Identification of a polycomb responsive region in human HoxA cluster and its long-range interaction with polycomb enriched genomic regions.

Polycomb and Trithorax group proteins (PcG, TrxG) epigenetically regulate developmental genes. These proteins bind with specific DNA elements, the Polycomb Response Element (PRE). Apart from mutations in polycomb/ trithorax proteins, altered cis-elements like PRE underlie the modified function and thus disease etiology. PREs are well studied in Drosophila, while only a few human PREs have been reported. We have identified a polycomb responsive DNA element, hPRE-HoxA3, in the intron of the HoxA3 gene. The hPRE-HoxA3 represses luciferase reporter activity in a PcG-dependent manner. The endogenous hPRE-HoxA3 element recruits PcG proteins and is enriched with repressive H3K27me3 marks, demonstrating that hPRE-HoxA3 is a part of the PcG-dependent gene regulatory network. Furthermore, it interacts with D11-12, the well-known PRE in the human Hox cluster. hPRE-Hox3 is a part of the 3-dimensional chromosomal domain organization as it is involved in the long-range interaction with other PcG enriched regions of Hox A, B, C, and D clusters.

SIN-3 functions through multi-protein interaction to regulate apoptosis, autophagy, and longevity in Caenorhabditis elegans.

SIN3/HDAC is a multi-protein complex that acts as a regulatory unit and functions as a co-repressor/co-activator and a general transcription factor. SIN3 acts as a scaffold in the complex, binding directly to HDAC1/2 and other proteins and plays crucial roles in regulating apoptosis, differentiation, cell proliferation, development, and cell cycle. However, its exact mechanism of action remains elusive. Using the Caenorhabditis elegans (C. elegans) model, we can surpass the challenges posed by the functional redundancy of SIN3 isoforms. In this regard, we have previously demonstrated the role of SIN-3 in uncoupling autophagy and longevity in C. elegans. In order to understand the mechanism of action of SIN3 in these processes, we carried out a comparative analysis of the SIN3 protein interactome from model organisms of different phyla. We identified conserved, expanded, and contracted gene classes. The C. elegans SIN-3 interactome -revealed the presence of  well-known proteins, such as DAF-16, SIR-2.1, SGK-1, and AKT-1/2, involved in autophagy, apoptosis, and longevity. Overall, our analyses propose  potential mechanisms by which SIN3 participates in multiple biological processes and their conservation across species and identifies candidate genes for further experimental analysis.

Genome and transcriptome analysis of the mealybug Maconellicoccus hirsutus: Correlation with its unique phenotypes.

Mealybugs are aggressive pests with world-wide distribution and are suitable for the study of different phenomena like genomic imprinting and epigenetics. Genomic approaches facilitate these studies in absence of robust genetics in this system. We sequenced, de novo assembled, annotated Maconellicoccus hirsutus genome. We carried out comparative genomics it with four mealybug and eight other insect species, to identify expanded, specific and contracted gene classes that relate to pesticide and desiccation resistance. We identified horizontally transferred genes adding to the mutualism between the mealybug and its endosymbionts. Male and female transcriptome analysis indicates differential expression of metabolic pathway genes correlating with their physiology and the genes for sexual dimorphism. The significantly lower expression of endosymbiont genes in males relates to the depletion of endosymbionts in males during development.

Correlation of drug resistance with single nucleotide variations through genome analysis and experimental validation in a multi-drug resistant clinical isolate of M. tuberculosis.

BACKGROUND: Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. RESULT: By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase ß gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. CONCLUSION: The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

Epigenetic regulation and transcriptional memory in development; selection facilitating prudence.

The epigenetic mechanisms regulating developmental gene expression are examples of a strategy to generate unique expression profiles with global regulators controlling several genes. In a simplified view, a common set of tools, that include DNA motif recognizing proteins (recruiters), binding/interacting surfaces (ARPs- actin related proteins), epigenetic writers (histone methyltransferases, acetylases), readers (chromatin remodeling proteins, PRC1 members) and erasers (demethylases, deacetylases) form complexes which not only regulate transcription, but also retain the transcriptional memory through mitosis. There are two arms of epigenetic regulation: covalent modification of DNA and the post-translational modification of histones. In this review, we discuss both of these aspects briefly to illustrate functional diversity. We discuss our efforts at utilization of the genome sequence data for de novo identification of new players and their functional validation in this remarkable process.

