Elevated polyamines induce c-MYC overexpression by perturbing quadruplex–WC duplex equilibrium

The biological role of quadruplexes and polyamines has been independently associated with cancer. However, quadruplex-polyamine mediated transcriptional regulation remain unaddressed. Herein, using c-MYC quadruplex model, we have attempted to understand quadruplex–polyamine interaction and its role in transcriptional regulation. We initially employed biophysical approach involving CD, UV and FRET to understand the role of polyamines (spermidine and spermine) on conformation, stability, molecular recognition of quadruplex and to investigate the effect of polyamines on quadruplex–Watson Crick duplex transition. Our study demonstrates that polyamines affect the c-MYC quadruplex conformation, perturb its recognition properties and delays duplex formation. The relative free energy difference (ΔΔG°) between the duplex and quadruplex structure indicate that polyamines stabilize and favor c-MYC quadruplex over duplex. Further, we investigated the influence of polyamine mediated perturbation of this equilibrium on c-MYC expression. Our results suggest that polyamines induce structural transition of c-MYC quadruplex to a transcriptionally active motif with distinctive molecular recognition property, which drives c-MYC expression. These findings may allow exploiting quadruplex–polyamines interaction for developing antiproliferative strategies to combat aberrant gene expression

Quadruplex-single nucleotide polymorphisms (Quad-SNP) influence gene expression difference among individuals

Non-canonical guanine quadruplex structures are not only predominant but also conserved among bacterial and mammalian promoters. Moreover recent findings directly implicate quadruplex structures in transcription. These argue for an intrinsic role of the structural motif and thereby posit that single nucleotide polymorphisms (SNP) that compromise the quadruplex architecture could influence function. To test this, we analysed SNPs within quadruplex motifs (Quad-SNP) and gene expression in 270 individuals across four populations (HapMap) representing more than 14 500 genotypes. Findings reveal significant association between quadruplex-SNPs and expression of the corresponding gene in individuals (P < 0.0001). Furthermore, analysis of Quad-SNPs obtained from population-scale sequencing of 1000 human genomes showed relative selection bias against alteration of the structural motif. To directly test the quadruplex-SNP-transcription connection, we constructed a reporter system using the RPS3 promoter—remarkable difference in promoter activity in the ‘quadruplex-destabilized’ versus ‘quadruplex-intact’ promoter was noticed. As a further test, we incorporated a quadruplex motif or its disrupted counterpart within a synthetic promoter reporter construct. The quadruplex motif, and not the disrupted-motif, enhanced transcription in human cell lines of different origin. Together, these findings build direct support for quadruplex-mediated transcription and suggest quadruplex-SNPs may play significant role in mechanistically understanding variations in gene expression among individuals

Evidence of genome-wide G4 DNA-mediated gene expression in human cancer cells

Guanine-rich DNA of a particular sequence adopts four-stranded structural forms known as G-quadruplex or G4 DNA. Though in vitro formation of G4 DNA is known for several years, in vivo presence of G4 DNA was only recently noted in eukaryote telomeres. Recent bioinformatics analyses showing prevalence of G4 DNA within promoters of human and related species seems to implicate G4 DNA in a genome-wide cis-regulatory role. Herein we demonstrate that G4 DNA may present regulatory sites on a genome-wide scale by showing widespread effect on gene expression in response to the established intracellular G4 DNA-binding ligands. This is particularly relevant to genes that harbor conserved potential G4 DNA (PG4 DNA) forming sequence across human, mouse and rat promoters of orthologous genes. Genes with conserved PG4 DNA in promoters show co-regulated expression in 79 human and 61 mouse normal tissues (z-score > 3.5; P < 0.0001). Conservation of G4 DNA across related species also emphasizes the biological importance of G4 DNA and its role in transcriptional regulation of genes; shedding light on a relatively novel mechanism of regulation of gene expression in eukaryotes

