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Pushpavalli S.N.C.V.L.,Functional Genomics and Gene Silencing Group | Pushpavalli S.N.C.V.L.,Indian Institute of Chemical Technology | Pushpavalli S.N.C.V.L.,Acharya N.G. Ranga Agricultural University | Sarkar A.,Indian Institute of Chemical Technology | And 6 more authors.
FASEB Journal | Year: 2014

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs-miR-981 and miR-317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle. © FASEB.


Pushpavalli S.N.C.V.L.,Indian Institute of Chemical Technology | Sarkar A.,Indian Institute of Chemical Technology | Ramaiah M.J.,Indian Institute of Chemical Technology | Chowdhury D.R.,Functional Genomics and Gene Silencing Group | And 2 more authors.
BMC Molecular Biology | Year: 2013

Background: In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established.Results: Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks.Conclusion: mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway. © 2013 Pushpavalli et al.; licensee BioMed Central Ltd.


Sarma P.,Indian Institute of Chemical Technology | Ramaiah M.J.,Indian Institute of Chemical Technology | Kamal A.,Indian Institute of Chemical Technology | Bhadra U.,Functional Genomics and Gene Silencing Group | Pal Bhadra M.,Indian Institute of Chemical Technology
Cancer Biology and Therapy | Year: 2014

DNA damage response (DDR) that includes cell cycle check points, DNA repair, apoptosis, and senescence is intimately linked with cancer. It shields an organism against cancer development when genomic integrity fails. DNA repair pathways protect the cells from tumor progression caused as a result of DNA damage induced by irradiation or due to chemotherapeutic treatment. Many promising anticancer agents have been identified that target specific DNA repair pathways in response to DNA damage thereby leading to apoptosis. here we identified a novel bisindole-PBD conjugate that possess potent anticancer activity in breast cancer cells. Further studies aimed at understanding the mechanism of action of the molecule showed its role in DNA damage induced apoptosis via inhibition of DNA repair pathway. Trypan blue and BrdU assay exhibited a dose-dependent effect. single-stranded DNA damage was observed by COMET assay. In addition DNA damage induced ROS generation with simultaneous activation of ATM and ATR upon compound treatment was observed. Further downregulation of Bcl-XL and activation of Bax showed DNA damage induced apoptosis in MCF-7 and MDAMB-231 cells. In conclusion, it can be summarized that bisindole-PBD conjugate induces DNA damage in a dose dependent (2, 4, and 8 μM) manner by inhibiting the DNA repair genes. © 2014 Landes Bioscience.


Shinde S.,Functional Genomics and Gene Silencing Group | Arora N.,Functional Genomics and Gene Silencing Group | Bhadra U.,Functional Genomics and Gene Silencing Group
International Journal of Genomics | Year: 2013

Amyotrophic Lateral Sclerosis (ALS) is a rare neurological disease affecting mainly motor neurons and often leads to paralysis and death in extreme cases. For exploring the role of microRNAs in genes regulation in ALS disease, miRanda was employed for prediction of target sites of miRNAs expressed in various parts of brain and CNS on 35 genes associated with ALS. Similar search was conducted using TargetScan and PicTar for prediction of target sites in 3 ′ UTR only. 1456 target sites were predicted using miRanda and more target sites were found in 5 ′ UTR and CDS region as compared to 3 ′ UTR. 11 target sites were predicted to be common by all the algorithms and, thus, these represent the most significant sites. Target site hotspots were identified and were recognized as hotspots for multiple miRNAs action, thus, acting as favoured sites of action for the repression of gene expression. The complex interplay of genes and miRNAs brought about by multiplicity and cooperativity was explored. This investigation will aid in elucidating the mechanism of action of miRNAs for the considered genes. The intrinsic network of miRNAs expressed in nervous system and genes associated with ALS may provide rapid and effective outcome for therapeutic applications and diagnosis. © 2013 Santosh Shinde et al.


Shinde S.,Functional Genomics and Gene Silencing Group | Bhadra U.,Functional Genomics and Gene Silencing Group
BioMed Research International | Year: 2015

Small noncoding regulatory RNA exist in wide spectrum of organisms ranging from prokaryote bacteria to humans. In human, a systematic search for noncoding RNA is mainly limited to the nuclear and cytosolic compartments. To investigate whether endogenous small regulatory RNA are present in cell organelles, human mitochondrial genome was also explored for prediction of precursor microRNA (pre-miRNA) and mature miRNA (miRNA) sequences. Six novel miRNA were predicted from the organelle genome by bioinformatics analysis.The structures are conserved in other five mammals including chimp, orangutan, mouse, rat, and rhesus genome. Experimentally, six human miRNA are well accumulated or deposited in human mitochondria.Three of them are expressed less prominently inNorthern analysis. To ascertain their presence in human skeletalmuscles, total RNAwas extracted from enriched mitochondria by an immunomagnetic method. The expression of six novel pre-miRNA and miRNA was confirmed by Northern blot analysis; however, low level of remaining miRNA was found by sensitive Northern analysis. Their presence is further confirmed by real time RT-PCR.Thesix miRNA find theirmultiple targets throughout the human genome in three different types of software.The luciferase assay was used to confirm that MT-RNR2 gene was the potential target of hsa-miR-mit3 and hsamiR-mit4. Copyright © 2015 S. Shinde and U. Bhadra.


