Vani Brahmachari

Mining literature for a comprehensive pathway analysis: A case study for retrieval of homocysteine related genes for genetic and epigenetic studies

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.

Detection of altered global DNA methylation in coronary artery disease patients.

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 muM), 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.

Epigenetic regulation of cytomegalovirus major immediate-early promoter activity in transgenic mice.

The expression of genes in transgenic mice is known to be influenced by the site of integration even when they carry their own promoter elements and transcription factor binding sites. The cytomegalovirus (CMV) promoter, a strong promoter often used for transgene expression in mammalian cells in culture, is known to be silenced by DNA methylation and histone deacetylation but there is no report on the role of histone methylations in its regulation. We generated two transgenic lines carrying green fluorescence protein coding gene as reporter driven by cytomegalovirus major immediate-early promoter/enhancer. We observe that silencing of CMV promoter is dependent on the site of transgene integration, except in testis, and the nature of DNA and histone methylations strongly correlate with the expression status of the reporter. We find that silenced CMV promoter interacts in vivo, with Methyl CpG binding protein 2 (MeCP2), a recruiter of histone deacetylases (HDACs) and histone (H3K9) methyl transferase. Histone H3K4methylation, the active chromatin mark, is also associated with silenced promoter, suggesting bivalent marking of the promoter and its susceptibility to reactivation on induction.

Analysis of Expression Profile of Mammalian Cell Entry (mce) Operons of Mycobacterium tuberculosis

ABSTRACT The sequencing of the complete genome of M. tuberculosis H37Rv has resulted in the recognition of four mce operons in its genome by in silico analysis. In an attempt to understand the significance of the redundancy of mce operons, we analyzed the expression profile of mce operons after different periods of growth in culture as well as during in vivo infection. Our results strongly suggest that mce1 is expressed as a polycistronic message. In culture from day 8 to day 12, expression of only mce1 was observed, but as the cultures progress towards stationary phase the expression profile of mce operons was altered; the transcripts of the mce1 operon were barely detected while those of the mce4 operon were prominent. In an analysis of the expression of mce operons in tubercle material collected from infected animal tissues, we detected the expression of mce1, -3 and -4. Our results imply that mce operons other than mce1 are also expressed during infection and that it is necessary to examine their role in pathogenesis.

Genome wide DNA methylation profiling for epigenetic alteration in coronary artery disease patients

BACKGROUND The alteration in the epigenome forms an interface between the genotype and the environment. Epigenetic alteration is expected to make a significant contribution to the development of cardiovascular disease where environmental interactions play a key role in disease progression. We had previously shown that global DNA hypermethylation per se is associated with coronary artery disease (CAD) and is further accentuated by high levels of homocysteine, a thiol amino acid which is an independent risk factor for cardiovascular disease and is also a key modulator of macromolecular methylation. RESULTS We have identified 72 differentially methylated regions (DMRs) that were hypermethylated in CAD patients in the background of varying homocysteine levels. Following deep bisulfite sequencing of a few of the selected DMRs, we found significantly higher methylation in CAD cases. We get six CpG sites in three DMRs that included the intronic region of C1QL4 gene and upstream region of CCDC47 and TGFBR3 genes. CONCLUSION To the best of our knowledge, this is the first study to identify hypermethylated regions across the genome in patients with coronary artery disease. Further validation in different populations is necessary for this information to be used for disease risk assessment and management.

Characterization of Mce4A protein of Mycobacterium tuberculosis: role in invasion and survival

BackgroundThe mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far.ResultsBy transmission electron microscopy we have demonstrated that recombinant Mce4A protein facilitates the invasion of non-pathogenic strain of E. coli into non-phagocytic HeLa cells. We observe that mce4A gene has a role comparable to mce1A in the survival of recombinant E. coli in human macrophages. Using antibodies raised against Mce4A protein, we show that the protein is localized in the cell wall fraction of M. tuberculosis H37Rv stationary phase culture only.ConclusionMce4A protein is expressed during the stationary phase of broth culture and localizes in the cell wall fraction of M. tuberculosis. Mce4A protein expressed in non-pathogenic E. coli enables it to enter and survive within HeLa cells and the macrophages. As Mce4A protein is expressed during later phase of mycobacterial growth, our results raise the possibility of it playing a role in maintenance of persistent tubercular infection.

Differential serum cytokine levels are associated with cytokine gene polymorphisms in north Indians with active pulmonary tuberculosis.

