Expression of vascular endothelial growth factor (VEGF) is induced in cells exposedUnder normal physiologic conditions, each of the approximately 10 14 cells in the adult human body is provided with an adequate supply of O 2 to meet its metabolic demands through the concerted function of the pulmonary, hematopoietic, and cardiovascular systems. O 2 is transported through the circulation by erythrocytes, the production of which is controlled by the glycoprotein hormone/growth factor erythropoietin (EPO) (reviewed in references 20, 23, and 43). Cells in the liver and kidney that produce EPO are able to sense O 2 concentration and respond to systemic hypoxia with increased EPO gene transcription (8,15,46). A hypoxia-inducible enhancer element was identified in the 3Ј-flanking region of the human and mouse EPO genes (2,3,33,42,48,50). Hypoxia-inducible factor 1 (HIF-1) was detected in nuclear extracts of hypoxic Hep3B cells (exposed to 1% O 2 for 4 h) and was undetectable in extracts from nonhypoxic cells (maintained at 20% O 2 ). HIF-1 bound to the EPO enhancer, and mutations that eliminated HIF-1 binding also eliminated enhancer function (50). Exposure of hypoxic cells to inhibitors of protein synthesis (cycloheximide) or phosphorylation (2-aminopurine) inhibited the induction of both EPO mRNA and HIF-1 DNA-binding activity, and other inducers of EPO expression (CoCl 2 and desferrioxamine) also induced HIF-1 activity (50,58,60). Methylation interference analysis revealed that HIF-1 bound to the EPO enhancer sequence 5Ј-TACGTGCT-3Ј by making major groove contacts with both guanine residues on each strand (59).Protein purification indicated that HIF-1 was a heterodimeric protein (61). Peptide and nucleic acid sequence analysis demonstrated that both subunits were basic helixloop-helix (bHLH) proteins (57). HIF-1␣ was a novel 826-amino-acid polypeptide, whereas HIF-1␤ was identical to the 774-and 789-amino-acid products of the ARNT (aryl hydrocarbon receptor nuclear translocator) gene previously shown to heterodimerize with the aryl hydrocarbon receptor (AHR) (57). HIF-1␣, HIF-1␤ (ARNT), and AHR are all members of a subfamily of bHLH proteins that contain a conserved PAS domain following the bHLH motif (4,18,57). In all three polypeptides, the basic domain is required for DNA binding following heterodimerization mediated by the HLH and PAS domains, and the C terminus contains one or more transactivation domains (6,21,29,32,44,63). Forced expression of HIF-1␣ and HIF-1␤ (ARNT) in cultured cells transfected with a reporter plasmid containing the EPO enhancer resulted in significantly higher levels of transcription, both at 1% and at 20% O 2 , than in cells transfected with the reporter plasmid alone, demonstrating that transcriptional activation via the EPO enhancer is mediated by HIF-1 (21).In contrast to systemic hypoxia, which elicits increased EPO synthesis, hypoxia can also be restricted to cells within a localized region of a specific organ, usually as a result of insufficient perfusion, as in the case of myocard...

Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia-inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1a −/− embryonic stem cells that did not express the O 2 -regulated HIF-1␣ subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxic Hif1a −/− embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1␣ resulted in developmental arrest and lethality by E11 of Hif1a −/− embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1a +/+ embryos, HIF-1␣ expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1a −/− embryos. These results demonstrate that HIF-1␣ is a master regulator of cellular and developmental O 2 homeostasis.

Hypoxia-inducible factor 1 (HIF-1) is a basic helixloop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5-RCGTG-3 and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.Multiple homeostatic mechanisms are employed by mammals to respond to chronic hypoxia. In the case of systemic hypoxia due to decreased environmental O 2 (hypobaric hypoxia) or decreased blood O 2 -carrying capacity (anemia), erythropoiesis is stimulated by the production of erythropoietin (EPO).

Hypoxia-inducible factor 1 (HIF-1) binds to cis-acting hypoxia-response elements within the erythropoietin, vascular endothelial growth factor, and other genes to activate transcription in hypoxic cells. HIF-1 is a basic helix-loop-helix transcription factor composed of HIF-1␣ and HIF-1␤ subunits. Here, we demonstrate that HIF-1␣ contains two transactivation domains located between amino acids 531 and 826. When expressed as GAL4 fusion proteins, the transcriptional activity of these domains increased in response to hypoxia. Fusion protein levels were unaffected by changes in cellular O 2 tension. Two minimal transactivation domains were localized to amino acid residues 531-575 and 786 -826. The transcriptional activation domains were separated by amino acid sequences that inhibited transactivation. Deletion analysis demonstrated that the gradual removal of inhibitory domain sequences (amino acids 576 -785) was associated with progressively increased transcriptional activity of the fusion proteins, especially in cells cultured at 20% O 2 . Transcriptional activity of GAL4/HIF-1␣ fusion proteins was increased in cells exposed to 1% O 2 , cobalt chloride, or desferrioxamine, each of which also increased levels of endogenous HIF-1␣ protein but did not affect fusion protein levels. These results indicate that increased transcriptional activity mediated by HIF-1 in hypoxic cells results from both increased HIF-1␣ protein levels and increased activity of HIF-1␣ transactivation domains.Human cells require O 2 for essential metabolic processes, most notably oxidative phosphorylation. Hypoxia is a significant pathophysiologic component of many cardiovascular, hematologic, and pulmonary disorders (reviewed in Ref.