Banerjee P.,Manipal University India | Surendran H.,Manipal University India | Chowdhury D.R.,Functional Genomics and Gene Silencing Group | Prabhakar K.,Manipal Hospital | Pal R.,Manipal University India
Journal of Molecular Medicine | Year: 2016

Abstract: Epithelial-mesenchymal transition (EMT) is one of the key biological phenomena behind cancer and metastasis. Clinical studies suggest that patients undergoing metformin therapy are less predisposed to cancer but the underlying mechanism is far from clear. Given that metformin also acts as TGF-β inhibitor, we sought to explore whether and how metformin could modulate EMT in a cancer like microenvironment. Our data using human cell lines revealed that metformin induced a distinct change from stromal-shaped mesenchymal cells to cuboidal-shaped epithelial cells with upregulation of epithelial markers and mitigation of their invasive property. One of the key regulatory pathways, which intersect tumorigenesis and metformin activity, is AMPK. We demonstrated that metformin attenuates ERK signaling by activating AMPK pathway leading to suppression of Snail and Slug resulting in upregulation of crucial tumor suppressor gene E-cadherin. ChIP assay confirmed insufficient binding of repressors like Slug to the E-cadherin promoter. Further, our data revealed reduction in HDAC activity prompting hypomethylation of E-cadherin promoter thus reflecting an epigenetic modification. To expand the translational significance of the study we verified these findings in diabetic patients undergoing metformin treatment. To our knowledge this is the first report representing an inverse relationship of AMPK and ERK signaling axis in promoting mesenchymal to epithelial transition (MET) via re-expression of E-cadherin upon metformin treatment thus rationalizing lower incidence of cancer in metformin-administered patients. Key message: Metformin promotes reversal of the epithelial-mesenchymal transition.Metformin attenuates ERK signaling by activating AMP kinase.Metformin induces hypomethylation of the E-cadherin gene promoter.Epigenetic modification of the E-cadherin promoter was observed in leukocytes from diabetic subjects.These findings provide a potential basis for decreased cancer incidence in metformin-treated subjects. © 2016 Springer-Verlag Berlin Heidelberg


Chandrasekhar S.,Indian Institute of Chemical Technology | Pushpavalli S.N.C.V.L.,Functional Genomics and Gene Silencing Group | Chatla S.,Functional Genomics and Gene Silencing Group | Mukhopadhyay D.,Functional Genomics and Gene Silencing Group | And 6 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2012

aza-Flavanones have been identified as a new class of selective microRNA inhibitors. These compounds were found to arrest cell cycle via a novel cross species microRNA-dependent regulatory pathway interpreting an unexpected link between cell cycle arrest and microRNA mediated control in cancer. © 2011 Elsevier Ltd. All rights reserved.


Pushpavalli S.N.C.V.L.,Functional Genomics and Gene Silencing Group | Bag I.,Indian Institute of Chemical Technology | Pal-Bhadra M.,Indian Institute of Chemical Technology | Bhadra U.,Functional Genomics and Gene Silencing Group
Chromosome Research | Year: 2012

Argonaute-1 (Ago-1) plays a crucial role in gene regulation and genome stability via biogenesis of small non-coding RNAs. Two "Argonaute" family genes, piwi and Ago-2 in Drosophila are involved in multiple silencing mechanisms in the nucleus, transgene cosuppression, long-distant chromosome interaction, nuclear organization and heterochromatin formation. To investigate whether Ago-1 also plays a similar role, we have generated a series of Ago-1 mutations by excising P element, inserted in the Ago-1 promoter (Ago-1 k08121). AGO-1 protein is distributed uniformly in the nucleus and cytosol in early embryos but accumulated predominantly in the cytoplasm during the gastrulation stage. Repeat induced silencing produced by the mini-white (mw) array and transcriptional cosuppression of non-homologous transgenes Adh-w/w- Adh was disrupted by Ago-1 mutation. These effects of Ago-1 are distict from its role in microRNA processing because Dicer-1, a critical enzyme for miRNA biogenesis, has no role on the above silencing. Reduction of AGO-1 protein dislodged the POLYCOMB, EZ (enhancer of zeste) and H3me3K27 binding at the cosuppressed Adh-w transgene insertion sites suggesting its role in Polycomb dependent cosuppression. An overall reduction of methylated histone H3me2K9 and H3me3K27 from the polytene nuclei precisely from the mw promoters was also found that leads to concomitant changes in the chromatin structure. These results suggest a prominent role of Ago-1 in chromatin organization and transgene silencing and demonstrate a critical link between transcriptional transgene cosuppression, heterochromatin formation and chromatin organization. We propose Drosophila Ago-1 as a multifunctional RNAi component that interconnects at least two unrelated events, chromatin organization in the nucleus and microRNA processing in the cytoplasm, which may be extended to the other systems. © 2012 Springer Science+Business Media B.V.


