Epigenetics of the non-coding RNA nc886 across blood, adipose tissue and skeletal muscle in offspring exposed to diabetes in pregnancy.

BACKGROUND: Diabetes in pregnancy is associated with increased risk of long-term metabolic disease in the offspring, potentially mediated by in utero epigenetic variation. Previously, we identified multiple differentially methylated single CpG sites in offspring of women with gestational diabetes mellitus (GDM), but whether stretches of differentially methylated regions (DMRs) can also be identified in adolescent GDM offspring is unknown. Here, we investigate which DNA regions in adolescent offspring are differentially methylated in blood by exposure to diabetes in pregnancy. The secondary aim was to characterize the RNA expression of the identified DMR, which contained the nc886 non-coding RNA. METHODS: To identify DMRs, we employed the bump hunter method in samples from young (9-16 yr, n = 92) offspring of women with GDM (O-GDM) and control offspring (n = 94). Validation by pyrosequencing was performed in an adult offspring cohort (age 28-33 years) consisting of O-GDM (n = 82), offspring exposed to maternal type 1 diabetes (O-T1D, n = 67) and control offspring (O-BP, n = 57). RNA-expression was measured using RT-qPCR in subcutaneous adipose tissue and skeletal muscle. RESULTS: One significant DMR represented by 10 CpGs with a bimodal methylation pattern was identified, located in the nc886/VTRNA2-1 non-coding RNA gene. Low methylation status across all CpGs of the nc886 in the young offspring was associated with maternal GDM. While low methylation degree in adult offspring in blood, adipose tissue, and skeletal muscle was not associated with maternal GDM, adipose tissue nc886 expression was increased in O-GDM compared to O-BP, but not in O-T1D. In addition, adipose tissue nc886 expression levels were positively associated with maternal pre-pregnancy BMI (p = 0.006), but not with the offspring's own adiposity. CONCLUSIONS: Our results highlight that nc886 is a metastable epiallele, whose methylation in young offspring is negatively correlated with maternal obesity and GDM status. The physiological effect of nc886 may be more important in adipose tissue than in skeletal muscle. Further research should aim to investigate how nc886 regulation in adipose tissue by exposure to GDM may contribute to development of metabolic disease.

DNA Methylation and Gene Expression in Blood and Adipose Tissue of Adult Offspring of Women with Diabetes in Pregnancy-A Validation Study of DNA Methylation Changes Identified in Adolescent Offspring.

Maternal gestational diabetes and obesity are associated with adverse outcomes in offspring, including increased risk of diabetes and cardiovascular diseases. Previously, we identified a lower DNA methylation degree at genomic sites near the genes ESM1, MS4A3, and TSPAN14 in the blood cells of adolescent offspring exposed to gestational diabetes and/or maternal obesity in utero. In the present study, we aimed to investigate if altered methylation and expression of these genes were detectable in blood, as well in the metabolically relevant subcutaneous adipose tissue, in a separate cohort of adult offspring exposed to gestational diabetes and obesity (O-GDM) or type 1 diabetes (O-T1D) in utero, compared with the offspring of women from the background population (O-BP). We did not replicate the findings of lower methylation of ESM1, MS4A3, and TSPAN14 in blood from adults, either in O-GDM or O-T1D. In contrast, in adipose tissue of O-T1D, we found higher MS4A3 DNA methylation, which will require further validation. The adipose tissue ESM1 expression was lower in O-GDM compared to O-BP, which in turn was not associated with maternal pre-pregnancy BMI nor the offspring's own adiposity. Adipose tissue TSPAN14 expression was slightly lower in O-GDM compared with O-BP, but also positively associated with maternal pre-pregnancy BMI, as well as offspring's own adiposity and HbA1c levels. In conclusion, the lower DNA methylation in blood from adolescent offspring exposed to GDM could not be confirmed in the present cohort of adult offspring, potentially due to methylation remodeling with increased aging. In offspring adipose tissue, ESM1 expression was associated with maternal GDM, and TSPAN14 expression was associated with both maternal GDM, as well as pre-pregnancy BMI. These altered expression patterns are potentially relevant to the concept of developmental programming of cardiometabolic diseases and require further studies.

Ablation of DNA-methyltransferase 3A in skeletal muscle does not affect energy metabolism or exercise capacity.

In response to physical exercise and diet, skeletal muscle adapts to energetic demands through large transcriptional changes. This remodelling is associated with changes in skeletal muscle DNA methylation which may participate in the metabolic adaptation to extracellular stimuli. Yet, the mechanisms by which muscle-borne DNA methylation machinery responds to diet and exercise and impacts muscle function are unknown. Here, we investigated the function of de novo DNA methylation in fully differentiated skeletal muscle. We generated muscle-specific DNA methyltransferase 3A (DNMT3A) knockout mice (mD3AKO) and investigated the impact of DNMT3A ablation on skeletal muscle DNA methylation, exercise capacity and energy metabolism. Loss of DNMT3A reduced DNA methylation in skeletal muscle over multiple genomic contexts and altered the transcription of genes known to be influenced by DNA methylation, but did not affect exercise capacity and whole-body energy metabolism compared to wild type mice. Loss of DNMT3A did not alter skeletal muscle mitochondrial function or the transcriptional response to exercise however did influence the expression of genes involved in muscle development. These data suggest that DNMT3A does not have a large role in the function of mature skeletal muscle although a role in muscle development and differentiation is likely.

