Epigenetics is a field that encompasses chemical modifications of DNA and histone proteins, both of which alter gene expression without changing the underlying nucleotide sequence. DNA methylation and modifications of histone tails have been studied in detail and are now known to be global gene regulatory mechanisms. An analogous post-transcriptional modification is chemical modification of specific nucleotides in RNA. Study of RNA modifications is a nascent field as yet, and the significance of these marks in controlling cell growth and differentiation is just beginning to be appreciated. The addition of a methyl group to adenosine (N-methyl-6-adenosine) or m6A is the most abundant modification in mammalian mRNAs. Though identified four decades ago, interest in this particular modification was set off by the discovery that the obesity gene FTO was an RNA demethylase. Since then, many studies have investigated m6A modification in different species. In this review, we summarize the current literature and hypotheses about the presence and function of this ubiquitous RNA modification in mammals, viruses, yeast and plants in terms of the consensus sequence and the methyltransferase/demethylation machinery identified thus far. We discuss its potential role in regulating molecular and physiological processes in each of these organisms, especially its role in RNA splicing, RNA degradation and development. We also enlist the methodologies developed so far, both locus-specific and transcriptome-wide, to study this modification. Lastly, we discuss whether m6A alterations have consequences on modulating disease aetiology, and speculate about its potential role in cancer.

Purpose Accurate detection of human papillomavirus (HPV) in oral cavity squamous cell carcinoma (OSCC) is essential to understanding the role of HPV in disease prognosis and management of patients. We used different analytes and methods to understand the true prevalence of HPV in a cohort of patients with OSCC with different molecular backgrounds, and we correlated HPV data with patient survival. Methods We integrated data from multiple analytes (HPV DNA, HPV RNA, and p16), assays (immunohistochemistry, polymerase chain reaction [PCR], quantitative PCR [qPCR], and digital PCR), and molecular changes (somatic mutations and DNA methylation) from 153 patients with OSCC to correlate p16 expression, HPV DNA, and HPV RNA with HPV incidence and patient survival. Results High prevalence (33% to 58%) of HPV16/18 DNA did not correlate with the presence of transcriptionally active viral genomes (15%) in tumors. Eighteen percent of the tumors were p16 positive and only 6% were both HPV DNA and HPV RNA positive. Most tumors with relatively high copy number HPV DNA and/or HPV RNA, but not with HPV DNA alone (irrespective of copy number), were wild-type for TP53 and CASP8 genes. In our study, p16 protein, HPV DNA, and HPV RNA, either alone or in combination, did not correlate with patient survival. Nine HPV-associated genes stratified the virus-positive from the virus-negative tumor group with high confidence ( P < .008) when HPV DNA copy number and/or HPV RNA were considered to define HPV positivity, and not HPV DNA alone, irrespective of copy number ( P < .2). Conclusion In OSCC, the presence of both HPV RNA and p16 is rare. HPV DNA alone is not an accurate measure of HPV positivity and therefore may not be informative. HPV DNA, HPV RNA, and p16 do not correlate with patients’ outcome.

Laryngo-pharyngeal squamous cell carcinomas are one of the most common head and neck cancers. Despite the presence of a large body of information, molecular biomarkers are not currently used in the diagnosis, treatment and management of patients for this group of cancer. Here, we have profiled expression of genes and microRNAs of larynx and hypopharynx tumors using high-throughput sequencing experiments. We found that matrix metalloproteinases along with SCEL, CRNN, KRT4, SPINK5, and TGM3 among others have significantly altered expression in these tumors. Alongside gene expression, the microRNAs hsa-miR-139, hsa-miR-203 and the hsa-miR-424/503 cluster have aberrant expression in these cancers. Using target genes for these microRNAs, we found the involvement of pathways linked to cell cycle, p53 signaling, and viral carcinogenesis significant (P-values 10−13, 10−9 and 10−7 respectively). Finally, using an ensemble machine-learning tool, we discovered a unique 8-gene signature for this group of cancers that differentiates the group from the other tumor subsites of head and neck region. We investigated the role of promoter methylation in one of these genes, WIF1, and found no correlation between DNA methylation and down-regulation of WIF1. We validated our findings of gene expression, 8-gene signature and promoter methylation using q-PCR, data from TCGA and q-MSP respectively.Data presented in this manuscript has been submitted to the NCBI Geo database with the accession number GSE67994.

21Purpose: The prevalence of human papillomavirus (HPV) in oral cavity squamous cell carcinoma 22 (OSCC) varies significantly based on assay sensitivity and patient geography. Accurate detection is 23 essential to understand the role of HPV in disease prognosis and management of patients with 24 OSCC. 25Methods: We generated and integrated data from multiple analytes (HPV DNA, HPV RNA, and 26 p16), assays (immunohistochemistry, PCR, qPCR and digital PCR) and molecular changes (somatic 27 mutations and DNA methylation) from 153 OSCC patients to correlate p16 expression, HPV DNA, 28 and HPV RNA with HPV incidence and patient survival. 29Results: High prevalence (33-58%) of HPV16/18 DNA did not correlate with the presence of 30 transcriptionally active viral genomes (15%) in tumors. Eighteen percent of the tumors were p16 31 positive. and only 6% were both HPV DNA and RNA positive. Most tumors with relatively high-32 copy HPV DNA, and/or HPV RNA, but not with HPV DNA alone (irrespective of copy number), 33were wild-type for TP53 and CASP8 genes. In our study, p16 protein, HPV DNA and HPV RNA, 34 either alone or in combinations, did not correlate with patient survival. Nine HPV-associated genes 35 stratified the virus +ve from the -ve tumor group with high confidence (p<0.008) when HPV DNA 36 copy number and/or HPV RNA were considered to define HPV positivity and not HPV DNA alone 37 irrespective of their copy number (p < 0.2). 38Conclusions: In OSCC, the presence of both HPV RNA and p16 are rare. HPV DNA alone is not an 39 accurate measure of HPV positivity and therefore not informative. Moreover, HPV DNA, RNA or 40 p16 don't correlate with outcome. 41 42

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.

We developed a targeted DNA sequencing method that is capable of detecting a comprehensive panel of DNA mutations including small DNA mutations and large DNA deletions with unknown/flexible boundaries. The method directly identifies the large DNA deletions (Cat-D) without relying on sequencing coverage to make the genotype calls. We performed the method to simultaneously detect 10 small DNA mutations in β-thalassemia and 2 large genomic deletions in α-thalassemia from 10 genomic DNA samples. Cat-D was performed on 8 genomic DNA samples in duplicate. The 18 Cat-D samples were combined in one sequencing run. In total, 216 genotype calls were made, and 215 of the genotype calls were accurate. No false negative genotype calls were made. One false positive genotype call was made on one target mutation in one experimental duplicate from a genomic DNA sample. In summary, Cat-D can be developed into a robust, high-throughput and cost-effective method suitable for population-based carrier screens.

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