Gandhi S.G.,Functional Genomics and Gene Silencing Group | Bag I.,Functional Genomics and Gene Silencing Group | Bag I.,Indian Institute of Chemical Technology | Sengupta S.,Indian Institute of Chemical Technology | And 2 more authors.
FEBS Journal | Year: 2015

Glioma amplified sequence41 (Gas41) is a highly conserved putative transcription factor that is frequently abundant in human gliomas. Gas41 shows oncogenic activity by promoting cell growth and viability. In the present study, we show that Gas41 is required for proper functioning of RNA interference (RNAi) machinery in the nuclei, although three basic structural domains of RNAi components PAZ, PIWI and dsRNA with respect to binding are absent in the structural sequences. Variations of structural domains are highly conserved among prokaryotes and eukaryotes. Gas41 interacts with cytological RNase III enzyme Dicer1 both biochemically and genetically. However, Drosophila Gas41 functions as chromatin remodeler and interacts with different heterochromatin markers and repeat-induced transgene silencing by modulating position effect variegation. We also show that transcriptional inactive Gas41 mutant interferes with the functional assembly of heterochromatin-associated proteins, dimethylated lysine 9 of histone H3 and heterochromatic protein 1 in developing embryos. A reduction of heterochromatic markers is accompanied by the mini-w promoter sequence in Gas41 mutants. These findings suggest that Drosophila Gas41 guides the repeat associated gene silencing and the Dicer1 interaction, thereby depicting a new role for Gas41. Gas41 is a critical RNAi component. In Drosophila, Gas41 plays a dual role. On the one hand, it appears to participate with Dicer 1 in the RNAi pathway and, alternatively, it also participates in repeat-induced gene silencing by accumulating heterochromatin proteins at the mini-w array promoters. Therefore, it represents an intriguing and apparently paradoxical new finding in RNA technology with respect to the process of heterochromatin gene silencing. Oncogene Glioma amplified sequence41 (Gas41) is required for functioning RNAi machinery in the nuclei without three proper RNAi structural domains. Gas-41 interacts with Dicer1 in Drosophila. Gas 41 functions as chromatin remodeler interacting with HP1, H3K9 proteins and participates in siRNA pathways. Therefore Gas 41 is a new addition of RNAi machinery, which process heterochromatin gene silencing in the Drosophila. © 2014 FEBS.


Bhadra U.,Functional Genomics and Gene Silencing Group | Gandhi S.G.,Functional Genomics and Gene Silencing Group | Palaparthi R.,Functional Genomics and Gene Silencing Group | Balyan M.K.,Functional Genomics and Gene Silencing Group | Pal-Bhadra M.,Indian Institute of Chemical Technology
FEBS Journal | Year: 2016

The loss of the entire X chromosome in Drosophila males generates a genome-wide aneuploid effect. We have employed a systems biology approach (microarray) to investigate the global aneuploid effect of the maleless (mle) mutation that disrupts the binding of male specific lethal (MSL) proteins that function in dosage compensation. A large number of the genes (144) that encode a broad spectrum of cellular transport proteins and transcription factors are located mainly in the autosomes of Drosophila melanogaster. We found several such targets to be sensitive to the aneuploid effect and conserved with the X chromosome in primitive Drosophila species and Anopheles gambiae. During evolution, they shifted gradually from their X-chromosomal positions to the autosomes in the species D. melanogaster, suggesting that the counteraction of trans-acting regulatory modifiers and their targets in two separate chromosomes is evolutionarily advantageous. These findings suggest a remarkable and previously unexpected level of complexity favoring natural compensation for the aneuploid effect. We propose that the MSL complex functions in dosage compensation in two separate steps. Initially, X-linked genes including X-linked trans-acting modifiers are hyperactivated in dosage compensated males. Later, these compensated modifiers eventually mitigate inverse aneuploid effects for the equality in expression of their autosomal targets in males and females. Database: Microarray data are available at the Gene Expression Omnibus (GEO) web deposit of National Centre for Biotechnology Information (NCBI) with the accession number GSE78227. © 2016 Federation of European Biochemical Societies

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