Mining histone methyltransferases and demethylases from whole genome sequence.

Epigenetic regulation through post-translational modification of histones, especially methylation, is well conserved in evolution. Although there are several insect genomes sequenced, an analysis with a focus on their epigenetic repertoire is limited. We have utilized a novel work-flow to identify one or more domains as highpriority domain (HPD), if present in at least 50% of the genes of a given functional class in the reference genome, namely, that of Drosophila melanogaster. Based on this approach, we have mined histone methyltransferases and demethylases from the whole genome sequence of Aedes aegypti (Diptera), the pea aphid Acyrthosiphon pisum, the triatomid bug Rhodnius prolixus (Hemiptera), the honeybee Apis mellifera (Hymenoptera), the silkworm Bombyx mori (Lepidoptera) and the red flour beetle Tribolium castaneum (Coleoptera). We identified 38 clusters consisting of arginine methyltransferases, lysine methyltransferases and demethylases using OrthoFinder, and the presence of HPD was queried in these sequences using InterProScan. This approach led us to identify putative novel members and currently inaccurate ones. Other than the highpriority domains, these proteins contain shared and unique domains that can mediate protein-protein interaction. Phylogenetic analysis indicates that there is different extent of protein sequence similarity; average similarity between histone lysine methyltransferases varies from 41% (for active mark) to 48% (for repressive mark), arginine methyltransferases is 51%, and demethylases is 52%. The method utilized here facilitates reliable identification of desired functional class in newly sequenced genomes.

The regulatory function of dIno80 correlates with its DNA binding activity.

The INO80 complex, including the Ino80 protein, forms a highly conserved canonical complex that remodels chromatin in the context of multiple cellular functions. The Drosophila homologue, dIno80, is involved in homeotic gene regulation during development as a canonical Pho-dIno80 complex. Previously, we found that dIno80 regulates homeotic genes by interacting with epigenetic regulators, such as polycomb and trithorax, suggesting the occurrence of non-canonical Ino80 complexes. Here using spectroscopic methods and gel retardation assays, we identified a set of consensus DNA sequences that DNA binding domain of dIno80 (DBINO) interacts with having differential affinity and high specificity. Testing these sequences in reporter assays, showed that this interaction can positively regulate transcription. These results suggest that, dIno80 has a sequence preference for interaction with DNA leading to transcriptional changes.

In praise of mealybugs.

The fascinating chromosomal cycle leading to facultative heterochromatization in the mealybugs has been a challenging system for mechanistic understanding of the phenomenon of genomic imprinting and epigenetics. The elegant cytological dissection of the various processes reported in the literature is equally fascinating for the researchers of current molecular age. Presently, a two way approach is being pursued; continued efforts of utilizing elegant cytology, in combination with the molecular probes to decipher molecular correlates on one hand and on the other, the de novo biochemical/molecular analysis for the identification of the molecular players using genomic tools. The hope is to uncover novel players in genomic imprinting and epigenetic regulation in the mealybug system which shows differential regulation of the entire genome, with 50% of its genome being transcriptionally inactivated in a parental-origin-specific and sex specific manner. In addition to being a model for epigenetic regulation, the mealybugs are being utilized for the analysis of radiation resistance as well as metabolic interactions between the microbiome and the host. The overview presented here is an attempt to bring out some of the work carried out in these directions. We also discuss the areas that remain poorly explored in this system, such as the role/involvement of noncoding RNA in male-specific inactivation and the molecular dissection of heterochromatin, the cytological manifestation of the inactive state of genes and chromosome.

Correlation between desiccation stress response and epigenetic modifications of genes in Drosophila melanogaster: An example of environment-epigenome interaction.

