Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1A) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma (HCC) cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing.

Chromatin remodeling is an active process, which represses or enables the access of transcription machinery to genes in response to external stimuli, including hypoxia. However, in hypoxia, the specific requirement, as well as the molecular mechanism by which the chromatin-remodeling complexes regulate gene expression, remains unclear. In this study, we report that the Brahma (BRM) and Brahma-related gene 1 (BRG1) ATPase-containing SWI/SNF chromatin-remodeling complexes promote the expression of the hypoxia-inducible transcription factor 1␣ (HIF1␣) and HIF2␣ genes and also promote hypoxic induction of a subset of HIF1 and HIF2 target genes. We show that BRG1 or BRM knockdown in Hep3B and RCC4T cells reduces hypoxic induction of HIF target genes, while reexpression of BRG1 or BRM in BRG1/BRM-deficient SW13 cells increases HIF target gene activation. Mechanistically, HIF1 and HIF2 increase the hypoxic induction of HIF target genes by recruiting BRG1 complexes to HIF target gene promoters, which promotes nucleosome remodeling of HIF target gene promoters in a BRG1 ATPase-dependent manner. Importantly, we found that the function of BRG1 complexes in hypoxic SW13 and RCC4T cells is dictated by the HIF-mediated hypoxia response and could be opposite from their function in normoxic SW13 and RCC4T cells.H ypoxia (Hx) is a common characteristic of many solid tumors. The Hx intratumoral microenvironment stabilizes hypoxia-inducible transcription factor 1␣ (HIF1␣) and HIF2␣, which are normally degraded under normoxia (Nx). The stabilized HIF1␣ and HIF2␣ proteins translocate to the nucleus, where they dimerize with the constitutive nuclear protein ARNT (the aryl hydrocarbon receptor nuclear translocator, also called HIF1␤) to form HIF1␣/ARNT (HIF1) and HIF2␣/ARNT (HIF2) heterodimers. HIF1 and HIF2 bind to HIF binding sites (HBS) on HIF target gene promoters and/or enhancers and transactivate genes involved in neovascularization, glycolysis, cellular proliferation, and metastasis. Thus, the HIF-mediated Hx transcriptional response is critical for tumor progression by allowing cancer cells to adapt to a low-oxygen environment (1-4). However, recent reports indicate that the HIF2-and particularly the HIF1-mediated Hx response can activate tumor-suppressive genes, such as Scgb3a, Bnip3, Bnip3L, Nix, MYC inhibitor Mxi, p21, and p27, in a cell-type-specific manner (5-10).It is well established that multiple transcription factors (TFs) are required to achieve maximal activation of target genes in response to a specific stimulus such as Hx. This multifactorial transcription complex has been termed the "enhanceosome." Individual TFs in the enhanceosome complex may promote transcription by recruiting RNA polymerase II/general TFs and/or by recruiting chromatin-modifying enzymes such as histone acetylase, CBP and p300, and the chromatin-remodeling SWI/SNF complex. In the context of the enhanceosome associated with the Hx response, two additional TFs, STAT3 and USF2, are required for maximal Hx response (11,12). STAT3 and USF2 function...

Exposure to crude oil or its individual constituents can have detrimental impacts on fish species, including impairment of the immune response. Increased observations of skin lesions in northern Gulf of Mexico fish during the 2010 Deepwater Horizon oil spill indicated the possibility of oil-induced immunocompromisation resulting in bacterial or viral infection. This study used a full factorial design of oil exposure and bacterial challenge to examine how oil exposure impairs southern flounder (Paralichthys lethostigma) immune function and increases susceptibility to the bacteria Vibrio anguillarum, a causative agent of vibriosis. Fish exposed to oil prior to bacterial challenge exhibited 94.4% mortality within 48 hours of bacterial exposure. Flounder challenged with V. anguillarum without prior oil exposure had <10% mortality. Exposure resulted in taxonomically distinct gill and intestine bacterial communities. Mortality strongly correlated with V. anguillarum levels, where it comprised a significantly higher percentage of the microbiome in Oil/Pathogen challenged fish and was nearly non-existent in the No Oil/Pathogen challenged fish bacterial community. Elevated V. anguillarum levels were a direct result of oil exposure-induced immunosuppression. Oil-exposure reduced expression of immunoglobulin M, the major systemic fish antibody, and resulted in an overall downregulation in transcriptome response, particularly in genes related to immune function, response to stimulus and hemostasis. Ultimately, sediment-borne oil exposure impairs immune function, leading to increased incidences of bacterial infections. This type of sediment-borne exposure may result in long-term marine ecosystem effects, as oil-bound sediment in the northern Gulf of Mexico will likely remain a contamination source for years to come.