Luminal lncRNAs Regulation by ERα-Controlled Enhancers in a Ligand-Independent Manner in Breast Cancer Cells.

Estrogen receptor-α (ERα) is a ligand-inducible protein which mediates estrogenic hormones signaling and defines the luminal BC phenotype. Recently, we demonstrated that even in absence of ligands ERα (apoERα) binds chromatin sites where it regulates transcription of several protein-coding and lncRNA genes. Noteworthy, apoERα-regulated lncRNAs marginally overlap estrogen-induced transcripts, thus representing a new signature of luminal BC genes. By the analysis of H3K27ac enrichment in hormone-deprived MCF-7 cells, we defined a set of Super Enhancers (SEs) occupied by apoERα, including one mapped in proximity of the DSCAM-AS1 lncRNA gene. This represents a paradigm of apoERα activity since its expression is largely unaffected by estrogenic treatment, despite the fact that E2 increases ERα binding on DSCAM-AS1 promoter. We validated the enrichment of apoERα, p300, GATA3, FoxM1 and CTCF at both DSCAM-AS1 TSS and at its associated SE by ChIP-qPCR. Furthermore, by analyzing MCF-7 ChIA-PET data and by 3C assays, we confirmed long range chromatin interaction between the SE and the DSCAM-AS1 TSS. Interestingly, CTCF and p300 binding showed an enrichment in hormone-depleted medium and in the presence of ERα, elucidating the dynamics of the estrogen-independent regulation of DSCAM-AS1 expression. The analysis of this lncRNA provides a paradigm of transcriptional regulation of a luminal specific apoERα regulated lncRNA.

Association of GSTT1 null, XPD 751 CC and XPC 939 CC genotypes with increased levels of genomic damage among hospital pathologists.

CONTEXT: Hospital workers are at risk for genotoxic damage following occupationally exposure to xenobiotics. Pathologists are exposed to chemicals during their use in health care environments, particularly throughout inhalation of airborne agents, absorption through skin or contact with the patient's body fluids. OBJECTIVE: We evaluated the level of genomic damage in a sample of 61 hospital pathologists (occupationally exposed to antineoplastic drugs and sterilizing agents) and 60 control subjects. MATERIALS AND METHODS: Lymphocytes were analyzed by SCEs and CAs assays and genotyped for GSTT1, GSTM1, CYP1A1 Ile/Val, XPD (A751C) and XPC (A939C) gene polymorphisms. RESULTS: Pathologists showed significantly higher frequencies of SCEs and CAs with respect to control subjects. GSTT1 null genotype was found to be associated with higher SCEs and CAs frequencies, whereas XPD 751 CC and XPC 939 CC genotypes only with a higher level of SCEs. DISCUSSION AND CONCLUSIONS: The SCEs and CAs results are consistent with other published data, placing hospital workers as a category at risk for genotoxic damage caused by chronic exposure to xenobiotics. The higher levels of cytogenetic damage observed among GSTT1 null, XPD 751 and XPC 939 CC homozygote subjects confirm the importance of the genetic polymorphisms analysis associated to genotoxicological studies.

Relationships between cytokine (IL-6 and TGF-ß1) gene polymorphisms and chromosomal damage in hospital workers.

Cytokine gene polymorphisms have been found to be associated with a pre-disposition to a variety of diseases, including inflammatory and cancer diseases. The present study evaluated the influence of six cytokine gene polymorphisms on the level of genomic damage observed in peripheral blood lymphocytes from hospital pathologists chronically exposed to low doses of different xenobiotics. Lymphocytes from 50 pathologists and 50 control subjects were recruited and analyzed in Sister Chromatid Exchange (SCE) and Chromosomal Aberrations (CA) assays. The frequencies of six cytokine gene polymorphisms and their relationships with the cytogenetic damage levels were also evaluated. The results indicated that significant differences were found between pathologists and controls in terms of SCE frequency (p < 0.001) and RI values (p < 0.001), as well as in terms of CA and cells with aberrations (p < 0.001). No associations were found between all analyzed cytokine gene polymorphisms and CA frequency in both pathologists and control groups. Vice versa, among pathologists, homozygote individuals for the IL-6 G allele showed a significantly (p = 0.017) lower frequency of SCE with respect to heterozygote subjects. Similarly, for TGFß1 codon 10 locus, homozygote for T allele and heterozygote TC subjects showed a significantly (p = 0.021) lower frequency of SCE with respect to homozygote CC individuals. Among controls, no significant differences were found in the frequency of SCE between genotypes at all loci. Based on these results, we speculate that high circulating levels of a pro-inflammatory cytokine like IL-6 and lower levels of the immunosuppressant cytokine TGFß1 could be associated directly with a longer duration and/or greater intensity of inflammatory processes, and indirectly with significantly higher levels of genomic damage.