Animals from different phyla including arthropods tolerate water stress to different extent. This tolerance is accompanied by biochemical changes which in turn are due to transcriptional alteration. The changes in transcription can be an indirect effect on some of the genes, ensuing from the effect of stress on the regulators of transcription including epigenetic regulators. Within this paradigm, we investigated the correlation between stress response and epigenetic modification underlying gene expression modulation during desiccation stress in Canton-S. We report altered resistance of flies in desiccation stress for heterozygote mutants of PcG and TrxG members. Pc/+ mutant shows lower survival, while ash1/+ mutants show higher survival under desiccation stress as compared to Canton-S. We detect expression alteration in stress related genes as well the genes of the Polycomb and trithorax complex in Canton-S subjected to desiccation stress. Concomitant with this, there is an altered enrichment of H3K27me3 and H3K4me3 at the upstream regions of the stress responsive genes. The enrichment of activating mark, H3K4me3, is higher in non-stress condition. H3K27me3, the repressive mark, is more pronounced under stress condition, which in turn, can be correlated with the binding of Pc. Our results show that desiccation stress induces dynamic switching in expression and enrichment of PcG and TrxG in the upstream region of genes, which correlates with histone modifications. We provide evidence that epigenetic modulation could be one of the mechanisms to adapt to the desiccation stress in Drosophila. Thus, our study proposes the interaction of epigenome and environmental factors.

Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections.

The present study describes the immunotoxic effect of chronic fluoride exposure on adult zebrafish (Danio rerio). Zebrafish were exposed to fluoride (71.12 mg/L; 1/10 LC50) for 30 d and the expression of selected genes studied. We observed significant elevation in the detoxification pathway gene cyp1a suggesting chronic exposure to non-lethal concentration of fluoride is indeed toxic to fish. Fluoride mediated pro-oxidative stress is implicated with the downregulation in superoxide dismutase 1 and 2 (sod1/2) genes. Fluoride affected DNA repair machinery by abrogating the expression of the DNA repair gene rad51 and growth arrest and DNA damage inducible beta a gene gadd45ba. The upregulated expression of casp3a coupled with altered Bcl-2 associated X protein/B-cell lymphoma 2 ratio (baxa/bcl2a) clearly suggested chronic fluoride exposure induced the apoptotic cascade in zebrafish. Fluoride-exposed zebrafish when challenged with non-lethal dose of fish pathogen A. hydrophila revealed gross histopathology in spleen, bacterial persistence and significant mortality. We report that fluoride interferes with system-level output of pro-inflammatory cytokines tumour necrosis factor-α, interleukin-1ß and interferon-γ, as a consequence, bacteria replicate efficiently causing significant fish mortality. We conclude, chronic fluoride exposure impairs the redox balance, affects DNA repair machinery with pro-apoptotic implications and suppresses pro-inflammatory cytokines expression abrogating host immunity to bacterial infections.

Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc.

In vivo and in silico studies to identify mechanisms associated with Nurr1 modulation following early life exposure to permethrin in rats.

The present work was designed to study the mechanisms associated with Nurr1 modulation following early life permethrin (PERM) treatment during rat's life span. Here we demonstrate that PERM exposure in rats, at a dose close to No Observed Adverse Effect Level (NOAEL) for 15days during neonatal brain development leads to its accumulation long after exposure. In striatum from adolescent rats we detected an increase in DNA methyltransferases (DNMTs) such as DNMT1, DNMT3a, Tyrosine hydroxylase, monomeric and aggregated α-synuclein protein levels. Adult rats showed enhanced DNMT3b and α-synuclein aggregation compared to the control group, while with aging a significant decrease in all biomarkers studied was observed. No changes in Nurr1 promoter methylation in adolescent, adult and old rats were found. In silico studies showed clear evidence of a strong binding interaction between PERM and its metabolite 3-phenoxybenzoic acid with the nuclear orphan receptor Nurr1. These findings suggest that an additional interference with the dopaminergic neuron pathway could occur in situ during PERM accumulation in brain. Therefore, Nurr1 modulation in early life PERM-treated rats, depends on age-related adaptive responses in animals.

Human PRE-PIK3C2B, an intronic cis-element with dual function of activation and repression.