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix protein that activates transcription of hypoxia-inducible genes, including those encoding: erythropoietin, vascular endothelial growth factor, heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1. Hypoxia response elements from these genes consist of a HIF-1 binding site (that contains the core sequence 5'-CGTG-3') as well as additional DNA sequences that are required for function, which in some elements include a second HIF-1 binding site. HIF-1 is a heterodimer. The HIF-1 alpha subunit is unique to HIF-1, whereas HIF-1 beta (ARNT) can dimerize with other bHLH-PAS proteins. Structural analysis of HIF-1 alpha revealed that dimerization with HIF-1 beta (ARNT) requires the HLH and PAS domains, DNA binding is mediated by the basic domain, and that HIF-1 alpha contains a carboxyl-terminal transactivation domain. Co-transfection of HIF-1 alpha and HIF-1 beta (ARNT) expression vectors and a reporter gene containing a wild-type hypoxia response element resulted in increased transcription in non-hypoxic cells and a superinduction of transcription in hypoxic cells, whereas HIF-1 expression vectors had no effect on the transcription of reporter genes containing a mutation in the HIF-1 binding site. HIF-1 alpha and HIF-1 beta (ARNT) protein levels were induced by hypoxia in all primary and transformed cell lines examined. In HeLa cells, the levels of HIF-1 alpha and HIF-1 beta protein and HIF-1 DNA-binding activity increased exponentially as cellular oxygen tension decreased, with maximum values at 0.5% oxygen and half-maximal values at 1.5 to 2% oxygen. HIF-1 alpha and HIF-1 beta (ARNT) mRNAs were detected in all human, mouse, and rat organs assayed and mRNA expression was modestly induced in rodents subjected to hypoxia. HIF-1 alpha protein levels were induced in vivo when animals were subjected to anemia or hypoxia. The HIF1A gene was mapped to human chromosome 14q21-q24 and mouse chromosome 12.

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BackgroundAging and sun exposure are the leading causes of skin cancer. It has been shown that epigenetic changes, such as DNA methylation, are well established mechanisms for cancer, and also have emerging roles in aging and common disease. Here, we directly ask whether DNA methylation is altered following skin aging and/or chronic sun exposure in humans.ResultsWe compare epidermis and dermis of both sun-protected and sun-exposed skin derived from younger subjects (under 35 years old) and older subjects (over 60 years old), using the Infinium HumanMethylation450 array and whole genome bisulfite sequencing. We observe large blocks of the genome that are hypomethylated in older, sun-exposed epidermal samples, with the degree of hypomethylation associated with clinical measures of photo-aging. We replicate these findings using whole genome bisulfite sequencing, comparing epidermis from an additional set of younger and older subjects. These blocks largely overlap known hypomethylated blocks in colon cancer and we observe that these same regions are similarly hypomethylated in squamous cell carcinoma samples.ConclusionsThese data implicate large scale epigenomic change in mediating the effects of environmental damage with photo-aging.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0644-y) contains supplementary material, which is available to authorized users.

IMPORTANCE Given the widespread use of systemic antibiotics for treatment of moderate to severe acne, it is important to understand the associations of such antibiotic use with changes not only in Cutibacterium acnes (formerly Propionibacterium acnes) but also in the complete bacterial community of the skin. OBJECTIVE To examine the composition, diversity, and resilience of skin microbiota associated with systemic antibiotic perturbation in individuals with acne. DESIGN, SETTING, AND PARTICIPANTS This longitudinal cohort study conducted at an academic referral center in Maryland from February 11 to September 23, 2014, included 4 female participants who had received a recent diagnosis of acne vulgaris, showed comedonal and inflammatory acne on the face, were at least 18 years old, and had no recent use of systemic or topical treatments for acne, including antibiotics and retinoids. Data analysis was performed between July 5, 2017, and November 7, 2018. INTERVENTIONS Participants were prescribed oral minocycline, 100 mg, twice daily for 4 weeks. Skin areas on the forehead, cheek, and chin were sampled for 16S ribosomal RNA gene sequencing at baseline, 4 weeks after starting minocycline treatment, and then 1 week and 8 weeks after discontinuation of treatment. MAIN OUTCOMES AND MEASURES Skin microbiota examined with respect to relative abundance of bacterial taxa, a diversity (represents within-sample microbial diversity), and β diversity (represents between-sample microbial diversity). Acne status evaluated with photography and lesion count. RESULTS Of the 4 patients included in this study, 2 were 25 years old, 1 was 29 years old, and 1 was 35 years old; 2 were white women, 1 was an African American woman, and 1 was an Asian woman. Across all 4patients, antibiotic treatment was associated with a 1.4-fold reduction in the level of Cacnes (difference, −10.3%; 95% CI, −19.9% to −0.7%; P = .04) with recovery following cessation of treatment. Distinct patterns of change were identified in multiple bacterial genera, including a transient 5.6-fold increase in the relative abundance of Pseudomonas species (difference, 2.2%; 95% CI, 0.9%−3.4%; P < .001) immediately following antibiotic treatment, as well asa persistent 1.7-fold increase in the relative abundance of Streptococcus species (difference, 5.4%; 95% CI, 0.3%−10.6%; P = .04) and a 4.7-fold decrease in the relative abundance of Lactobacillus species (difference, −0.8%; 95% CI, −1.4% to −0.2%; P = .02) 8 weeks following antibiotic treatment withdrawal. In general, antibiotic administration was associated with an initial decrease from baseline of bacterial diversity followed by recovery. Principal coordinates analysis results showed moderate clustering of samples by patient (analysis of similarity, R = 0.424; P = .001) and significant clustering of samples by time in one participant (analysis of similarity, R = 0.733; P = .001). CONCLUSIONS AND RELEVANCE In this study, systemic antibiotic treatment of acne was associated with changes in the composition an...