Chinese Herbs Antagonize The Effects of Bisphenol A On Kiss1 Expression Through Epigenetic Regulation In The Promoter Region

Background: Bisphenol A (BPA), an environmental endocrine disruptor, is involved in precocious puberty. Chinese Herbs for Nourishing Yin and Purging Fire (NYPF) have been effective in delaying early onset of puberty. However, the underline mechanism is unknown. As epigenetics plays an important role in advanced puberty caused by extraneous factors. We intend to investigate the epigenetic impact of BPA and NYPF herbs on Kiss1 gene, a vital gene driving puberty onset, in hypothalamic neuron cells.Methods: GT1-7 cells derived from GnRH neurons were used in the study. A series of concentration of BPA and serum containing NYPF herbs were prepared in advance. GT1-7 cells were administrated with BPA or vehicle first, followed by NYPF herbs or normal saline (NS) for 24 hours. Afterward, quantification of Kiss1 expression was performed through Realtime-PCR and Weston-blot methods. In addition, epigenetic modifications at Kiss1 promoter were examined through Bisulphite Sequencing (BSP) and Chromatin immunoprecipitation combined real-time PCR（Chip-qPCR）assays.Results: Compared to control group, GT1-7 cells with BPA 10mg/l administration presented lower methylation status (61.25% Vs. 76.56%) concomitantly with enhanced abundance of mixed-lineage leukemia 1(MLL1) (1.05%±0.15 Vs. 0.27%±0.01) and Tri-Methyl-Histone 3 at lysine 4 (H3K4me3) (0.77%±0.51 Vs. 0.02%±0.04) at Kiss1 promoter, leading to elevated expression of Kiss1. On the contrary, TCM for NYPF promoted increased DNA methylation (83.75% Vs. 72.5%), accompanied with less deposition of MLL1/H3K4me3 (MLL1: 0.14%±0.02 Vs. 0.97%±0.09; H3K4me3: 0.01%±0.00 Vs. 0.57%±0.30) at Kiss1 promoter than BPA 10mg/l group, which resulted in markedly inhibition of Kiss1 expression. Conclusions: BPA may induce elevated expression of Kiss1 through influencing DNA methylation and histone modification at Kiss1 promoter. And NYPF herbs reverse the adverse effects of BPA on Kiss1 gene through epigenetic modification as well.

The Mouse Papillomavirus Epigenetic Signature is Characterised by DNA Hypermethylation after Lesion Regression

Papillomaviruses (PVs) are double-stranded DNA tumour viruses that can infect cutaneous and mucosal epidermis. Human papillomavirus (HPV) types have been linked to the causality of cutaneous squamous cell carcinoma (cSCC); however, HPV DNA is not always detected in the resultant tumour. DNA methylation is an epigenetic change that can contribute to carcinogenesis. We hypothesise that the DNA methylation pattern in cells is altered following PV infection. We tested if DNA methylation was altered by PV infection in the mouse papillomavirus (MmuPV1) model. Immunosuppressed mice were infected with MmuPV1 on cutaneous tail skin. Immunosuppression was withdrawn for some mice, causing lesions to spontaneously regress. Reduced representation bisulphite sequencing was carried out on DNA from the actively infected lesions, visibly regressed lesions, and mock-infected control mice. DNA methylation libraries were generated and analysed for differentially methylated regions throughout the genome. The presence of MmuPV1 sequences was also assessed. We identified 834 predominantly differentially hypermethylated fragments in regressed lesions, and no methylation differences in actively infected lesions. The promoter regions of genes associated with tumorigenicity, including the tumour suppressor protein DAPK1 and mismatch repair proteins MSH6 and PAPD7, were hypermethylated. Viral DNA was detected in active lesions and in some lesions that had regressed. This is the first description of the genome-wide DNA methylation landscape for active and regressed MmuPV1 lesions. We propose that the DNA hypermethylation in the regressed lesions that we report here may increase the susceptibility of cells to ultraviolet-induced cSCC.

Genome-Wide Bisulphite Sequencing Uncovered the Contribution of DNA Methylation to Rice Short-Term Drought Memory Formation

Rice is the important crop for more than half of the world population. However, drought can have a devastating impact on rice growth and reduce yield drastically. Understanding the response of rice to drought stress is especially important for crop breeding. Previously we found that rice enhanced its tolerance to drought stresses via stress memory mechanisms. Numerous memory genes were identified to play important roles in the process. DNA methylation was reported to mediate tolerance via regulating gene expression and enhances the survival rate of rice encountering drought stress. However, how DNA methylation involved in stress memory is still not clear. In this study, genome-wide bisulphite sequencing at a single base resolution methylome profiling level was performed and analyzed in rice cultivar under recurrent drought stresses and recovery treatments. We found that rice drought stress memory-related differentially methylated regions (DMRs) showed dynamic and distinct patterns. The drought-memory DMRs may regulate Transposable elements and gene expression to cope with short-term repeated drought stresses. Our findings of drought-memory DMRs can explain mechanisms of rice drought stress memory in a new perspective on global methylome details. Using epigenetic markers to breed drought-resistant rice would become a feasible way in the future research.

