Characterization of Regulatory Elements in the 5′-Upstream Region of the Human Gαq Gene in a Human Megakaryocytic Cell Line.

GTP-binding protein Gαq plays a major role in platelet signal transduction. We studied the transcriptional regulation of the human Gαq gene in a megakaryocytic cell line, human erythroleukemia (HEL). 10 nM phorbal myristate acetate (PMA) was used to induce megakaryocytic lineage in HEL cells. Western blot analysis on untreated vs PMA-treated HEL lysates showed enhanced Gαq expression with PMA. Firefly luciferase reporter gene constructs carrying various lengths of the Gαq gene upstream promoter region −1/−2727 bp from ATG codon, were transiently transfected into PMA-treated HEL cells. Gαq promoter driven luciferase expression was measured by Dual Luciferase Reporter Assay system (Promega). These studies indicated that the Gαq gene is regulated by positive and negative elements. Two positive regulatory sites were identified in the proximal region (−138/−238 bp) and distal region (−731/−1116 bp); a negative regulatory site was observed further upstream (−1117/−2727 bp). The proximal region −138/−238 was resolved into a repressor (−207/−238) and a positive (−138/−207) site. Consensus sequences for two well recognized megakaryocytic transcription factors (TFs) PU.1 (−225/−230) and GATA-1 (−208/−211) in the repressor site were deleted; this had no effect on promoter activity. The positive region (−138/−207) was further resolved into repressor (−162/−186) and positive (−187/−207) regions by functional studies. In the repressor region a CCACC (−175/−179) consensus motif for Sp/XKLF family of TF was found and deleted; the repressor activity was lost indicating a functional effect of the CCACC-motif. The positive region contained a Sp1/AP2 consensus sites between −152 and −203. Gel shift and super shift experiments on double stranded DNA oligo 1 (−175/−203) and oligo 2 (−152/−174) using HEL extracts demonstrated nuclear protein binding to these regions. However, antibodies against Sp1 and AP2 or the consensus oligos did not alter the binding. Gel shift mutant analysis was performed on both oligos to define the functional sequences mediating the nuclear protein binding. Mutations made at −190/−194, which has a consensus sequence for EGR1/EGR2 and, at −180/−184 and at −156/−160, abolished the protein binding suggesting that they are preferred sequences for DNA-protein interaction. The distal upstream positive region −731/−1116 also revealed positive and negative elements. Mutation in the EGR-2 consensus sequence (−909/−919) decreased the promoter activity to 50% and mutation in the AP2/EGR-1/Sp1 consensus sequence (−885/−893) abolished the promoter activity. Our results suggest that the sequences −156/−160, −180/−184, and −190/−194 in the proximal region, and −885/−893 and −909/−919 in the distal region have a critical role in the transcriptional regulation of Gαq in megakaryocytic cells.