Epigenetic modifications, especially alteration in DNA methylation, are increasingly being recognized as a key factor in the pathogenesis of complex disorders, including atherosclerosis. However, there are limited data on the epigenetic changes in the coronary artery disease (CAD) patients. In the present study we evaluated the methylation status of genomic DNA from peripheral lymphocytes in a cohort of 287 individuals: 137 angiographically confirmed CAD patients and 150 controls. The differential susceptibility of genomic DNA to methylation-sensitive restriction enzymes was utilized to assess the methylation status of the genome. We observed that the genomic DNA methylation in CAD patients is significantly higher than in controls ( p < 0.05). Since elevated homocysteine levels are known to be an independent risk factor for CAD and a key modulator of macromolecular methylation, we investigated the probable correlation between plasma homocysteine levels and global DNA methylation. We observed a significant positive correlation of global DNA methylation with plasma homocysteine levels in CAD patients ( p = 0.001). Further, within a higher range of serum homocysteine levels (≥12–50 μM), global DNA methylation was significantly higher in CAD patients than in controls. The alteration in genomic DNA methylation associated with cardiovascular disease per se appears to be further accentuated by higher homocysteine levels.

Homocysteine is an independent risk factor for cardiovascular diseases. It is also known to be associated with a variety of complex disorders. While there are a large number of independent studies implicating homocysteine in isolated pathways, the mechanism of homocysteine induced adverse effects are not clear. Homocysteine-induced modulation of gene expression through alteration of methylation status or by hitherto unknown mechanisms is predicted to lead to several pathological conditions either directly or indirectly. In the present manuscript, using literature mining approach, we have identified the genes that are modulated directly or indirectly by an elevated level of homocysteine. These genes were then placed in appropriate pathways in an attempt to understand the molecular basis of homocysteine induced complex disorders and to provide a resource for selection of genes for polymorphism screening and analysis of mutations as well as epigenetic modifications in relation to hyperhomocysteinemia. We have identified 135 genes in 1137 abstracts that either modulate the levels of homocysteine or are modulated by elevated levels of homocysteine. Mapping the genes to their respective pathways revealed that an elevated level of homocysteine leads to the atherosclerosis either by directly affecting lipid metabolism and transport or via oxidative stress and/or Endoplasmic Reticulum (ER) stress. Elevated levels of homocysteine also decreases the bioavailability of nitric oxide and modulates the levels of other metabolites including S-adenosyl methionine and S-adenosyl homocysteine which may result in cardiovascular or neurological disorders. The ER stress emerges as the common pathway that relates to apoptosis, atherosclerosis and neurological disorders and is modulated by levels of homocysteine. The comprehensive network collated has lead to the identification of genes that are modulated by homocysteine indicating that homocysteine exerts its effect not only through modulating the substrate levels for various catalytic processes but also through regulation of expression of genes involved in complex diseases.

Multiple Sclerosis (MS) is an autoimmune disease associated with abnormal expression of a subset of cytokines, resulting in inappropriate T-lymphocyte activation and uncontrolled immune response. A key issue in the field is the need to understand why these cytokines are transcriptionally activated in the patients. Here, we have examined several transcription units subject to pathological reactivation in MS, including the TNFα and IL8 cytokine genes and also several Human Endogenous RetroViruses (HERVs). We find that both the immune genes and the HERVs require the heterochromatin protein HP1α for their transcriptional repression. We further show that the Peptidylarginine Deiminase 4 (PADI4), an enzyme with a suspected role in MS, weakens the binding of HP1α to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. The resulting de-repression of both cytokines and HERVs can be reversed with the PADI-inhibitor Cl-amidine. Finally, we show that in peripheral blood mononuclear cells (PBMCs) from MS patients, the promoters of TNFα, and several HERVs share a deficit in HP1α recruitment and an augmented accumulation of histone H3 with a double citrulline 8 tri-methyl lysine 9 modifications. Thus, our study provides compelling evidence that HP1α and PADI4 are regulators of both immune genes and HERVs, and that multiple events of transcriptional reactivation in MS patients can be explained by the deficiency of a single mechanism of gene silencing. In patients suffering from Multiple Sclerosis (MS), T lymphocytes express abnormally high levels of a subset of cytokines. The same cells also transcribe a series of vestigial retroviral sequences normally silenced by chromatin factors. In this study, we have searched for regulatory mechanisms shared between the cytokines and the retroviral sequences. We find that the repressor protein HP1α is present on the promoter of both types of transcription units in normal cells and that the recruitment of this protein to these promoters is decreased in MS patients. Furthermore, we show that the delocalization of HP1α from these promoters can be caused by citrullination of histone H3, and we provide evidence indicating that levels of this histone modification is augmented in MS patients. Together our data provide a possible explanation for the simultaneously increased transcriptional activity of cytokines and endogenous retroviruses in MS-patient T lymphocytes and suggest that inhibitors of the enzyme responsible for the increased citrullination of histone H3 could help restore normal levels of cytokine activity in the patients.

Cytosine methylation in mammals is important for epigenetic control of the transcriptome. Although altered methylation is frequently encountered in disease situations, particularly cancer, the relationship between genome-wide methylation and DNA structure is poorly understood. It is now evident that alternative DNA forms are functionally relevant in replication, recombination and transcription. Herein, we researched the role of alternative DNA structure in cytosine methylation using quadruplex DNA as a case study. Our findings from analysis of 2.1 million CpGs in humans, across 12 tissues from the Human Epigenome Project (HEP), revealed a striking correlation within each tissue: CpGs with low methylation were enriched (P = 5.24E⁻²⁰) whereas CpGs with high methylation were relatively depleted (P = 9.28E⁻¹⁵), within quadruplex-forming regions. This was further substantiated on considering 1.07E⁸ methylcytosines from genome-wide sequencing within embryonic stem cells and differentiated fibroblasts. To further test the predictions we experimentally determined methylation in >600 000 CpGs across 18 individuals using bisulfite mapping and found significantly low methylation of CpGs within quadruplex-forming regions (P = 1.36E⁻⁰⁸). Together, these suggest the role of guanine-quadruplexes in CpG methylation and directly impact our understanding of the inter-relationship between DNA conformation and global cytosine methylation.