Sanjeev Khosla

A novel nucleoid-associated protein of Mycobacterium tuberculosis is a sequence homolog of GroEL

The Mycobacterium tuberculosis genome sequence reveals remarkable absence of many nucleoid-associated proteins (NAPs), such as HNS, Hfq or DPS. In order to characterize the nucleoids of M. tuberculosis, we have attempted to identify NAPs, and report an interesting finding that a chaperonin-homolog, GroEL1, is nucleoid associated. We report that M. tuberculosis GroEL1 binds DNA with low specificity but high affinity, suggesting that it might have naturally evolved to bind DNA. We are able to demonstrate that GroEL1 can effectively function as a DNA-protecting agent against DNase I or hydroxyl-radicals. Moreover, Atomic Force Microscopic studies reveal that GroEL1 can condense a large DNA into a compact structure. We also provide in vivo evidences that include presence of GroEL1 in purified nucleoids, in vivo crosslinking followed by Southern hybridizations and immunofluorescence imaging in M. tuberculosis confirming that GroEL1: DNA interactions occur in natural biological settings. These findings therefore reveal that M. tuberculosis GroEL1 has evolved to be associated with nucleoids.

DNA Methylation Profile at the DNMT3L Promoter: A Potential Biomarker for Cervical Cancer

Epigenetic events play a prominent role during cancer development. This is evident from the fact that almost all cancer types show aberrant DNA methylation. These abnormal DNA methylation levels are not restricted to just a few genes but affect the whole genome. Previous studies have shown genome-wide DNA hypomethylation and gene-specific hypermethylation to be a hallmark of most cancers. Molecules like DNA methyltransferase act as effectors of epigenetic reprogramming. In the present study we have examined the possibility that the reprogramming genes themselves undergo epigenetic modifications reflecting their changed transcriptional status during cancer development. Comparison of DNA methylation status between the normal and cervical cancer samples was carried out at the promoters of a few reprogramming molecules . Our study revealed statistically significant DNA methylation differences within the promoter of DNMT3L. A regulator of de novo DNA methyltransferases DNMT3A and DNMT3B, DNMT3L promoter was found to have lost DNA methylation to varying levels in 14 out of 15 cancer cervix samples analysed. The present study highlights the importance of DNA methylation profile at DNMT3L promoter not only as a promising biomarker for cervical cancer, which is the second most common cancer among women worldwide, but also provides insight into the possible role of DNMT3L in cancer development.

The DNA methyltranferase Dnmt2 participates in RNA processing during cellular stress.

The strong evolutionary conservation of the DNA methyltransferase, Dnmt2, is at odds with the absence of phenotypic defects in organisms lacking Dnmt2. The cellular processes where Dnmt2 has a role to play also remain largely undiscovered. Here we show that Dnmt2 is a part of RNA processing machinery during cellular stress. In addition to interacting with proteins involved in RNA processing and cellular stress, Dnmt2 exhibits nucleo-cytoplasmic shuttling in response to cellular stress. Normally present in the nucleus, under conditions of stress, Dnmt2 relocalises to the cytoplasmic Stress Granules and RNA processing bodies. Surprisingly, for a DNA methyltransferase, knockout of which showed no phenotypic defects in several species, our results show that transient transfection of Dnmt2 in mammalian cells causes cell lethality. Interestingly, Dnmt2 overexpression altered the expression of several genes involved in viral infection. Taking into consideration its recently identified role in retrotransposon silencing, the role of Dnmt2 in stress granules could represent a primitive cellular defense mechanism against viral infection.

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.

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.

Reprogramming of HeLa cells upon DNMT3L overexpression mimics carcinogenesis

Previously we had discovered loss of DNA methylation at the DNMT3L promoter, an enzymatically-inactive DNA methyltransferase, in squamous cell carcinoma of cervix indicating association between cancer and DNMT3L. This study extends this correlation further by identifying the role of DNMT3L in nuclear reprogramming, an event central to the process of carcinogenesis. We show that in cervical cancer cell lines, overexpression of DNMT3L, which functions by regulating the activity of DNMT3A and DNMT3B, increased cellular proliferation and anchorage-independent growth. Importantly, increased DNMT3L expression resulted in changed morphology of cells but this change was gradual and observed only after several passages. Interestingly, confluent cultures of DNMT3L-overexpressing HeLa cell colonies had characteristics of iPS cells. Concomitant with the morphological changes, expression pattern of genes important in nuclear reprogramming, development and cell cycle were observed to have significantly changed. Many imprinted genes, the known targets of DNMT3L, were downregulated. The slow nature of morphological changes and genome-wide nuclear reprogramming observed upon DNMT3L overexpression reinforces its role in carcinogenesis.

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.

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.

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

ABSTRACT 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.

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.