Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter

The human oncogene c-myc is regulated by G-quadruplex formation within the nuclease hypersensitive element (NHE III(I)) in the c-myc promoter, making the quadruplex a strong anti-cancer target. With respect to this, the competing equilibrium between intramolecular quadruplex folding and bimolecular duplex formation is poorly understood and very few techniques have addressed this problem. We present a method for simultaneously determining the kinetic constants for G-quadruplex folding/unfolding and hybridization in the presence of the complementary strand from a single reaction using an optical biosensor based on surface plasmon resonance (SPR). Using this technique, we demonstrate for the first time that quadruplex formation in the c-myc promoter is favored at low strand concentrations. Our results indicate favorable quadruplex folding (equilibrium folding constant K(F) of 2.09 calculated from the kinetic parameters: folding rate constant, k(f) = 1.65 × 10(−2) s(−1) and unfolding rate constant, k(u) = 7.90 × 10(−3) s(−1)) in 150 mM K(+). The hybridization rate constants detected concurrently gave a bimolecular association constant, k(a) = 1.37 × 10(5) M(−1) s(−1) and dissociation constant, k(d) = 4.94 × 10(−5) s(−1). Interestingly, in the presence of Na(+) we observed that G-quadruplex folding was unfavorable (K(F) = 0.54). Implication of our results on the c-myc transcription activation model is discussed in light of aberrant c-myc expression observed on destabilization of the G-quadruplex

QuadBase2: web server for multiplexed guanine quadruplex mining and visualization

DNA guanine quadruplexes or G4s are non-canonical DNA secondary structures which affect genomic processes like replication, transcription and recombination. G4s are computationally identified by specific nucleotide motifs which are also called putative G4 (PG4) motifs. Despite the general relevance of these structures, there is currently no tool available that can allow batch queries and genome-wide analysis of these motifs in a user-friendly interface. QuadBase2 (quadbase.igib.res.in) presents a completely reinvented web server version of previously published QuadBase database. QuadBase2 enables users to mine PG4 motifs in up to 178 eukaryotes through the EuQuad module. This module interfaces with Ensembl Compara database, to allow users mine PG4 motifs in the orthologues of genes of interest across eukaryotes. PG4 motifs can be mined across genes and their promoter sequences in 1719 prokaryotes through ProQuad module. This module includes a feature that allows genome-wide mining of PG4 motifs and their visualization as circular histograms. TetraplexFinder, the module for mining PG4 motifs in user-provided sequences is now capable of handling up to 20 MB of data. QuadBase2 is a comprehensive PG4 motif mining tool that further expands the configurations and algorithms for mining PG4 motifs in a user-friendly way

BreCAN-DB: a repository cum browser of personalized DNA breakpoint profiles of cancer genomes

BreCAN-DB (http://brecandb.igib.res.in) is a repository cum browser of whole genome somatic DNA breakpoint profiles of cancer genomes, mapped at single nucleotide resolution using deep sequencing data. These breakpoints are associated with deletions, insertions, inversions, tandem duplications, translocations and a combination of these structural genomic alterations. The current release of BreCAN-DB features breakpoint profiles from 99 cancer-normal pairs, comprising five cancer types. We identified DNA breakpoints across genomes using high-coverage next-generation sequencing data obtained from TCGA and dbGaP. Further, in these cancer genomes, we methodically identified breakpoint hotspots which were significantly enriched with somatic structural alterations. To visualize the breakpoint profiles, a next-generation genome browser was integrated with BreCAN-DB. Moreover, we also included previously reported breakpoint profiles from 138 cancer-normal pairs, spanning 10 cancer types into the browser. Additionally, BreCAN-DB allows one to identify breakpoint hotspots in user uploaded data set. We have also included a functionality to query overlap of any breakpoint profile with regions of user's interest. Users can download breakpoint profiles from the database or may submit their data to be integrated in BreCAN-DB. We believe that BreCAN-DB will be useful resource for genomics scientific community and is a step towards personalized cancer genomics

Use of Technology in Segregating Occupational risks of Migrant and linking them with Services: Experiences from National AIDS Control Program for Migrants