A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro

SP1 is a ubiquitous transcription factor that is involved in the regulation of various house-keeping genes. It is known that it acts by binding to a double-stranded consensus motif. Here, we have discovered that SP1 binds also to a non-canonical DNA structure, a G-quadruplex, with high affinity. In particular, we have studied the SP1 binding site within the promoter region of the c-KIT oncogene and found that this site can fold into an anti-parallel two-tetrad G-quadruplex. SP1 pull-down experiments from cellular extracts, together with biophysical binding assays revealed that SP1 has a comparable binding affinity for this G-quadruplex structure and the canonical SP1 duplex sequence. Using SP1 ChIP-on-chip data sets, we have also found that 87% of SP1 binding sites overlap with G-quadruplex forming sequences. Furthermore, while many of these immuoprecipitated sequences (36%) even lack the minimal SP1 consensus motif, 5′-GGGCGG-3′, we have shown that 77% of them are putative G-quadruplexes. Collectively, these data suggest that SP1 is able to bind both, canonical SP1 duplex DNA as well as G-quadruplex structures in vitro and we hypothesize that both types of interactions may occur in cells

Metastases suppressor NM23-H2 interaction with G-quadruplex DNA within c-MYC promoter nuclease hypersensitive element induces c-MYC expression

Regulatory influence of the G-quadruplex or G4 motif present within the nuclease hypersensitive element (NHE) in the promoter of c-MYC has been noted. On the other hand, association of NM23-H2 to the NHE leads to c-MYC activation. Therefore, NM23-H2 interaction with the G4 motif within the c-MYC NHE presents an interesting mechanistic possibility. Herein, using luciferase reporter assay and chromatin immunoprecipitation we show NM23-H2 mediated c-MYC activation involves NM23-H2-G4 motif binding within the c-MYC NHE. G4 motif complex formation with recombinant NM23-H2 was independently confirmed using fluorescence energy transfer, which also indicated that the G4 motif was resolved to an unfolded state within the protein-bound complex. Taken together, this supports transcriptional role of NM23-H2 via a G4 motif

A reference human induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field

Elevated polyamines induce c-MYC overexpression by perturbing quadruplex–WC

duplex equilibriu

Zika Virus: An Emerging Public Health Challenge

Emerging infectious diseases comprise a substantial proportion of global morbidity and mortality. The world has been hit by Zika virus (ZIKV) after it was able to surmount an effective public health response for its control. ZIKV disease is an emerging mosquitoborne disease which occurred as large outbreaks in Yap since 2007, Polynesia in 2013 and Brazil in 2015. ZIKV infection in pregnant women has been observed to be associated with congenital microcephaly with neurological and autoimmune sequelae in general population of Brazil. The incubation period of ZIKV varies from few days to weeks. Only 20% of infected cases have symptoms like any other arboviral illness. ZIKV is diagnosed using RT-PCR (reverse transcriptase-polymerase chain reaction) and virus isolation from blood samples. The treatment comprises of relief of symptoms by conservative management with no specific vaccine being available. The prevention and control of ZIKV is based on reduction of vector density by Integrated Vector Management and personal protection measures. As per Indian scenario, Ministry of Health had issued guidelines based on effective surveillance, risk communication, laboratory and travel regulations. Approaches to such a potential global health security threat should be consistent, proactive, and should involve coordinated, multi-pronged, multilateral collaborative efforts since the concern is at the highest and immediate because of Global epidemic, Rio de Janeiro Olympic Games starting from Aug 5-21, 2016 and strong association with microcephaly. Most importantly the need of the hour is the development of vaccine for protection especially the young women who are in the reproductive age groups. The research for which is ongoing as far as the current situation of global epidemic response is concerned

A novel G-quadruplex motif modulates promoter activity of human thymidine kinase 1

G-quadruplex motifs constitute unusual DNA secondary structures formed by stacking of planar hydrogen-bonded G-tetrads. Recent genome-wide bioinformatics and experimental analyses have suggested the interesting possibility that G-quadruplex motifs could be cis-regulatory elements. Here, we identified a characteristic potential G-quadruplex-forming sequence element within the promoter of human thymidine kinase 1 (TK1). Our NMR, UV and CD spectroscopy and gel electrophoresis data suggested that this sequence forms a novel intramolecular G-quadruplex with two G-tetrads in K+ solution. The results presented here indicate the role of this G-quadruplex motif in transcription of TK1 in cell-based reporter assays. Specific nucleotide substitutions designed to destabilize the G-quadruplex motif resulted in increased promoter activity, supporting direct involvement of the G-quadruplex motif in transcription of TK1. These studies suggest that the G-quadruplex motif may be an important target for controlling critical biological processes, such as DNA synthesis, mediated by TK1