Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli.

The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA ( dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp0 dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli.

The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA ( dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp0 dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Hepatocyte nuclear factor 4alpha transactivates the mitochondrial alanine aminotransferase gene in the kidney of Sparus aurata

Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5′-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L-alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.

The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation.

Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity to other reported enzymes known to catalyze these reactions, but instead are similar to oxygenases acting on aromatic compounds. Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on 54, is mediated by the Ntr system. In contrast, neither NtrC nor NAC is involved in the nitrogen control of hpxDE, which is dependent on 70 for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N4-GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.