DNMT3L interacts with Piwi and modulates the expression of piRNAs in transgenic Drosophila.

Aim: To explore the role of Piwi protein and piRNAs in DNMT3L-mediated epigenetic inheritance. Materials & methods: Transgenic Drosophila were used to examine the effect of ectopically expressed DNMT3L on the profile of piRNAs by sequencing of small RNAs. Results & conclusion: Our previous work showed accumulation and inheritance of epimutations across multiple generations in transgenic DNMT3L Drosophila. Here, we show interaction of DNMT3L with Piwi and a significant alteration in the piRNA profile across multiple generations in transgenic Drosophila. In the light of its interaction with histone H1, we propose that in addition to its role of modulating core histone modifications, DNMT3L allows for inheritance of epigenetic information through its collaboration with Piwi, piRNAs and histone H1.

The Mycobacterium tuberculosis methyltransferase Rv2067c manipulates host epigenetic programming to promote its own survival.

Mycobacterium tuberculosis has evolved several mechanisms to counter host defense arsenals for its proliferation. Here we report that M. tuberculosis employs a multi-pronged approach to modify host epigenetic machinery for its survival. It secretes methyltransferase (MTase) Rv2067c into macrophages, trimethylating histone H3K79 in a non-nucleosomal context. Rv2067c downregulates host MTase DOT1L, decreasing DOT1L-mediated nucleosomally added H3K79me3 mark on pro-inflammatory response genes. Consequent inhibition of caspase-8-dependent apoptosis and enhancement of RIPK3-mediated necrosis results in increased pathogenesis. In parallel, Rv2067c enhances the expression of SESTRIN3, NLRC3, and TMTC1, enabling the pathogen to overcome host inflammatory and oxidative responses. We provide the structural basis for differential methylation of H3K79 by Rv2067c and DOT1L. The structures of Rv2067c and DOT1L explain how their action on H3K79 is spatially and temporally separated, enabling Rv2067c to effectively intercept the host epigenetic circuit and downstream signaling.

Detection of SARS-CoV-2 in the air in Indian hospitals and houses of COVID-19 patients.

To understand the transmission characteristics of severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) through air, samples from different locations occupied by coronavirus disease (COVID-19) patients were analyzed. Three sampling strategies were used to understand the presence of virus in the air in different environmental conditions. In the first strategy, which involved hospital settings, air samples were collected from several areas of hospitals like COVID-intensive-care units (ICUs), nurse-stations, COVID-wards, corridors, non-COVID-wards, personal protective equipment (PPE) doffing areas, COVID rooms, out-patient (OP) corridors, mortuary, COVID casualty areas, non-COVID ICUs and doctors' rooms. Out of the 80 air samples collected from 6 hospitals from two Indian cities- Hyderabad and Mohali, 30 samples showed the presence of SARS-CoV-2 nucleic acids. In the second sampling strategy, that involved indoor settings, one or more COVID-19 patients were asked to spend a short duration of time in a closed room. Out of 17 samples, 5 samples, including 4 samples collected after the departure of three symptomatic patients from the room, showed the presence of SARS-CoV-2 nucleic acids. In the third strategy, involving indoor settings, air samples were collected from rooms of houses of home-quarantined COVID-19 patients and it was observed that SARS-CoV-2 RNA could be detected in the air in the rooms occupied by COVID-19 patients but not in the other rooms of the houses. Taken together, we observed that the air around COVID-19 patients frequently showed the presence of SARS-CoV-2 RNA in both hospital and indoor residential settings and the positivity rate was higher when 2 or more COVID-19 patients occupied the room. In hospitals, SARS-CoV-2 RNA could be detected in ICUs as well as in non-ICUs, suggesting that the viral shedding happened irrespective of the severity of the infection. This study provides evidence for the viability of SARS-CoV-2 and its long-range transport through the air. Thus, airborne transmission could be a major mode of transmission for SARS-CoV-2 and appropriate precautions need to be followed to prevent the spread of infection through the air.

Epigenetic interaction of microbes with their mammalian hosts.

The interaction of microbiota with its host has the ability to alter the cellular functions of both, through several mechanisms. Recent work, from many laboratories including our own, has shown that epigenetic mechanisms play an important role in the alteration of these cellular functions. Epigenetics broadly refers to change in the phenotype without a corresponding change in the DNA sequence. This change is usually brought by epigenetic modifications of the DNA itself, the histone proteins associated with the DNA in the chromatin, non-coding RNA or the modifications of the transcribed RNA. These modifications, also known as epigenetic code, do not change the DNA sequence but alter the expression level of specific genes. Microorganisms seem to have learned how to modify the host epigenetic code and modulate the host transcriptome in their favour. In this review, we explore the literature that describes the epigenetic interaction of bacteria, fungi and viruses, with their mammalian hosts.

