. However, several critical questions remain including the identification of mechanisms that transmit the signals from the social environment to the DNA methylation/demethylation enzymes.Key words: DNA methylation, psychiatry, development, epidemiology, environment     

Heritability and role for the environment in DNA methylation in AXL receptor tyrosine kinase

DNA methylation in AXL, a receptor tyrosine kinase relevant in cancer and immune function, is reportedly highly heritable. We present evidence to suggest that heritability of DNA methylation in AXL is variable, dependent on population characteristics and cell type studied. Moreover, environmental exposures in utero, particularly exposure to maternal smoking, contributes to variation in DNA methylation of select CpG loci that can affect calculations of heritability. Children exposed to maternal smoking in utero had a 2.3% increase (95 % CI 0.3, 4.2) in DNA methylation in AXL, which was magnified in girls as compared to boys. These results present compelling evidence that environmental exposure to tobacco smoke during pregnancy may alter DNA methylation levels in subtle but potentially important ways, and that these changes are persistent years after birth.     

Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction

The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.     

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.     

DNA methylation in the fungi

A systematic study on the incidence and patterns of cytosine methylation in the fungi has been carried out by restriction and nearest-neighbor analysis of DNAs isolated from undifferentiated cells of several fungal species. With respect to DNA modification, the fungi appear to be a heterogeneous group, with a 5-methylcytosine content ranging from undetectable levels (less than or equal to 0.1% of cytosine residues methylated in 18 out of 20 species tested) to low but detectable levels (e.g. congruent to 0.2 and congruent to 0.5% of the total cytosines methylated in Sporotrichum dimorphosporum and Phycomyces blakesleeanus, respectively). In the species where it has been detected, 5-methylcytosine is located mostly at CpG doublets, and the methylated sites are clustered in long tracts (10-30 kilobase pairs) separated from essentially unmethylated regions. This methylated compartment, which comprises a small fraction (1-11%) of the total DNA, contains at least a specific set of repetitive sequences. These results contrast with the higher 5-methylcytosine content found in the fungus Physarum polycephalum and in vertebrates and higher plants.     

Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation

The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,^{1, 2, 3} lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.^{4, 5} It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.^{6, 7} It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.^{8, 9, 10, 11, 12, 13} As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,^{14, 15} TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.^{13, 16} Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,^{17, 18} supporting the key roles of miRs in controlling cell fate.^{19, 20, 21, 22} Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.^{7, 23, 24} As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).^{7, 25} We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,^{7, 13, 26, 27} and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.^{7, 13} These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers^{28, 29, 30, 31} and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.^{29, 30, 32} However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells.     

Effect of prolactin on DNA methylation in the liver and kidney of rat

Prolactin is an important growth modulatory hormone in fetal and adult tissues. It stimulates DNA synthesis and enzymatic markers of the G1 phase of cell cycle in rat liver and other tissues. In this study the effects of prolactin on 5-methyl cytosine content in liver and kidney of rats was studied using HPLC. Prolactin treatment caused hypomethylation of DNA in the liver and kidney of immature rats at 48 h after treatment and the effect remained even at 72 h. Prolactin also caused hypomethylation of DNA in the kidney and liver of adult rats at 48 h after treatment. These results indicate that prolactin probably regulates DNA methylation in the liver and kidney of immature and adult rats.     

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2‐propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.     

Clustered DNA methylation changes in polycomb target genes in early-stage liver cancer

Polycomb-group proteins mark specific chromatin conformations in embryonic and somatic stem cells that are critical for maintenance of their "stemness". These proteins also mark altered chromatin modifications identified in various cancers. In normal differentiated cells or advanced cancerous cells, these polycomb-associated loci are frequently associated with increased DNA methylation. It has thus been hypothesized that changes in DNA methylation status within polycomb-associated loci may dictate cell fate and that abnormal methylation within these loci may be associated with tumor development. To assess this, we examined the methylation states of four polycomb target loci -Trip10, Casp8AP2, ENSA, and ZNF484 - in liver cancer. These four targets were selected because their methylation levels are increased during mesenchymal stem cell-to-liver differentiation. We found that these four loci were hypomethylated in most early-stage liver cancer specimens. For comparison, two non-polycomb tumor suppressor genes, HIC1 and RassF1A, were also examined. Whereas the methylation level of HIC1 did not differ significantly between normal and tumor samples, RassF1A was significantly hypermethylated in liver tumor samples. Unsupervised clustering analysis classified the methylation changes within polycomb and non-polycomb targets to be independent, indicating independent epigenetic evolution. Thus, pre-deposited polycomb marks within somatic stem cells may contribute to the determination of methylation changes during hepatic tumorigenesis.     

DNA methylation of liver and HTC cells during corticosteroid induction

The status of DNA methylation, as measured by m⁵C² content of nuclear DNA, was examined during corticosteroid mediated TAT induction in rat liver and in dividing and nondividing HTC cells. The m⁵C content, determined by HPLC, was not significantly altered in HTC cells during TAT induction whether the cells were in the logarithmic or stationary growth phase. In the liver of adrenalectomized rats where the range of corticosteroid effects is greater than in HTC cells, a small but significant decline in genomic levels of m⁵C was detected between 1 to 8 hr post-induction. The alterations in DNA methylation did not fluctuate during induction by more than 8% in the liver or 7.5% in HTC cells. These results demonstrate that no gross change or elevation in m⁵C content is detected in two, different, hormonally responsive hepatocellular systems during gene activation.     

Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver

Background

Gene silencing due to aberrant DNA methylation is a frequent event in hepatocellular carcinoma (HCC) and also in hepatocellular adenoma (HCA). However, very little is known about epigenetic defects in fibrolamellar carcinoma (FLC), a rare variant of hepatocellular carcinoma that displays distinct clinical and morphological features.

Methodology/Principal Findings

We analyzed the methylation status of the APC, CDH1, cyclinD2, GSTπ1, hsa-mir-9-1, hsa-mir-9-2, and RASSF1A gene in a series of 15 FLC and paired normal liver tissue specimens by quantitative high-resolution pyrosequencing. Results were compared with common HCC arising in non-cirrhotic liver (n = 10). Frequent aberrant hypermethylation was found for the cyclinD2 (19%) and the RASSF1A (38%) gene as well as for the microRNA genes mir-9-1 (13%) and mir-9-2 (33%). In contrast to common HCC the APC and CDH1 (E-cadherin) genes were found devoid of any DNA methylation in FLC, whereas the GSTπ1 gene showed comparable DNA methylation in tumor and surrounding tissue at a moderate level. Changes in global DNA methylation level were measured by analyzing methylation status of the highly repetitive LINE-1 sequences. No evidence of global hypomethylation could be found in FLCs, whereas HCCs without cirrhosis showed a significant reduction in global methylation level as described previously.

Conclusions

FLCs display frequent and distinct gene-specific hypermethylation in the absence of significant global hypomethylation indicating that these two epigenetic aberrations are induced by different pathways and that full-blown malignancy can develop in the absence of global loss of DNA methylation. Only quantitative DNA methylation detection methodology was able to identify these differences.