Himanshu Narayan Singh

Interaction of adriamycin with a regulatory element of hmgb1: spectroscopic and calorimetric approach

HMGB1 is a non-histone nuclear protein which plays important role in transcription, variable, diverse and joining (VDJ) recombination, chromatin remodeling, and DNA repair, etc. and its over expression is directly correlated with various human malignancies and inflammatory diseases. Because of the clear association between HMGB1 and cancer, we studied the binding of adriamycin (ADM), a well-known anticancer drug with the promoter region (−165 to −183) of hmgb1 by using a variety of spectroscopic, calorimetric techniques, and in-silico molecular modeling. Changes in UV and CD spectral characteristics (intensity and wavelength) of ADM and DNA associated with an induced peak (300 nm) in CD spectrum of DNA and a high binding constant of 2.0 × 105 M−1 suggest a strong and stable complex formation between DNA and ADM. Scatchard analysis of spectroscopic data indicate that ADM binds to DNA in a non-cooperative nature. Further the quenching of fluorescence emission of ADM and isothermal titration calorimetry of ADM in presence of DNA points out to the intercalative mode of ADM binding to DNA which is enthalpically driven with additional small entropic contribution. Results from molecular modeling, Isothermal titration calorimetry, and Fourier transform infrared spectroscopy reveal that ADM has no marked preference between AT vs. GC base pair in binding to DNA. Therefore, hmgb1 can be considered as a novel potential chemotherapeutic target in treating cancers associated with HMGB1 upregulation.

Role of long purine stretches in controlling the expression of genes associated with neurological disorders.

Purine repeat sequences present in the human genome are known to act as hotspots for mutations leading to chromosomal imbalances. It is established that large purine repeats (PRs) form stable DNA triplex structure which can inhibit gene expression. Friedreichs ataxia (FRDA), the autosomal neurodegenerative disorder is the only human disease known so far, where a large purine (GAA) repeat in the FXN gene is known to inhibit the expression of frataxin protein. We explored the hidden purine repeats (PRn with n ≥ 200) if any, in the human genome to find out how they are associated with neurological disorders. The results showed 28 PRs, which are mostly restricted to the intronic regions. Interestingly, the transcriptome expression analysis of PR-carrying genes (PR-genes) revealed that most of them are down-regulated in neurological disorders (autism, Alzheimers disease, schizophrenia, epilepsy, mental retardation, Parkinsons disease, brain tumor) as compared to that in healthy controls. The altered gene expression in brain disorders can be interpreted in terms of a possible expansion of purine repeats leading to formation of very stable DNA-triplex and/or alleviation of the repair enzymes and/or other unknown cellular factors. Interactome analysis identified four PR-genes in signaling pathways whose dysregulation is correlated directly with pathogenesis: GRK5 and KLK6 in Alzheimers disease; FGF14 in craniosynostosis, mental retardation and FLT1 in neuroferritinopathy. By virtue of being mutational hotspots and their ability to form DNA-triplex, purine repeats in genome disturb the genome integrity and interfere with the transcriptional regulation. However, validation of the disease linkage of PR-genes can be validated using knock-out techniques.

A Possible Mechanism of Zika Virus Associated Microcephaly: Imperative Role of Retinoic Acid Response Element (RARE) Consensus Sequence Repeats in the Viral Genome.

Owing to the reports of microcephaly as a consistent outcome in the fetuses of pregnant women infected with ZIKV in Brazil, Zika virus (ZIKV)—microcephaly etiomechanistic relationship has recently been implicated. Researchers, however, are still struggling to establish an embryological basis for this interesting causal handcuff. The present study reveals robust evidence in favor of a plausible ZIKV-microcephaly cause-effect liaison. The rationale is based on: (1) sequence homology between ZIKV genome and the response element of an early neural tube developmental marker “retinoic acid” in human DNA and (2) comprehensive similarities between the details of brain defects in ZIKV-microcephaly and retinoic acid embryopathy. Retinoic acid is considered as the earliest factor for regulating anteroposterior axis of neural tube and positioning of structures in developing brain through retinoic acid response elements (RARE) consensus sequence (5′–AGGTCA–3′) in promoter regions of retinoic acid-dependent genes. We screened genomic sequences of already reported virulent ZIKV strains (including those linked to microcephaly) and other viruses available in National Institute of Health genetic sequence database (GenBank) for the RARE consensus repeats and obtained results strongly bolstering our hypothesis that ZIKV strains associated with microcephaly may act through precipitation of dysregulation in retinoic acid-dependent genes by introducing extra stretches of RARE consensus sequence repeats in the genome of developing brain cells. Additional support to our hypothesis comes from our findings that screening of other viruses for RARE consensus sequence repeats is positive only for those known to display neurotropism and cause fetal brain defects (for which maternal-fetal transmission during developing stage may be required). The numbers of RARE sequence repeats appeared to match with the virulence of screened positive viruses. Although, bioinformatic evidence and embryological features are in favor of our hypothesis, additional studies including animal models are warranted to validate our proposition. Such studies are likely to unfold ZIKV-microcephaly association and may help in devising methods to combat it.