Attaching Unique Molecular Identifiers (UMI) to RNA molecules in the first step of sequencing library preparation establishes a distinct identity for each input molecule. This makes it possible to eliminate the effects of PCR amplification bias, which is particularly important where many PCR cycles are required, for example, in single cell studies. After PCR, molecules sharing a UMI are assumed to be derived from the same input molecule. In our single cell RNA-Seq studies of Physcomitrella patens, we discovered that reads sharing a UMI, and therefore presumed to be derived from the same mRNA molecule, frequently map to different, but closely spaced locations. This behaviour occurs in all such libraries that we have produced, and in multiple other UMI-containing RNA-Seq data sets in the public domain. This apparent paradox, that reads of identical origin map to distinct genomic coordinates may be partially explained by PCR stutter, which is often seen in low-entropy templates and those containing simple tandem repeats. In the absence of UMI this artefact is undetectable. We show that the common assumption that sequence reads having different mapping coordinates are derived from different starting molecules does not hold. Unless taken into account, this artefact is likely to result in over-estimation of certain transcript abundances, depending on the counting method employed.

Adrenomedullin (ADM) is important for tumor angiogenesis, tumor cell growth and survival. Under normoxic conditions, the ADM gene was found to produce two alternative transcripts, a fully-spliced transcript that produces AM and PAMP peptides and a intron-3-retaining transcript that produces a less functionally significant PAMP peptide only. ADM is a well-established hypoxia inducible gene; however, it is not clear which ADM isoform is induced by hypoxia. In this study, it was determined that various cancer and normal cells express two predominant types of ADM transcripts, a AM/PAMP peptide producing FL transcript in which all introns are removed, and a non-protein producing I1-3 transcript in which all introns are retained. Interestingly, hypoxia preferentially induced the FL isoform. Moreover, HIFs, but not hypoxia per se are necessary and sufficient to increase splicing of ADM pre-mRNA. ADM splicing reporters confirmed that transcriptional activation by HIF or other transcription factors is sufficient to enhance splicing. However, HIFs are more potent in enhancing ADM pre-mRNA splicing than other transcriptional activators. Thus, ADM intron retention is not a consequence of abnormal splicing, but is an important mechanism to regulate ADM expression. These results demonstrate a novel function of HIFs in regulating ADM expression by enhancing its pre-mRNA splicing. Importantly, using endogenous and cloned ADM gene, further evidence is provided for the coupling of transcription and RNA splicing.

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Studies since 2004 have shown that the cytocompatibility of ultrafine grain (UG) commercial purity (CP) titanium exceeds that of coarse grain (CG) CP titanium (Ti) by 30% to 20-fold. To isolate the factors affecting this large reported variability of CP titanium's cytocompatibility, discs of UG and CG titanium were fabricated with controlled texture and roughness. The discs were seeded with MC3T3-E1 pre-osteoblastic cells and cultured for 72 h. The proliferation of cells on polished UG-Ti exceeded unpolished CG-Ti 3.04-fold. Cell proliferation was found to correlate with a new biophysical parameter, the average grain boundary length per surface-attached cell. IMPACT STATEMENT By eliminating the influences of texture and roughness we have isolated the positive correlation between MC3TE-E1 cell proliferation on titanium and grain boundary length per surface-attached cell.

RNA viruses are infamous for their high rates of mutation, which produce swarms of genetic variants within individual hosts. To date, analyses of intrahost genetic diversity have focused on the primary genome sequence. However, virus phenotypes are shaped not only by primary sequence but also by the secondary structures into which this sequence folds. Such structures enable viral replication, translation, and binding of small RNAs, yet within-host variation at the structural level has not been adequately explored. We characterized the structural diversity of the 5′ untranslated region (UTR) of populations of West Nile virus (WNV) that had been subject to five serial passages in triplicate in each of three bird species. Viral genomes were sampled from host serum samples at each passage (n = 45 populations) and subjected to next-generation sequencing. For populations derived from passages 1, 3, and 5 (n = 9 populations), we predicted the impact of each mutation occurring at a frequency of ≥1% on the secondary structure of the 5′ UTR. As expected, mutations in double-stranded (DS) regions of the 5′ UTR stem structures caused structural changes of significantly greater magnitude than did mutations in single-stranded (SS) regions. Despite the greater impact of mutations in DS regions, mutations in DS and SS regions occurred at similar frequencies, with no evidence of enhanced selection against mutation in DS regions. In contrast, mutations in two regions that mediate genome cyclization and thereby regulate replication and translation, the 5′ cyclization sequence and the UAR flanking stem (UFS), were suppressed in all three hosts. IMPORTANCE The enzymes that copy RNA genomes lack proofreading, and viruses that possess RNA genomes, such as West Nile virus, rapidly diversify into swarms of mutant lineages within a host. Intrahost variation of the primary genomic sequence of RNA viruses has been studied extensively because the extent of this variation shapes key virus phenotypes. However, RNA genomes also form complex secondary structures based on within-genome nucleotide complementarity, which are critical regulators of the cyclization of the virus genome that is necessary for efficient replication and translation. We sought to characterize variation in these secondary structures within populations of West Nile virus during serial passage in three bird species. Our study indicates that the intrahost population of West Nile virus is a diverse assortment of RNA secondary structures that should be considered in future analyses of intrahost viral diversity, but some regions that are critical for genome cyclization are conserved within hosts. Besides potential impacts on viral replication, structural diversity can influence the efficacy of small RNA antiviral therapies.