Descripción: Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke. © 2010 Dekanty et al.

Descripción: Although hypoxia-inducible factor-α (HIFα) subunit-specific hydroxylation and proteolytic breakdown explain the binary switch between the presence (hypoxia) and absence (normoxia) of HIFs, little is known of the mechanisms that fine-tune HIF activity under constant, rather than changing, oxygen tensions. Here, we report that the Drosophila HIFα homolog, the basic helix-loop-helix/PAS protein Sima (Similar), in hypoxic cultures of SL2 cells is expressed in full-length (fl) and splice variant (sv) isoforms. The following evidence supports the role of flSima as functional HIFα and the role of SL2 HIF as a transcriptional activator or suppressor. The pO2 dependence of Sima abundance matched that of HIF activity. HIF-dependent changes in candidate target gene expression were detected through variously effective stimuli: hypoxia (strong) > iron chelation, e.g. desferrioxamine (moderate) ≪ transition metals, e.g. cobalt ≃ normoxia (ineffective). Sima overexpression augmented hypoxic induction or suppression of different targets. In addition to the full-length exon 1-12 transcript yielding the 1510-amino acid HIFα homolog, the sima gene also expressed, specifically under hypoxia, an exon 1-7/12 splice variant, which translated into a 426-amino acid Sima truncation termed svSima. svSima contains basic helix-loop-helix and PAS sequences identical to those of flSima, but, because of deletion of exons 8-11, lacks the oxygen-dependent degradation domain and nuclear localization signals. Overexpressed svSima failed to transactivate reporter genes. However, it attenuated HIF (Sima-Tango)-stimulated reporter expression in a dose-dependent manner. Thus, svSima has the potential to regulate Drosophila HIF function under steady and hypoxic pO2 by creating a cytosolic sink for the Sima partner protein Tango.

Descripción: We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mM concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.

Descripción: p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B trans-activation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.