Silencing of toxic gene expression by Fis

Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.

Silencing of toxic gene expression by Fis

Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mumodifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fisacts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.

Primary Microcephaly Gene MCPH1 Shows Signatures of Tumor Suppressors and Is Regulated by miR-27a in Oral Squamous Cell Carcinoma

Mutations in the MCPH1 (microcephalin 1) gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS) gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC) samples, and observed that 14/71 (19.72%) informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22%) and 19/25 (76%) OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10%) tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival

Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M.tuberculosis topoisomerase I (TopoI(Mt)) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M.tuberculosis growth and open up new avenues for targeting the enzyme.

Methylation Silencing of ULK2, an Autophagy Gene, Is Essential for Astrocyte Transformation and Tumor Growth

Glioblastoma (GBM) is the most aggressive type of brain tumor and shows very poor prognosis. Here, using genome-wide methylation analysis, we show that G-CIMP+ and G-CIMP-subtypes enrich distinct classes of biological processes. One of the hypermethylated genes in GBM, ULK2, an upstream autophagy inducer, was found to be down-regulated in GBM. Promoter hypermethylation of ULK2 was confirmed by bisulfite sequencing. GBM and glioma cell lines had low levels of ULK2 transcripts, which could be reversed upon methylation inhibitor treatment. ULK2 promoter methylation and transcript levels showed significant negative correlation. Ectopic overexpression of ULK2-induced autophagy, which further enhanced upon nutrient starvation or temozolomide chemotherapy. ULK2 also inhibited the growth of glioma cells, which required autophagy induction as kinase mutant of ULK2 failed to induce autophagy and inhibit growth. Furthermore, ULK2 induced autophagy and inhibited growth in Ras-transformed immortalized Baby Mouse Kidney (iBMK) ATG5(+/+) but not in autophagy-deficient ATG5(-/-) cells. Growth inhibition due to ULK2 induced high levels of autophagy under starvation or chemotherapy utilized apoptotic cell death but not at low levels of autophagy. Growth inhibition by ULK2 also appears to involve catalase degradation and reactive oxygen species generation. ULK2 overexpression inhibited anchorage independent growth, inhibited astrocyte transformation in vitro and tumor growth in vivo. Of all autophagy genes, we found ULK2 and its homologue ULK1 were only down-regulated in all grades of glioma. Thus these results altogether suggest that inhibition of autophagy by ULK1/2 down-regulation is essential for glioma development.

Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by New Cationic Gemini Tocopheryl Lipids

Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.

Structural study of CUG-repeating small RNAs complexed with silencing suppressor P19

The study is focused on structural aspects of interaction between silencing suppressor p19 and CUG-repeating small RNAs. The work involves crystal structure determination of a protein-unbound RNA form and RNA fragments of various lengths (19, 20, 21 nucleotides) complexed with p19-suppressor. Results prove the ability of silencing suppressor p19 to bind CUG-repeating small RNAs, as well as reveal features of U•U mismatches flanked by Watson-Crick C•G base pairs in p19-bound and p19-unbound states. In addition, structural data reveal a p19 specific site for anchoring extra nucleotides in small RNAs. In general, the study extends our knowledge about the mechanism of small RNA recognition by silencing suppressor p19.

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.

Microbial inactivation of Pseudomonas putida and Pichia pastoris using gene silencing.

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.