Differential regulation of GPR54 transcription by specificity protein-1 and partial estrogen response element in mouse pituitary cells

Abstract

Precise spatial and temporal expression of the recently identified G-protein coupled receptor GPR54 is critical for proper reproductive function and metastasis suppression. However, regulatory factors that control GPR54 expression remain unknown. Thus, the identification of these cis-acting DNA elements can provide insight into the role of GPR54 in reproduction and cancer. Using luciferase reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we demonstrate that three SP1 sites and a partial estrogen response element modulate mouse GPR54 (mGPR54) promoter activity. Supporting experiments show transcription factor SP1 binds directly to the mGPR54 promoter region and activates gene expression. In conclusion, these novel findings now identify factors that regulate activity of the mGPR54 promoter, and these factors are highly conserved across multiple mammalian species.

Figure 1. Analysis of the mGPR54 gene

A, Mus musculus chromosome 10qC1 genomic arrangement is presented. The gene order is conserved between mouse and human (Med16, C19orf22, GPR54, and Arid3a). Two CpG islands are contained within the GPR54 sequence, one in the promoter region and one in the fifth exon. The sequence 1752 bp 5’ to the ATG of mouse GPR54 was chosen as the putative mGPR54 promoter; this region spans the sequence from the 5’ end of C19orf22 to the start site of translation for mGPR54 (N.B. C19orf22 is on opposite strand of DNA). Preliminary analysis identified the following promoter elements: four SP1 sites, one E box, one AP-1 site, and one partial estrogen response element (ERE). The SP1 and partial ERE sites are conserved in both the mouse and human promoters. (not shown here, see Sup. Fig. 2.). B, Activity of mGPR54 truncation constructs were examined using luciferase reporter assays. Activity was normalized to % of full-length construct in each assay, (n = 2-5, 3 replicates each, and expressed as average ± SEM).

Figure 2. Contribution of SP1 elements and partial ERE to mGPR54 promoter activity

A, Activities of mGPR54 deletion constructs were examined in AtT-20 cells. Data are normalized to % of activity observed using full-length mGRP54, (n = 2-3, 3 replicates each and expressed as average ± SEM). B Activity of full-length mGPR54 promoter construct or SP1Δ3 construct co-transfected with 0 or 400 ng SP1 in AtT-20 cells. Data are normalized to % of activity observed using full-length promoter, (n=3, 3 replicates each and expressed as average ± SEM).

Figure 3. AtT-20 Nuclear Extract binds to mGPR54 gene

A, Nucleotide sequence of the SP1 probe used for EMSA. B, Increasing concentrations of AtT-20 nuclear extract (NE) were tested to determine optimal concentration for maximum EMSA shift. (-) does not contain AtT-20 NE.

Figure 4. SP1 binds directly to mGPR54 gene

A, SP1 probe was incubated without (-) or with (+) 5 ug of AtT-20 nuclear extract (NE) containing SP1 antibody (SP1) or IgY antibody (IgY). EMSA reveals a supershift in the presence of SP1 antibody, but not IgY. DNA-protein complex indicated by unlabeled arrow. B, SP1 probe was incubated without (-) or with (+) 5 ug of AtT-20 NE containing wild type (WT), nonspecific (NS), or mutant SP1 sites (MT) cold probe. 10X and 25X concentrations of cold competitors were used where indicated. C, Chromatin isolated from AtT-20 cells was incubated with no antibody or SP1 antibody, Enrichment at SP1 sites was calculated as % of input. Data presented are from 2-3 replicates within one chromatin preparation and representative of 2 experiments.