Structural aspects of the interaction of anticancer drug Actinomycin-D to the GC rich region of hmgb1 gene.

The high mobility group box 1 protein has been identified as a key player in chromatin homeostasis including transcription regulation, recombination, repair, and chromatin remodeling. Emerging findings indicate HMGB1 protein over expression in nearly all types of human cancers and inflammatory disorders. Thus it is considered as a potential therapeutic target for treating various malignancies. We screened the promoter region of hmgb1 gene and selected a positive regulatory element of 25 base pair duplex (25RY) (-165 to -183) as a potential target for chemotherapeutic intervention. The molecular interaction of actinomycin (ACT) with the regulatory region of hmgb1 gene was characterized by spectroscopic, calorimetric and molecular docking studies. The hypochromic and bathochromic shift in the absorption spectrum, stabilization of 25RY duplex against thermal denaturation, perturbation of CD spectrum of duplex and enhancement of fluorescence intensity of actinomycin indicate strong binding of actinomycin to the hmgb1 promoter region (25RY).The energetics was characterized to be endothermic and entropy driven. All these results are in good agreement with in silico investigation that suggest minor groove binding with effective intercalation at GC bases of actinomycin to 25RY. This study identifies hmgb1 gene promoter region a potential target for the anticancer therapautiucs.

Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer

Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich’s ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed “PuRepeatFinder.pl” algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, n ≥ 200. A total of 1158 EPRs were identified in the genome which followed Wakeby distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein–protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.

Assessment of binding properties of Actinomycin-D to 21nt DNA segment of hmgb1 gene promoter using spectroscopic and calorimetric techniques.

High mobility group box 1 protein is primarily a nuclear protein that acts as an architectural transcription factor to facilitate transcription, replication, recombination, repair, nucleosome remodeling etc. in the genome. Under pathological condition HMGB1 is released out of the cell and trigger inflammatory response by binding to its high affinity receptors such as RAGE (Receptor for advance glycation end product) and TLR (Toll like receptors). The cytokine activity of HMGB1 regulate immune response, apoptosis, autophagy, differentiation etc. (Lohani & Rajeswari, 2016). Moreover, overexpression of HMGB1 level has been reported in almost all type of human cancer and inflammatory diseases such as sepsis, shock, autoimmune disorders, viral and bacterial infections etc. Thus HMGB1 is an attractive therapeutic target for drug designing to treat various diseases associated with high HMGB1 expression. Actinomycin-D (Act-D) is a well known potent anticancer drug produced by Streptomyces bacteria. The high toxicity caused by Act-D is attributed to its ability to inhibit transcription of several important gene such as c-myc, c-met, myotonic Dystrophy type 1 etc. Importantly, it has been approved by FDA for multiple tumor, Wilms’ tumor and gestational choriocarcinoma and in combination with other drugs to treat high risk tumor in chemotherapy regime (Schink, Singh, Rademaker, Miller, & Lurain, 1992). The chemical structure of Act-D comprise planar phenoxazone ring (Figure 1(a)) and two cyclic pentapeptide lactones. Numerous reports have shown that Act-D inhibit transcription by intercalation between base pair of double stranded duplex DNA or by binding to selected sites in single stranded DNA (but not RNA) in GpC rich sequence. However, other investigation has proposed other binding mode. In the present study we explore the characteristic binding mode and energetics of Act-D binding to 21nt mixed GpC sequence in the positive regulatory region of hmgb1 gene to identify areas of pressing experimental need, and provide promising future therapeutic options. The binding of Act-D to 21nt duplex was studied using UV-visible absorption spectroscopy, fluorescence spectrophotometer, circular dichroism (CD), and Isothermal titration calorimetric technique.

Gene regulation by long purine tracks in brain related diseases

Purine repeats are randomly distributed in the human genome, however, they show potential role in the transcriptional deregulation of genes. Presence of long tracks of purine repeats in the genome can disturb its integrity and interfere with the cellular behavior by introducing mutations and/or triple stranded structure formation in DNA. Our data revealed interesting finding that a majority of genes carrying purine repeats, of length n≥200, were down regulated and found to be linked with several brain related diseases [1]. The unique feature of the purine repeats found in the present study clearly manifests their significant application in developing therapeutics for neurological diseases.

DNA trinucleotide (GAA) repeats in human genome: hint for disease pathogenesis?

