Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous ...cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(-) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(-) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(-) cell lines. Differentially methylated loci between HPV(+) and HPV(-) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(-) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(-) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(-) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.
Curcumin is a potential agent for both the prevention and treatment of cancers. Curcumin treatment alone, or in combination with piperine, limits breast stem cell self-renewal, while remaining ...non-toxic to normal differentiated cells. We paired fluorescence-activated cell sorting with RNA sequencing to characterize the genome-wide changes induced specifically in normal breast stem cells following treatment with these compounds. We generated genome-wide maps of the transcriptional changes that occur in epithelial-like (ALDH+) and mesenchymal-like (ALDH−/CD44+/CD24−) normal breast stem/progenitor cells following treatment with curcumin and piperine. We show that curcumin targets both stem cell populations by down-regulating expression of breast stem cell genes including
ALDH1A3
,
CD49f
,
PROM1
, and
TP63
. We also identified novel genes and pathways targeted by curcumin, including downregulation of
SCD
. Transient siRNA knockdown of SCD in MCF10A cells significantly inhibited mammosphere formation and the mean proportion of CD44+/CD24− cells, suggesting that SCD is a regulator of breast stemness and a target of curcumin in breast stem cells. These findings extend previous reports of curcumin targeting stem cells, here in two phenotypically distinct stem/progenitor populations isolated from normal human breast tissue. We identified novel mechanisms by which curcumin and piperine target breast stem cell self-renewal, such as by targeting lipid metabolism, providing a mechanistic link between curcumin treatment and stem cell self-renewal. These results elucidate the mechanisms by which curcumin may act as a cancer-preventive compound and provide novel targets for cancer prevention and treatment.
Large sets of genomic regions are generated by the initial analysis of various genome-wide sequencing data, such as ChIP-seq and ATAC-seq experiments. Gene set enrichment (GSE) methods are commonly ...employed to determine the pathways associated with them. Given the pathways and other gene sets (e.g., GO terms) of significance, it is of great interest to know the extent to which each is driven by binding near transcription start sites (TSS) or near enhancers. Currently, no tool performs such an analysis. Here, we present a method that addresses this question to complement GSE methods for genomic regions. Specifically, the new method tests whether the genomic regions in a gene set are significantly closer to a TSS (or to an enhancer) than expected by chance given the total list of genomic regions, using a non-parametric test. Combining the results from a GSE test with our novel method provides additional information regarding the mode of regulation of each pathway, and additional evidence that the pathway is truly enriched. We illustrate our new method with a large set of ENCODE ChIP-seq data, using the
Bioconductor package. The results show that our method is a powerful complementary approach to help researchers interpret large sets of genomic regions.
The primary aim of this investigation was to evaluate the effect of training on the immune activation in skeletal muscle in response to an acute bout of resistance exercise (RE). Seven young healthy ...men and women underwent a 12-wk supervised progressive unilateral arm RE training program. One week after the last training session, subjects performed an acute bout of bilateral RE in which the trained and the untrained arm exercised at the same relative intensity. Muscle biopsies were obtained 4 h postexercise from the biceps brachii of both arms and assessed for global transcriptom using Affymetrix U133 plus 2.0 microarrays. Significantly regulated biological processes and gene groups were analyzed using a logistic regression-based method following differential (trained vs. untrained) gene expression testing via an intensity-based Bayesian moderated t-test. The results from the present study suggest that training blunts the transcriptional upregulation of immune activation by minimizing expression of genes involved in monocyte recruitment and enhancing gene expression involved in macrophage anti-inflammatory polarization. Additionally, our data suggest that training blunts the transcriptional upregulation of the stress response and the downregulation of glucose metabolism, mitochondrial structure, and oxidative phosphorylation, and it enhances the transcriptional upregulation of the extracellular matrix and cytoskeleton development and organization and the downregulation of gene transcription and muscle contraction. This study provides novel insight into the molecular processes involved in the adaptive response of skeletal muscle following RE training and the cellular and molecular events implicating the protective role of training on muscle stress and damage inflicted by acute mechanical loading.
Lead (Pb) is a well-known toxicant that interferes with the development of a child's nervous and metabolic systems and increases the risk of developing diseases later in life. Although studies have ...investigated epigenetic effects associated with Pb exposure, knowledge of genome-wide changes with
low dose perinatal Pb exposure in multiple tissues is limited. Within the Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET II) consortium, we utilized a mouse model to investigate tissue- and sex-specific DNA methylation. Dams were assigned to control or Pb-acetate water, respectively. Exposures started 2 weeks prior to mating and continued until weaning at post-natal day 21 (PND21). Liver and blood were collected from PND21 mice, and the DNA methylome was assessed using enhanced reduced representation bisulfite sequencing (ERRBS). We identified ∼1000 perinatal Pb exposure related differentially methylated cytosines (DMCs) for each tissue- and sex-specific comparison, and hundreds of tissue- and sex-specific differentially methylated regions (DMRs). Several mouse imprinted genes were differentially methylated across both tissues in males and females. Overall, our findings demonstrate that perinatal Pb exposure can induce tissue- and sex-specific DNA methylation changes and provide information for future Pb studies in humans.
The impact of the oral microbiome on head and neck cancer pathogenesis and outcomes requires further study. 16s rRNA was isolated and amplified from pre-treatment oral wash samples for 52 cases and ...102 controls. The sequences were binned into operational taxonomic units (OTUs) at the genus level. Diversity metrics and significant associations between OTUs and case status were assessed. The samples were binned into community types using Dirichlet multinomial models, and survival outcomes were assessed by community type. Twelve OTUs from the phyla Firmicutes, Proteobacteria, and Acinetobacter were found to differ significantly between the cases and the controls. Beta-diversity was significantly higher between the cases than between the controls (
< 0.01). Two community types were identified based on the predominant sets of OTUs within our study population. The community type with a higher abundance of periodontitis-associated bacteria was more likely to be present in the cases (
< 0.01), in older patients (
< 0.01), and in smokers (
< 0.01). Significant differences between the cases and the controls in community type, beta-diversity, and OTUs indicate that the oral microbiome may play a role in HNSCC.
Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the ...underrepresentation of both the 5' and 3' ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 5' and 3' ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 5' ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the 'full-length' transcriptome with a single library.
ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and isocitrate dehydrogenase (IDH)-mutant grade 2/3 adult glioma. Previous work has shown that ...ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We find that ATRX binds the regulatory elements of cell-cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to the early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell-cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intracranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wild-type controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display Chk1-mediated dysregulation of cell-cycle phase transitions, which opens a window for therapies targeting this phenotype.
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•ATRX binds regulatory elements of CHEK1 in glioma and glioma precursor cells•ATRX loss is associated with loss of the cell-cycle regulator Chk1•Chk1 loss increases reliance on ATM, an alternate cell-cycle checkpoint modulator•ATM inhibition may sensitize ATRX-deficient gliomas to radiation therapy
Qin et al. show that ATRX binds the gene regulatory elements and promotes expression of the cell-cycle regulator of CHEK1 in glioma and glioma precursor cells. ATRX loss in high-grade glioma results in decreased Chk1, dysregulation of cell-cycle checkpoints after radiation, and enhanced radio-sensitization with ATM inhibitors.
The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on ...the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.
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