The Polycomb/Trithorax Responsive Elements (PRE/TREs) are the cis-regulatory sequences that interact with both repressive (PcG) as well as activating (TrxG) complexes. However, most of the mammalian PREs are demonstrated to interact with the repressive polycomb (PcG) complexes only. We have carried out an unbiased search for proteins interacting with human PRE-PIK3C2B (hPRE-PIK3C2B) based on DNA affinity purification followed by mass spectrometry and identified MLL, MLL4 and WDR87 among other proteins in three biological replicates in HEK, U87 and HeLa cell lines. The hPRE-PIK3C2B interacts with the members of multiple activating complexes (COMPASS-like). The increase in the interaction of MLL and MLL4 on depletion of YY1 and the increase in the enrichment of YY1 and EZH2 upon MLL knockdown at the hPRE-PIK3C2B indicate the dual occupancy and suggest a concentration dependent enrichment of the activator or the repressor complex at hPRE-PIK3C2B. Further, we show that the hPRE-PIK3C2B interacts with the Drosophila homologues of PcG and TrxG proteins in transgenic flies. Here, we found that there is an increased enrichment of Pc (Polycomb) in comparison to Trx (TrxG protein) at hPRE-PIK3C2B in the Drosophila transgenic flies and this seems to be the default state while the balance is tipped towards the trithorax complex in PcG mutants. To the best of our knowledge, this is one of the early demonstrations of human PRE acting as a TRE without any sequence alteration.

Interaction of the Chromatin Remodeling Protein hINO80 with DNA.

The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity in vitro. Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes.

The functional diversity of Drosophila Ino80 in development.

Ino80 is well known as a chromatin remodeling protein with the catalytic function of DNA dependent ATPase and is highly conserved across phyla. Ino80 in human and Drosophila is known to form the Ino80 complex in association with the DNA binding protein Ying-Yang 1 (YY1)/Pleiohomeotic (Pho) the Drosophila homologue. We have earlier reported that Ino80 sub-family of proteins has two functional domains, namely, the DNA dependent ATPase and the DNA binding domain. In the background of the essential role of dIno80 in development, we provide evidence of Pho independent function of dIno80 in development and analyze the dual role of dIno80 in activation as well as repression in the context of the homeotic gene Scr (sex combs reduced) in imaginal discs. This differential effect of dIno80 in different imaginal discs suggests the contextual function of dIno80 as an Enhancer of Trithorax and Polycomb (ETP). We speculate on the role of dIno80 as a chromatin remodeler on one hand and a potential recruiter of epigenetic regulatory complexes on the other.

The Mycobacterium tuberculosis recombinant LprN protein of mce4 operon induces Th-1 type response deleterious to protection in mice.

Lipoproteins are known to be effective immunogens and affect both innate and adaptive immunity. The lprN gene of Mycobacterium tuberculosis has been predicted to encode for a putative lipoprotein in silico. Here, we studied its function as an immunogen by in vivo studies in mice. The recombinant LprN protein, expressed and purified in Escherichia coli, triggered a cell-mediated immune response in BALB/c mice. This was observed by significantly higher T-cell proliferation and increased production of TNF-α and IFN-γ cytokines. However, pre-exposure to LprN protein failed to provide protection in mice after challenge with a virulent strain of M. tuberculosis. Histological examination showed an increase in tissue destruction in experimental animals, indicating an immunogenic potential for LprN protein that enhanced the virulence of bacilli.

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.

Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach.

Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy.

Pharmacoepigenomics: an interplay of epigenetic modulation of drug response and modulation of the epigenome by drugs.

Epigenetic modulation captures the lack of correlation between the genotype and the phenotype. It also provides an interface between environment and the genotype leading to functional plasticity of the genome. While drug response can be modulated by the epigenome, the therapeutic intervention by drugs can also be considered as an environmental cue for epigenetic alterations. The effect of genetic polymorphism has accrued considerable interest and population polymorphism leading to variation in drug response is being studied extensively. The available data on the epigenetic marking of the whole genome in different contexts implies that no biological pathway or process in the mammalian system is free of epigenetic influence and thus, drug metabolism would not be an exception. In the light of the fact that the epigenome is not only variable between individuals, but that it also varies between different tissues of the same individual and with the age of the individual, it is still a long journey to transit from the correlation to causal relationship between drug response and the epigenomic variations. The present review is focused on recent developments in the area and a brief discussion of the future prospects and challenges.