Role of genomic imprinting in mammalian development.

Non-mendelian inheritance refers to the group of phenomena and observations related to the inheritance of genetic information that cannot be merely explained by Mendel's laws of inheritance. Phenomenon including Genomic imprinting, X-chromosome Inactivation, Paramutations are some of the best studied examples of non-mendelian inheritance. Genomic imprinting is a process that reversibly marks one of the two homologous loci, chromosome or chromosomal sets during development, resulting in functional non-equivalence of gene expression. Genomic imprinting is known to occur in a few insect species, plants, and placental mammals. Over the years, studies on imprinted genes have contributed immensely to highlighting the role of epigenetic modifications and the epigenetic circuitry during gene expression and development. In this review, we discuss the phenomenon of genomic imprinting in mammals and the role it plays especially during fetoplacental growth and early development.

The putative Neuronatin imprint control region is an enhancer that also regulates the Blcap gene.

AIM: To investigate the regulatory potential of the Nnat second intron within the Nnat/Blcap micro-imprinted domain. MATERIALS & METHODS: Mice with deletion of Nnat second intron at the endogenous Nnat/Blcap micro-imprinted domain were used to examine the effect of Nnat second intron on the transcriptional regulation of the Nnat and Blcap genes. RESULTS & CONCLUSION: Deletion of Nnat second intron affected Nnat expression in cis leading to the loss of Nnat expression from the active paternal allele. Nnat second intron was found to have the characteristics of an imprint control region including allele-specific DNA methylation and histone modifications and it also regulated the epigenetic profile of Nnat promoter by acting as an enhancer. Nnat second intron was also found to be regulating the expression of the Blcap transcripts.

Biochemical and dynamic basis for combinatorial recognition of H3R2K9me2 by dual domains of UHRF1.

UHRF1 is a multi-domain protein comprising of a tandem tudor (UHRF1 TTD), a PHD finger, and a SET and RING-associated domain. It is required for the maintenance of CG methylation, heterochromatin formation and DNA repair. Isothermal titration calorimetry binding studies of unmodified and methylated lysine histone peptides establish that the UHRF1 TTD binds dimethylated Lys9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that TTD-PHD of UHRF1 (UHRF1 TTD-PHD) preferentially recognizes dimethyl-lysine status. Importantly, we show that Asp145 in the binding pocket determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. Interestingly, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of K9me2 by the UHRF1 TTD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 by the UHRF1 TTD-PHD. We propose that subtle variations of key residues at the binding pocket determine status specific recognition of histone methyl-lysines by the reader domains.

Differentially regulated gene expression in quiescence versus senescence and identification of ARID5A as a quiescence associated marker.

In multicellular organisms majority of the cells remain in a non-dividing states of either quiescence (reversible) or senescence (irreversible). In the present study, gene expression signatures unique to quiescence and senescence were identified using microarray in osteosarcoma cell line, U2OS. It was noted that certain genes and pathways like NOD pathway was shared by both the growth arrest conditions. A major highlight of the present study was increased expression of number of chemokines and cytokines in both quiescence and senescence. While senescence-associated secretory phenotype (SASP) is well known, the quiescence-associated secretory phenotype (QASP) is relatively unknown and appeared novel in this study. ARID5A, a subunit of SWI/SNF complex was identified as a quiescence associated gene. The endogenous expression of ARID5A increased during serum starved condition of quiescence. Overexpression of ARID5A resulted in more number of cells in G0/G1 phase of cell cycle. Further ARID5A overexpressing cells when subjected to serum starvation showed a pronounced secretory phenotype. Overall, the present work has identified gene expression signatures which can distinguish quiescence from senescence.

Histone methyltransferase SUV39H1 participates in host defense by methylating mycobacterial histone-like protein HupB.

Host cell defense against an invading pathogen depends upon various multifactorial mechanisms, several of which remain undiscovered. Here, we report a novel defense mechanism against mycobacterial infection that utilizes the histone methyltransferase, SUV39H1. Normally, a part of the host chromatin, SUV39H1, was also found to be associated with the mycobacterial bacilli during infection. Its binding to bacilli was accompanied by trimethylation of the mycobacterial histone-like protein, HupB, which in turn reduced the cell adhesion capability of the bacilli. Importantly, SUV39H1-mediated methylation of HupB reduced the mycobacterial survival inside the host cell. This was also true in mice infection experiments. In addition, the ability of mycobacteria to form biofilms, a survival strategy of the bacteria dependent upon cell-cell adhesion, was dramatically reduced in the presence of SUV39H1. Thus, this novel defense mechanism against mycobacteria represents a surrogate function of the epigenetic modulator, SUV39H1, and operates by interfering with their cell-cell adhesion ability.