Trinucleotide repeats are the most common unstable disease-associated DNA repeats, however, other repeats such as tetranucleotides, pentanucleotides, mini-satellites were also observed to be involved with various human diseases (Castel, Cleary, & Pearson, 2010; Pearson, Edamura, & Cleary, 2005; Rajeswari, 2012). In normal conditions, triplet repeat stretches are short and stable and shows unchanged length. On the other hand, triplet repeat stretches were found to be longer and unstable, with a high tendency to add (expansion) rather than subtract (contraction) repeat units (Budworth & McMurray, 2013; Cherng et al., 2011; McMurray, 2010). These length changes are dynamic and are seen between individuals and also between and within tissues of the same individual. As longer repeat stretches are transmitted through generations, disease symptoms can become more severe and appear at an earlier age. As the triplet repeat number grows, the growing triplet tract alters gene expression and/or function of the gene product. Expansion of triplet repeat is lethal: if the triplet repeat is residing in a coding sequence of a gene typically produce a faulty protein, otherwise expansion in a noncoding gene region suppresses protein expression, alters its splicing, or may influence aspects of antisense regulation (Singh & Rajeswari, 2015, 2016; Wells, 2008). In addition to this, these repeat can also form non-B DNA structure such as DNA-triplex. And, structure-prone DNA repeat sequences are found to be linked to genomic instabilities that result in various hereditary disorders including cancer, neurological disorders (Sharma et al., 2004; Wells, 2008). It has long been known that DNA repeats are not only highly polymorphic in length but can also cause chromosomal fragility and stimulate gross chromosomal rearrangements, i.e. deletions, duplications, inversions, translocations, and more complex shuffles. It is also now found that these repeats can modulate the regulation of genes by non-B DNA structure formation (Mirkin, 2007; Shah & Mirkin, 2015; Wang & Vasquez, 2004). Friedreich’s ataxia (FRDA) is the first human disease where a stretch of large number of purines triplet repeat (GAA)n in fxn gene is shown to be associated with clinical appearances (Patel & Isaya, 2001). In normal conditions, the triplet ‘GAA’ is repeated 5–33 times within the FXN gene. However, in the FRDA affected people, the GAA segment is repeated 66–1800 times (Genetics Home Reference, 2015). The length of the GAA trinucleotide repeat appears to be related to the age at which the symptoms of Friedreich ataxia appear, how severe they are, and how quickly they progress. People with GAA segments repeated fewer than 300 times tend to have a later appearance of symptoms (after age 25) than those with larger GAA trinucleotide repeats. The abnormally long GAA trinucleotide repeat disrupts the production of frataxin by means of forming DNA-triplex, which severely reduces the amount of this protein in cells. Certain nerve and muscle cells cannot function properly with a shortage of frataxin, leading to the characteristic signs and symptoms of Friedreich ataxia (Genetics Home Reference, 2015). The genetic etiology involved in FRDA with the GAA triplet repeat suggests the possible GAA repeat expansion in other genes, which may be potentially involved with other diseases as well. Therefore, we made an attempt to explore the presence of GAA triplet repeat in human genome.

Commentary: A Possible Mechanism of Zika Virus Associated Microcephaly: Imperative Role of Retinoic Acid Response Element (RARE) Consensus Sequence Repeats in the Viral Genome

Citation: Faiq MA, Kumar A, Singh HN, Pareek V and Kumar P (2018) Commentary: A Possible Mechanism of Zika Virus Associated Microcephaly: Imperative Role of Retinoic Acid Response Element (RARE) Consensus Sequence Repeats in the Viral Genome. Front. Microbiol. 9:190. doi: 10.3389/fmicb.2018.00190 Commentary: A Possible Mechanism of Zika Virus Associated Microcephaly: Imperative Role of Retinoic Acid Response Element (RARE) Consensus Sequence Repeats in the Viral Genome

Altered Expression Of A Unique Set Of Genes Reveals Complex Etiology Of Schizophrenia

Purpose The etiology of schizophrenia is extensively debated, and multiple factors have been contended to be involved. A panoramic view of the contributing factors in a genome-wide study can be an effective strategy to provide a comprehensive understanding of its causality. Materials and Methods GSE53987 dataset downloaded from GEO-database, which comprised mRNA expression data of post-mortem brain tissue across three regions from control and age-matched subjects of schizophrenia (N= Hippocampus (HIP): C-15, T-18, Prefrontal cortex (PFC): C-15, T-19, Associative striatum (STR): C-18, T-18). Bio-conductor-affy-package used to compute mRNA expression, and further t-test applied to investigate differential gene expression. The analysis of the derived genes performed using PANTHER Classification System and NCBI database. Results A set of 40 genes showed significantly altered (p<0.01) expression across all three brain regions. The analyses unraveled genes implicated in biological processes and events, and molecular pathways relating basic neuronal functions. Conclusions The deviant expression of genes maintaining basic cell machinery explains compromised neuronal processing in SCZ. Abbreviations Schizophrenia (SCZ), Hippocampus (HIP), Associative striatum (STR), Prefrontal cortex (PFC)