Cloning of the Human MORG1 Promoter: Differential Regulation by Hypoxia and Prolyl-Hydroxylase Inhibitors.

Tzvetanka Bondeva,Gunter Wolf

doi:10.3390/genes13030427

Abstract

MAPK-organizer 1 (MORG1) is a molecular scaffold for prolyl-hydroxylase-3 containing a domain (PHD3) protein linking MORG1 to mechanisms of adaptation in hypoxic conditions. In this paper, we report the cloning of the promoter region of the murine and human MORG1 gene. Among other transcriptional factors binding sites, we identified that both (mouse and human) promoter regions contained several putative hypoxia-inducible factor binding motifs. Analyses of the human MORG1 promoter by reporter assays revealed that hypoxia and pharmacological inhibitors of prolyl-hydroxylases under in vitro conditions in HEK 293 cells differentially regulate the MORG1 promoter reporter activity. The exposure of the cells to 10% hypoxia showed inhibition of MORG1 promotor activity at 6 and 12 h, but stimulation after 24 h while treated with prolyl-hydroxylase inhibitors led to a time-independent MORG1 promoter activation. Mutational analyses of the individual HIF binding sites on human MORG1 promoter suggest that the binding sites work in a complex corporation because single mutations were not sufficient to abolish completely the MORG1 reporter activation by PHD inhibitors. Our data provide the first evidence that not only MORG1 regulate HIF stabilization through a PHD complex, but also that, vice versa, HIFs control MORG1 expression directly or indirectly by a complex regulatory mechanism.

Highlights

The MAPK organizer-1 (MORG1) protein belongs to the family of the WD40 repeat domain-containing proteins [1]
We found that the binding reactions of hypoxia-responsible element binding site (HBS) D oligos electrophoretic mobility shift assay (EMSA) show that both Hypoxia Inducible Factors (HIFs)-1α and HIF-2α transcription factor (TF) could associate with this hMORG1 promoterbinding site, as the addition of HIF-1α or HIF-2α antibodies to the binding reaction, showed a competitor-like effect, reducing the binding of the HBS D oligonucleotides to the nuclear extracts (NE)
Among other depicted putative TF binding site, several predicted hypoxia-responsible elements (HRE) binding motives were identified in both murine and human MORG1 promoter sequences corresponding to the canonical HRE binding sequence 5 -A/GCGTG-3

Summary

Introduction

The MAPK organizer-1 (MORG1) protein belongs to the family of the WD40 repeat domain-containing proteins [1]. The WD40 domain-containing proteins are devoid of enzymatic activities, but due to their multiple WD-repeat structure, play an important role in assembling functional protein complexes [2] and are involved in different cellular processes, such as transcriptional regulation [3], ubiquitin-dependent protein degradation [4] and chromatin modification [5]. MORG1 was first discovered as a binding partner of the MP1-protein, that is, a part of the extracellular signal regulated kinases (ERKs) module, and facilitates ERK activation [1]. PHDs are pivotal cellular regulators of the HIF transcription factors [7,8] Those findings suggest an important function of MORG1 as a scaffold protein for two signal-transduction pathways regulating proliferation via a MAPK-signaling module and hypoxia/normoxia regulation and adaptation to hypoxia via its complex with PHD3

Methods

Results

Conclusion