Co-organizador del evento. Novel mechanisms of HIF regulation; genetic lessons from Drosophila

PABLO WAPPNER

Oxford

Simposio; Keystone Symposia. Therapeutic targeting of hypoxia-sensitive pathways; 2018

Keystone Symposia

The HIF system is largely conserved in Drosophila melanogaster, being Sima and Tango the HIF and HIF homologues respectively, whereas Fatiga is the single HIF-Prolyl-4-hydroxylase that occurs in this species. We have carried out genetic screens in Drosophila to define novel regulators of HIF-dependent responses to hypoxia. An RNAi based genome-wide screen performed in Drosophila S2 cells in culture rendered 21 novel regulators of HIF, which included Argonaute1 and other components of the miRNA machinery, suggesting that one or more microRNAs (miRNAs) are required for maximal HIF-dependent transcription. To identify the relevant miRNAs, we performed an over-expression screen in vivo in a collection of transgenic fly lines, which expressed each one of the miRNAs encoded in the Drosophila genome. We focused on those miRNAs that, when overexpressed, provoked an enhancement of HIF-dependent transcription. The screen, led to the identification of 4 miRNAs that enhanced HIF-dependent transcription, and characterized in detain the mechanism of action of miR-190. miR-190 mediates inhibition of translation of the HIF-prolyl-4-hydroxylase Fatiga, thereby enhancing responses to hypoxia. Other HIF novel regulators defined in the RNAi screen comprised elements of the chromatin modifying complex TIP60.com. Tip60.com is an evolutionary conserved Histone Acetyltransferase (HAT) complex required both in Drosophila and human cells for HIF maximal activity. Tip60.com is recruited by HIF to promoters in an oxygen-dependent manner and is required for RNA Pol II recruitment and transcript elongation. Finally, the screen led to the identification of the translational repressor Musashi as a novel HIF regulator. Musashi binds the 3? unstranslated region (UTR) of Sima mRNA, and represses Sima translation in an oxygen-dependent manner. Thus, by taking advantage of the wide range of genetic tools available in Drosophila, we have identified several novel conserved regulators of HIF-dependent transcription, revealing that HIF is controlled by a complex array of transcriptional and translational mechanisms that operate at different levels.