Background: The migrant intervention in India was initiated during the National AIDS Control Program (NACP) Phase-2 (2002-2007). Even by the end of NACP Phase-3 (2010-11); the service uptake among migrants remained very low (14% referred for HIV testing, of which only 37% were tested). USAID PHFI-PIPPSE project in collaboration with the National AIDS Control Organization (NACO) developed a unique system called Migrant Service Delivery System (MSDS) to capture migrants profile with respect to their risk profile and to provide tailor made services to them.Description: MSDS is a web-based system, designed and implemented to increase service uptake among migrants through evidence based planning. 110 destination migrants Targeted Intervention (TI) from 11 states were selected for study with varied target populations in terms of occupations; to understand occupation related risk behaviors amongst the migrants. Occupation wise registration data of high risk vulnerable migrants were analyzed through MSDS for the period April 2014-June 2016. Analysis was made on specific indicators amongst these occupational groups to understand the risk behavior and their vulnerability to HIV and STI.Lessons Learned: Out of total migrants workers enrolled in MSDS HIV rate is found to be highest amongst Auto-Rickshaw (18.66%) followed by daily wage laborers (14.46%), loom workers (10.73%), industrial workers (10.04%) and construction workers (7.93%). With 45.14% positivity, industrial workers are found to be most vulnerable to Sexually Transmitted Infections (STIs) amongst all occupational categories followed by loom workers (16.28%), skilled worker (Furniture, Jeweler)(7.14%), daily wage laborers (5.45%) .Conclusion/Next Steps: MSDS is an effective tool to assess migrants’ risk and their vulnerability to HIV for designing evidence informed program. This system calls for a replication across all destination TIs by NACO for differential strategies for different occupation groups to ensure better yield through scientific planning of intervention among high risk and high vulnerable migrants.

QuadBase: genome-wide database of G4 DNA—occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes

Emerging evidence indicates the importance of G-quadruplex motifs as drug targets. [Stuart A. Borman, Ascent of quadruplexes—nucleic acid structures become promising drug targets. Chem. Eng. News, 2007;85, 12–17], which stems from the fact that these motifs are present in a surprising number of promoters wherein their role in controlling gene expression has been demonstrated for a few. We present a compendium of quadruplex motifs, with particular focus on their occurrence and conservation in promoters—QuadBase. It is composed of two parts (EuQuad and ProQuad). EuQuad gives information on quadruplex motifs present within 10 kb of transcription starts sites in 99 980 human, chimpanzee, rat and mouse genes. ProQuad contains quadruplex information of 146 prokaryotes. Apart from gene-specific searches for quadruplex motifs, QuadBase has a number of other modules. ‘Orthologs Analysis’ queries for conserved motifs across species based on a selected reference organism; ‘Pattern Search’ can be used to fetch specific motifs of interest from a selected organism using user-defined criteria for quadruplex motifs, i.e. stem, loop size, etc. ‘Pattern Finder’ tool can search for motifs in any given sequence. QuadBase is freely available to users from non-profit organization at http://quadbase.igib.res.in/

SITE CLASSIFICATION AND SEISMIC RESPONSE OF DHAKA CITY SOILS

ABSTRACT Dhaka, one of the fastest growing megacities of the world, is located in a seismic zone with an equivalent peak ground acceleration value of 0.15g. One of the major concerns during an earthquake in Dhaka is the presence of soft soils which may result in amplification of ground motion. Many low lying lands in the city area have been filled up for urban construction to meet the population growth, which have not been properly compacted. Soil boring data including Standard Penetration Test (SPT) values from 34 sites around Dhaka have been collected to estimate equivalent dynamic properties of the top 30 m soil. These sites can be classified into three groups S2, S3 and S4 as per the 1993 Bangladesh national building code. This code, based on UBC 1990, recognizes site amplification effects and specifies design response spectrum with maximum spectral acceleration ratio of 2.5 for soil types S1, S2 and S3. Soil type S4 needs site-specific analysis. Seismic site response of the top 30 m soil at eighteen selected sites of the city belonging to S2, S3 and S4 class is computed using the computer program 'SHAKE'. Seven different earthquake records scaled and suited to soil type S2 have been used. Results obtained suggest that the maximum spectral acceleration ratio of the response spectrum should be around 2.75 for soil type S2 and around 3.75 for soil type S3. The current building code is thus on the unsafe side. The results presented thus indicate the necessity of revising the existing national code