Aberrant epigenetic alteration of PAX1 expression contributes to parathyroid tumorigenesis

Study Design Primary hyperparathyroidism (PHPT) results from the hypersecretion of parathyroid hormone from parathyroid tumors. Transcription factor i.e. Paired box1 (PAX1) is active in the parathyroid gland development. In the present study, we analyzed the role of DNA methylation via bisulphite specific polymerase chain reaction (BSP) and histone modifications via chromatin immunoprecipitation (ChIP) in regulating the differential expression of PAX1 in parathyroid adenomas tissues. Results The results showed that mRNA and protein expression of PAX1 was significantly reduced in parathyroid adenomas. Bisulphite sequencing demonstrated hypermethylation in the promoter region of PAX1 (35%; 14/40) and lower levels of histone 3 lysine 9 acetylation (H3K9ac) were observed on the promoter region of PAX1 (6-fold; P< 0.004) in parathyroid adenomas. Furthermore, upon treatment with pharmacologic inhibitor i.e. 5’aza-2 deoxycytidine (DAC) in rat parathyroid continuous cells, we found re-expression of PAX1 gene. Conclusion Our study not only reveals expression of PAX1 is epigenetically deregulated but also paves a way for clinical and therapeutic implications in patients with PHPT.

Impacts of Mycoplasma agalactiae restriction-modification systems on pan-epigenome dynamics and genome plasticity

DNA methylation plays an important role in the biology of bacteria. Often associated with restriction-modification (RM) systems, they also provide a defence against foreign DNA. Little is known regarding the methylome of the mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, single molecule real-time (SMRT) and bisulphite sequencing combined with whole-genome analysis identified 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems in Mycoplasma agalactiae, a ruminant pathogen and a model organism. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenome landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.

Ultralow amounts of DNA from long-term archived serum samples produce high-quality methylomes

Background Long-term stored serum is considered challenging for epigenomic analyses: as there are no cells, circulating DNA is scarce, and amplification removes epigenetic signals. Additionally, pre-analytical treatments and storage might introduce biases and fragmentation to the DNA. In particular, starting with low-input DNA can result in low-diversity libraries. However, successful whole-genome bisulphite sequencing (WGBS) of such serum samples has the potential to open biobanks for epigenetic analyses and deliver novel prediagnostic biomarkers. Here, we perform WGBS using the Accel-NGS library preparation kit on ultralow amounts of DNA from long-term archived samples with diverse pretreatments from the Janus Serum Bank. Results Ninety-four of the 96 samples produced satisfactory methylation calls; an average of 578 M reads per sample generated a mean coverage of 17× and mean duplication level of 35%. Failed samples were related to poor bisulphite conversion rather than to sequencing or library preparation. We demonstrate the feasibility of WGBS on ultralow DNA yields from serum samples stored up to 48 years. Conclusions Our results show the potential of large serum biobank collections for future epigenomic studies and biomarker discovery.

Novel Insights Into Rheumatoid Arthritis Through Characterization of Concordant Changes in DNA Methylation and Gene Expression in Synovial Biopsies of Patients With Differing Numbers of Swollen Joints

In this study, we sought to characterize synovial tissue obtained from individuals with arthralgia and disease-specific auto-antibodies and patients with established rheumatoid arthritis (RA), by applying an integrative multi-omics approach where we investigated differences at the level of DNA methylation and gene expression in relation to disease pathogenesis. We performed concurrent whole-genome bisulphite sequencing and RNA-Sequencing on synovial tissue obtained from the knee and ankle from 4 auto-antibody positive arthralgia patients and thirteen RA patients. Through multi-omics factor analysis we observed that the latent factor explaining the variance in gene expression and DNA methylation was associated with Swollen Joint Count 66 (SJC66), with patients with SJC66 of 9 or more displaying separation from the rest. Interrogating these observed differences revealed activation of the immune response as well as dysregulation of cell adhesion pathways at the level of both DNA methylation and gene expression. We observed differences for 59 genes in particular at the level of both transcript expression and DNA methylation. Our results highlight the utility of genome-wide multi-omics profiling of synovial samples for improved understanding of changes associated with disease spread in arthralgia and RA patients, and point to novel candidate targets for the treatment of the disease.

Genome-wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline

Background Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming. Results Sows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic-activated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage. Conclusions Our findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.