Globally only 5-10% of people encountering Mycobacterium tuberculosis have a lifetime risk of active disease indicating a strong host genetic bias towards development of tuberculosis. In the current study we investigated genotype variants pertaining to five cytokine genes namely IFNG, TNFA, IL4, IL10 and IL12 in the north Indian population with active pulmonary tuberculosis (APTB) and correlated the serum cytokine levels with the corresponding genotypes. Twenty five single nucleotide polymorphisms (SNPs) including six loci examined for the first time in tuberculosis were selected for genotyping in 108 patients with APTB from north India and 48 healthy regional controls (HC). Applying exclusion criteria 12 SNPs passed all the filters and were analysed further. The serum cytokine concentrations were measured by ELISA. Compared to HC mean serum IFN-γ, IL-12, IL-4, and IL-10 levels were higher in APTB (p = 0.3661, p = 0.0186, p = 0.003, p = 0.7, respectively). In contrast the mean serum TNF-α level was higher in HC (p = 0.007). Comparison of genotypes and serum levels of the corresponding cytokine genes reveal that though IFN-γ and IL-4 levels were higher in APTB the genotype variants showed no difference between HC and APTB. In contrast the genotypes of the selected rsIDs in the TNFA, IL12 and IL10 genes showed significant association with the varying serum levels of corresponding cytokines. The variant of the TNFA gene at rs3093662, the IL12 gene at rs3213094 and rs3212220 and the IL10 gene at rs3024498 did show a strong indication to be of relevance to the immunity to tuberculosis. To our knowledge this is the first report from this region relating genotypes and serum cytokine levels in north Indian population.

Genomic imprinting in the mealybugs.

The coccid insects (Hemiptera; Sternorrhyncha; Aphidiformes; Coccoidea; Pseudococcidae) are well suited to study not only the mechanisms of genomic imprinting but also facultative heterochromatization, a phenomenon well exemplified by inactivation of the X chromosome in female mammals. Coccids show sex-specific heterochromatization of an entire set of chromosomes and transcriptional silencing of all the paternally contributed chromosomes in males. Thus, genomic imprinting and the resultant differential regulation operate on 50% of the genome in contrast to the single X chromosome in female mammals. A significant insight into the phenomenon of genomic imprinting has come from very elegant cytological analysis of the coccid system. Recently, efforts have been made to dissect out at the molecular level the phenomenon of genomic imprinting in these insects. The present review summarizes both of these aspects. In light of the accruing experimental evidence for chromatin-based differences in the maternal and paternal genomes, it appears that the mealybug system may provide evidence for stable maintenance of chromatin code not only through mitosis but also through meiosis.

Proximity of H2A.Z containing nucleosome to the transcription start site influences gene expression levels in the mammalian liver and brain

Nucleosome positioning maps of several organisms have shown that Transcription Start Sites (TSSs) are marked by nucleosome depleted regions flanked by strongly positioned nucleosomes. Using genome-wide nucleosome maps and histone variant occupancy in the mouse liver, we show that the majority of genes were associated with a single prominent H2A.Z containing nucleosome in their promoter region. We classified genes into clusters depending on the proximity of H2A.Z to the TSS. The genes with no detectable H2A.Z showed lowest expression level, whereas H2A.Z was positioned closer to the TSS of genes with higher expression levels. We confirmed this relation between the proximity of H2A.Z and expression level in the brain. The proximity of histone variant H2A.Z, but not H3.3 to the TSS, over seven consecutive nucleosomes, was correlated with expression. Further, a nucleosome was positioned over the TSS of silenced genes while it was displaced to expose the TSS in highly expressed genes. Our results suggest that gene expression levels in vivo are determined by accessibility of the TSS and proximity of H2A.Z.

Incomplete penetrance and variable expressivity: is there a microRNA connection?

Incomplete penetrance and variable expressivity are non‐Mendelian phenomena resulting in the lack of correlation between genotype and phenotype. Not withstanding the diversity in mechanisms, differential expression of homologous alleles within cells manifests as variations in penetrance and expressivity of mutations between individuals of the same genotype. These phenomena are seen most often in dominantly inherited diseases, implying that they are sensitive to concentration of the gene product. In this framework and the advances in understanding the role of microRNA (miRNA) in fine‐tuning gene expression at translational level, we propose miRNA‐mediated regulation as a mechanism for incomplete penetrance and variable expressivity. The presence of miRNA binding sites at 3′ UTR, co‐expression of target gene–miRNA pairs for genes showing incomplete penetrance and variable expressivity derived from available data lend support to our hypothesis. Single nucleotide polymorphisms in the miRNA target site facilitate the implied differential targeting of the transcripts from homologous alleles.