Cytosine methylation by DNMT2 facilitates stability and survival of HIV-1 RNA in the host cell during infection.

The enigmatic methyltransferase, DNMT2 (DNA methyltransferase 2), structurally resembles a DNA methyltransferase, but has been shown to be a tRNA methyltransferase targeting cytosine within a specific CpG in different tRNA molecules. We had previously shown that, during environmental stress conditions, DNMT2 is re-localized from the nucleus to the cytoplasmic stress granules (SGs) and is associated with RNA-processing proteins. In the present study, we show that DNMT2 binds and methylates various mRNA species in a sequence-independent manner and gets re-localized to SGs in a phosphorylation-dependent manner. Importantly, our results indicate that HIV-1 enhances its survivability in the host cell by utilizing this RNA methylation capability of DNMT2 to increase the stability of its own genome. Upon infection, DNMT2 re-localizes from the nucleus to the SGs and methylates HIV-1 RNA. This DNMT2-dependent methylation provided post-transcriptional stability to the HIV-1 RNA. Furthermore, DNMT2 overexpression increased the HIV-1 viral titre. This would suggest that HIV hijacks the RNA-processing machinery within the SGs to ensure its own survival in the host cell. Thus, our findings provide for a novel mechanism by which virus tries to modulate the host cell machinery to its own advantage.

Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status.

Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status.

Genome-wide non-CpG methylation of the host genome during M. tuberculosis infection.

A mammalian cell utilizes DNA methylation to modulate gene expression in response to environmental changes during development and differentiation. Aberrant DNA methylation changes as a correlate to diseased states like cancer, neurodegenerative conditions and cardiovascular diseases have been documented. Here we show genome-wide DNA methylation changes in macrophages infected with the pathogen M. tuberculosis. Majority of the affected genomic loci were hypermethylated in M. tuberculosis infected THP1 macrophages. Hotspots of differential DNA methylation were enriched in genes involved in immune response and chromatin reorganization. Importantly, DNA methylation changes were observed predominantly for cytosines present in non-CpG dinucleotide context. This observation was consistent with our previous finding that the mycobacterial DNA methyltransferase, Rv2966c, targets non-CpG dinucleotides in the host DNA during M. tuberculosis infection and reiterates the hypothesis that pathogenic bacteria use non-canonical epigenetic strategies during infection.

DNMT3L enables accumulation and inheritance of epimutations in transgenic Drosophila.

DNMT3L is an important epigenetic regulator in mammals, integrating DNA methylation and histone modification based epigenetic circuits. Here we show DNMT3L to be a part of the machinery that enables inheritance of epigenetic modifications from one generation to the next. Ectopic expression of DNMT3L in Drosophila, which lacks DNMT3L and its normal interacting partners DNMT3A and DNMT3B, lead to nuclear reprogramming that was gradual and progressive, resulting in melanotic tumors that were observed only when these flies were maintained for five generations. This global gene expression misregulation was accompanied by aberrations in the levels of H3K4me3 and H3K36me3, globally as well as at specific gene promoters. The levels of these epigenetic aberrations (epimutations) also increased progressively across successive generations. The accumulation and inheritance of epimutations across multiple generations recapitulates the important role of DNMT3L in intergenerational epigenetic inheritance in mammals.

Learning epigenetic regulation from mycobacteria.

In a eukaryotic cell, the transcriptional fate of a gene is determined by the profile of the epigenetic modifications it is associated with and the conformation it adopts within the chromatin. Therefore, the function that a cell performs is dictated by the sum total of the chromatin organization and the associated epigenetic modifications of each individual gene in the genome (epigenome). As the function of a cell during development and differentiation is determined by its microenvironment, any factor that can alter this microenvironment should be able to alter the epigenome of a cell. In the study published in Nature Communications (Yaseen 2015 Nature Communications 6:8922 doi: 10.1038/ncomms9922), we show that pathogenic Mycobacterium tuberculosis has evolved strategies to exploit this pliability of the host epigenome for its own survival. We describe the identification of a methyltransferase from M. tuberculosis that functions to modulate the host epigenome by methylating a novel, non-canonical arginine, H3R42 in histone H3. In another study, we showed that the mycobacterial protein Rv2966c methylates cytosines present in non-CpG context within host genomic DNA upon infection. Proteins with ability to directly methylate host histones H3 at a novel lysine residue (H3K14) has also been identified from Legionella pnemophilia (RomA). All these studies indicate the use of non-canonical epigenetic mechanisms by pathogenic bacteria to hijack the host transcriptional machinery.

Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3.

Mycobacteria are successful pathogens that modulate the host immune response through unclear mechanisms. Here we show that Rv1988, a secreted mycobacterial protein, is a functional methyltransferase that localizes to the host nucleus and interacts with chromatin. Rv1988 methylates histone H3 at H3R42 and represses the genes involved in the first line of defence against mycobacteria. H3R42me2, a non-tail histone modification, is present at the entry and exit point of DNA in the nucleosome and not within the regulatory sites in the N-terminal tail. Rv1988 deletion in Mycobacterium tuberculosis reduces bacterial survival in the host, and experimental expression of M. tuberculosis Rv1988 in non-pathogenic Mycobacterium smegmatis negatively affects the health of infected mice. Thus, Rv1988 is an important mycobacterial virulence factor, which uses a non-canonical epigenetic mechanism to control host cell transcription.

The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding.

To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation.

The CpG island encompassing the promoter and first exon of human DNMT3L gene is a PcG/TrX response element (PRE).

DNMT3L, a member of DNA methyltransferases family, is present only in mammals. As it provides specificity to the action of de novo methyltransferases, DNMT3A and DNMT3B and interacts with histone H3, DNMT3L has been invoked as the molecule that can read the histone code and translate it into DNA methylation. It plays an important role in the initiation of genomic imprints during gametogenesis and in nuclear reprogramming. With important functions attributed to it, it is imperative that the DNMT3L expression is tightly controlled. Previously, we had identified a CpG island within the human DNMT3L promoter and first exon that showed loss of DNA methylation in cancer samples. Here we show that this Differentially Methylated CpG island within DNMT3L (DNMT3L DMC) acts to repress transcription, is a Polycomb/Trithorax Response Element (PRE) and interacts with both PRC1 and PRC2 Polycomb repressive complexes. In addition, it adopts inactive chromatin conformation and is associated with other inactive chromatin-specific proteins like SUV39H1 and HP1. The presence of DNMT3L DMC also influences the adjacent promoter to adopt repressive histone post-translational modifications. Due to its association with multiple layers of repressive epigenetic modifications, we believe that PRE within the DNMT3L DMC is responsible for the tight regulation of DNMT3L expression and the aberrant epigenetic modifications of this region leading to DNMT3L overexpression could be the reason of nuclear programming during carcinogenesis.

DNA methylation and cancer.

Cancer has been considered a genetic disease with a wide array of well-characterized gene mutations and chromosomal abnormalities. Of late, aberrant epigenetic modifications have been elucidated in cancer, and together with genetic alterations, they have been helpful in understanding the complex traits observed in neoplasia. "Cancer Epigenetics" therefore has contributed substantially towards understanding the complexity and diversity of various cancers. However, the positioning of epigenetic events during cancer progression is still not clear, though there are some reports implicating aberrant epigenetic modifications in very early stages of cancer. Amongst the most studied aberrant epigenetic modifications are the DNA methylation differences at the promoter regions of genes affecting their expression. Hypomethylation mediated increased expression of oncogenes and hypermethylation mediated silencing of tumor suppressor genes are well known examples. This chapter also explores the correlation of DNA methylation and demethylation enzymes with cancer.

Epigenetic profile of the euchromatic region of human Y chromosome.

The genome of a multi-cellular organism acquires various functional capabilities in different cell types by means of distinct chromatin modifications and packaging states. Acquired during early development, the cell type-specific epigenotype is maintained by cellular memory mechanisms that involve epigenetic modifications. Here we present the epigenetic status of the euchromatic region of the human Y chromosome that has mostly been ignored in earlier whole genome epigenetic mapping studies. Using ChIP-on-chip approach, we mapped H3K9ac, H3K9me3, H3K27me3 modifications and CTCF binding sites while DNA methylation analysis of selected CpG islands was done using bisulfite sequencing. The global pattern of histone modifications observed on the Y chromosome reflects the functional state and evolutionary history of the sequences that constitute it. The combination of histone and DNA modifications, along with CTCF association in some cases, reveals the transcriptional potential of all protein coding genes including the sex-determining gene SRY and the oncogene TSPY. We also observe preferential association of histone marks with different tandem repeats, suggesting their importance in genome organization and gene regulation. Our results present the first large scale epigenetic analysis of the human Y chromosome and link a number of cis-elements to epigenetic regulatory mechanisms, enabling an understanding of such mechanisms in Y chromosome linked disorders.