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A close interconnection between nutrient metabolism and virulence factor expression contributes to the pathophysiology of Pseudomonas aeruginosa as a successful pathogen. P. aeruginosa fatty acid (FA) degradation is complicated with multiple acyl-CoA synthetase homologs (FadDs) expressed in vivo in lung tissue during cystic fibrosis infections. The promoters of two genetically linked P. aeruginosa fadD genes (fadD1 and fadD2) were mapped and northern blot analysis indicated they could exist on two different transcripts. These FadDs contain ATP/AMP signature and FA-binding motifs highly homologous to those of the Escherichia coli FadD. Upon introduction into an E. coli fadD-/fadR- double mutant, both P. aeruginosa fadDs functionally complemented the E. coli fadD-/fadR- mutant, allowing degradation of different chain-length FAs. Chromosomal mutagenesis, growth analysis, induction studies, and determination of kinetic parameters suggested that FadD1 has a substrate preference for long-chain FAs while FadD2 prefers shorter-chain FAs. When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities. Interestingly, fadD1 mutant showed only increased swarming motility. Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source. This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo. [ABSTRACT FROM AUTHOR]

Introduction: PRKCBP1 is known to cooperatively bind to acetylated and methylated nucleosomes through its bromo- and PHD domains. It can modulate transcription and cancer progression by histone methylation modifications affecting enhancer and promoter regions of genes. Mutations and dysregulation of PRKCBP1 have been found in multiple cancer types, however its role in the cardiac myocardium is unexplored. Hypoxia-inducible factor 1α (HIF-1α) upregulation and stabilization is a common feature of both cancer and myocardial ischemia, promoting cellular functions such as proliferation, glucose metabolism and angiogenesis. Here we investigated the role of PRKCBP1 in a model of cardiac HIF-1 activation. Hypothesis: PRKCBP1 inhibits the HIF-1 response in hypoxic heart by decreasing enhancer activity of HIF-1 target genes. Methods & Results: We expressed an inducible, cardiac-specific, oxygen-stable form of HIF-1α in mice that express increased PRKCBP1 (P+). Compared to wild-type (WT) mice, P+ mice did not exhibit the expected HIF-1 phenotype of increased heart weight to body weight (6.4±0.3 vs. 4.8±0.2 vs. 5.1±0.3 mg/g, WT vs. P+ vs. uninduced; p<0.05) with reduced ventricular function and associated chamber dilation. Semi-quantitative real-time PCR analysis of HL-1 and H9C2 cardiac cell lines transfected with CMV-driven expression plasmids for PRKCBP1 and oxygen-stable HIF-1α showed striking reduction of multiple HIF-1 target genes such as PDK1 (45% reduction) compared to the HIF-1α plasmid alone. RNAi mediated knockdown of PRKCBP1 removed this negative regulation (65% increase). Bioinformatic analysis of human PRKCBP1 and HIF-1α ChIP-seq data indicates that PRKCBP1 binds to the enhancer of 78% of HIF-1 regulated genes. Furthermore, global DNA 5-mC is substantially reduced in P+ heart cells compared to WT, suggesting an additional mechanism of gene regulation. Conclusion: We have discovered a new regulator of HIF-1 action that modifies the hypoxic response, likely through chromatin remodeling. This new form of regulation may modify the pathophysiology of ischemia and provide new targets for therapy. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates gene response during hypoxia, including regulating angiogenesis, the formation of new blood vessels, and glycolytic metabolism. This heterodimeric protein is composed of HIF-1α and HIF-1α subunits and consists of a basic helix-loop-helix-PAS domain. HIF-1α is expressed constitutively, whereas HIF-1α is regulated by oxygen concentration and inducible by hypoxia. HIF- 1α possesses an oxygen-dependent degradation domain, which causes it to be targeted for rapid proteosomal degradation during normoxia; hypoxia slows the destruction of HIF-1α. Since HIF-1 plays an important role in angiogenesis, it is a subject of interest in the heart where hypoxia occurs due to coronary artery disease, the leading cause of morbidity and mortality on the Western world. This research project involved the over expression of an oxygenstable form of HIF-1α in the murine heart, to identify the genes regulated by HIF-1. In doing so we are able to determine how HIF-1 directly regulates angiogenesis and glucose metabolism. The study hypothesizes that HIF-1 promotes the expression of genes to stimulate angiogenesis and shifts the citric acid cycle from aerobic to anaerobic glycolysis. To identify the genes, mouse models, chromatin immunoprecipitation (ChIP) assays and other validation techniques, such as electrophoresis mobility shift assay (EMSA) gels and computational analyses were used. Bi-transgenic mice were used as animal models, with a cardiac specific promoter and a mutated form of human HIF-1α, under Tet-off control. The Tet-off system controls gene expression with the use of Doxycycline as an inhibitor to promoter complex binding, preventing the transcription of the transgene. A ChIP assay was used to identify DNA regions that are bound to transcription factors, thereby determining which genes are directly regulated. Since HIF-1α is being over expressed, the DNA sequence identified is composed of the promoter regions regulated by HIF-1. One gene of particular interest that was identified via ChIP analysis was pkc binding protein 1 isoform 2, which may play a role in the regulation of HIF-1. This gene has not been fully characterized, but has been implicated in HIF regulation previously. Genes will be further verified by using additional EMSA and computational analyses. Identifying the genes that HIF-1 directly binds to will give insight to the role that HIF-1 plays in ischemia and coronary artery disease. This will provide important information to assist in the development of HIF-1 gene therapy. [ABSTRACT FROM AUTHOR]

The chromatin immunoprecipitation (ChIP) assay is a versatile technique used to evaluate the association of proteins with specific DNA regions both in vivo and in vitro. This assay can be used to identify proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein. The ChIP assay can also be used to analyze binding of transcription factors, transcription cofactors, DNA replication factors, and DNA repair proteins. Here we describe a useful ChIP-qPCR protocol to examine the interaction of NFkB with the VEGF promoter in adult rat primary cardiomyocytes that have been mechanically stretched after attaching to the extracellular matrix protein laminin.