Genome-wide study predicts promoter-G4 DNA motifs regulate selective functions in bacteria: radioresistance of D. radiodurans involves G4 DNA-mediated regulation

A remarkable number of guanine-rich sequences with potential to adopt non-canonical secondary structures called G-quadruplexes (or G4 DNA) are found within gene promoters. Despite growing interest, regulatory role of quadruplex DNA motifs in intrinsic cellular function remains poorly understood. Herein, we asked whether occurrence of potential G4 (PG4) DNA in promoters is associated with specific function(s) in bacteria. Using a normalized promoter-PG4-content (PG4P) index we analysed >60 000 promoters in 19 well-annotated species for (a) function class(es) and (b) gene(s) with enriched PG4P. Unexpectedly, PG4-associated functional classes were organism specific, suggesting that PG4 motifs may impart specific function to organisms. As a case study, we analysed radioresistance. Interestingly, unsupervised clustering using PG4P of 21 genes, crucial for radioresistance, grouped three radioresistant microorganisms including Deinococcus radiodurans. Based on these predictions we tested and found that in presence of nanomolar amounts of the intracellular quadruplex-binding ligand N-methyl mesoporphyrin (NMM), radioresistance of D. radiodurans was attenuated by ∼60%. In addition, important components of the RecF recombinational repair pathway recA, recF, recO, recR and recQ genes were found to harbour promoter-PG4 motifs and were also down-regulated in presence of NMM. Together these results provide first evidence that radioresistance may involve G4 DNA-mediated regulation and support the rationale that promoter-PG4s influence selective functions

Genome-wide distribution of histone H4 Lysine 16 acetylation sites and their relationship to gene expression

BACKGROUND: Histone post-translational modifications are critical determinants of chromatin structure and function, impacting multiple biological processes including DNA transcription, replication, and repair. The post-translational acetylation of histone H4 at lysine 16 (H4K16ac) was initially identified in association with dosage compensation of the Drosophila male X chromosome. However, in mammalian cells, H4K16ac is not associated with dosage compensation and the genomic distribution of H4K16ac is not precisely known. Therefore, we have mapped the genome-wide H4K16ac distribution in human cells. RESULTS: We performed H4K16ac chromatin immunoprecipitation from human embryonic kidney 293 (HEK293) cells followed by hybridization to whole-genome tiling arrays and identified 25,893 DNA regions (false discovery rate <0.005) with average length of 692 nucleotides. Interestingly, although a majority of H4K16ac sites localized within genes, only a relatively small fraction (~10%) was found near promoters, in contrast to the distribution of the acetyltransferase, MOF, responsible for acetylation at K16 of H4. Using differential gene expression profiling data, 73 genes (> ±1.5-fold) were identified as potential H4K16ac-regulated genes. Seventeen transcription factor-binding sites were significantly associated with H4K16ac occupancy (p < 0.0005). In addition, a consensus 12-nucleotide guanine-rich sequence motif was identified in more than 55% of the H4K16ac peaks. CONCLUSIONS: The results suggest that H4K16 acetylation has a limited effect on transcription regulation in HEK293 cells, whereas H4K16ac has been demonstrated to have critical roles in regulating transcription in mouse embryonic stem cells. Thus, H4K16ac-dependent transcription regulation is likely a cell type specific process

Epigenetic suppression of human telomerase ( hTERT ) is mediated by the metastasis suppressor NME2 in a G-quadruplex–dependent fashion

Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well-understood. Surprisingly, here we found occupancy of the metastasis suppressor non-metastatic 2 (NME2) within the hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells and NME2-mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in up-regulated hTERT expression. Mechanistically, additional results indicated that the RE1-silencing transcription factor (REST)–lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in an NME2-dependent way and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G-quadruplex–binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G-quadruplex promoter motif, which potentially can be targeted by tailored small molecules