Regulation of gonadotropin-releasing hormone-1 gene transcription by members of the purine-rich element-binding protein family

Abstract

Gonadotropin-releasing hormone-1 (GnRH1) controls reproduction by stimulating the release of gonadotropins from the pituitary. To characterize regulatory factors governing GnRH1 gene expression, we employed biochemical and bioinformatics techniques to identify novel GnRH1 promoter-binding proteins from the brain of the cichlid fish, Astatotilapia burtoni (A. burtoni). Using an in vitro DNA-binding assay followed by mass spectrometric peptide mapping, we identified two members of the purine-rich element-binding (Pur) protein family, Puralpha and Purbeta, as candidates for GnRH1 promoter binding and regulation. We found that transcripts for both Puralpha and Purbeta colocalize in GnRH1-expressing neurons in the preoptic area of the hypothalamus in A. burtoni brain. Furthermore, we confirmed in vivo binding of endogenous Puralpha and Purbeta to the upstream region of the GnRH1 gene in A. burtoni brain and mouse neuronal GT1-7 cells. Consistent with the relative promoter occupancy exhibited by endogenous Pur proteins, overexpression of Purbeta, but not Puralpha, significantly downregulated GnRH1 mRNA levels in transiently transfected GT1-7 cells, suggesting that Purbeta acts as a repressor of GnRH1 gene transcription.

Fig. 1.

Mapping DNA/protein-binding sites in the gonadotropin-releasing hormone-1 (GnRH1) upstream region by capturing nucleoprotein complexes with selected DNA fragments immobilized on magnetic beads. A: mass spectrometry-compatible silver stain of SDS-PAGE resolved DNA-binding proteins captured from Astatotilapia burtoni (A. burtoni) brain lysate. G1–G5, upstream DNA fragments of GnRH1 gene sequence; SB, SeeBlue prestained protein ladder; SB+, SeeBlue-plus prestained protein ladder (Invitrogen); Ctr, a control fragment from the cDNA coding sequence of PCNA; Pur, purine-rich element binding. Compared with the Ctr, distinct 41- (black arrows) and 38-kDa (white arrows) bands were observed in the gel. B: schematic representation of the GnRH1 upstream fragments (gray lines) and the deduced binding sites of the 41- and 38-kDa binding proteins.

Fig. 2.

Molecular cloning of Purα and Purβ from A. burtoni. Vector-NT software (Invitrogen) was used to generate multiple alignments of predicted protein sequences for A. burtoni Purα (A) or Purβ (B) with corresponding sequences from other fish species, mouse, and human. Conserved sequences required for DNA/RNA-binding are indicated by an open box, and peptides matched in mass mapping are underlined. C: the phylogenetic tree for Pur proteins was generated by Mega 3.1 using neighbor-joining and bootstrap test. Note that the A. burtoni sequence cosegregates with sequences from other fish species.

Fig. 3.

Localization of Purα and Purβ mRNA in A. burtoni. A: mRNA of Purα and Purβ in various A. burtoni tissues. SC, spinal cord; Br, brain; Re, retina; Pit, pituitary gland; Mu, muscle; Gill, gill; Sp, spleen; St, stomach; Gut, gut; Li, liver; Ki, kidney; Ov, ovary; Te, testicle; He, heart; Ctr, control (water). β-Actin was used as an internal control. B: in situ hybridization of Purα or Purβ mRNA and GnRH1 mRNA in the preoptic area of the A. burtoni brain. GnRH1-releasing neurons were stained by 3,3′-diaminobenzidine (brown; top, bright field) and Purα or Purβ mRNA were visualized by silver grains developed in the emulsion (black dots; top, bright field, or white dots; bottom, dark field). Cresyl violet staining (blue; top) was used to visualize cell bodies. C: confirmation of the molecular weights of Pur proteins. The apparent molecular weights of Pur proteins detected on Western blots are comparable with the 41- and 38-kDa GnRH1 upstream binding proteins identified in the in vitro binding assay (Fig. 1). Bands corresponding to endogenous A. burtoni (a)Purα and aPurβ in fish Re and Br are shown at left, whereas Myc-His-tagged aPurα and Myc-His-tagged aPurβ overexpressed in GT1–7 (Gt) cells are highlighted at right. The sizes of endogenous mouse Purα (m)Purα and mPurβ from Gt were compared with the fish Pur proteins side by side. M, protein molecular weight marker; Oe, overexpressed; αA, antibody against Purα (A149); αB, antibody against Purβ (B302).

Fig. 4.

In vivo binding of Purα and Purβ to the upstream region of the GnRH1 gene. After chromatin immunoprecipitation (ChIP), PCR was performed using specific primers against A. burtoni (A) or mouse (B) GnRH1 upstream sequence. GnRH1 DNA fragments were amplified from the immunocomplex pulled down by antibody against Purα and Purβ. PCR products were visualized by electrophoresis (arrows). Bg, bacteria genomic DNA; NA, sample without antibody; MK, 1 KB + DNA Marker (Invitrogen).

Fig. 5.

Repression of mouse GnRH1 gene transcription by overexpressing Purα and Purβ in GnRH-releasing mouse neuronal GT1–7 cells. Ctr, aPurα, aPurβ, mPurα, and mPurβ plasmids were transfected into GT1–7 cells for 48 h. A: mGnRH1 mRNA level was analyzed by real-time PCR and normalized using mouse β-actin mRNA (n = 5). Two-tailed homoscedastic t-test (2 samples assuming equal variance) was used for statistical analysis. **Significant difference was found between the experimental group and control group (P < 0.01). Western blots were performed to verify the overexpression of Purα (B) and Purβ (C). Mouse β-actin was used as an internal control. αActin, antibody against mouse β-actin.

Fig. 6.

Schematic representation of A. burtoni and mouse GnRH1 genes with putative Pur protein-binding sites. GnRH1 genes were analyzed, and putative Purα and Purβ binding sites (GGGAGA and GGHGGH) were plotted. Symbols for binding sites on the top side of the gene indicate that they are on the sense strand. Bottom side indicates the antisense strand. Double-stranded DNA fragments used for the in vitro DNA/protein-binding assay are indicated by the gray lines, and the possible binding sites for Purα and Purβ were deduced from the in vitro DNA/protein-binding assay (Supplemental Table S2 and Fig. 1).