ARP-1 Regulates the Transcriptional Activity of the Aromatase Gene in the Mouse Brain

Abstract

An important function of aromatase in the brain is conversion of testosterone secreted from the testis into estradiol. Estradiol produced in the brain is thought to be deeply involved in the formation of sexually dimorphic nuclei and sexual behavior as a neurosteroid. We analyzed the brain-specific promoter to elucidate the control mechanisms of brain aromatase expression that may be highly involved in sexual differentiation of the brain. The 202-bp upstream region of the brain-specific exon 1 has three types of cis-acting elements, aro-AI, AII, and B. We isolated ARP-1 as an aro-AII-binding protein by yeast one-hybrid screening from a cDNA library of mouse fetal brains. ARP-1 is a member of the nuclear receptor superfamily and functions as an orphan-type transcription factor. ARP-1 protein synthesized in vitro showed the same binding property to the aro-AII site as nuclear extract from fetal brains. To determine how the promoter is involved in brain-specific transcription of the aromatase gene, we first detected the in vivo occupancy of the aro-AII site by ARP-1 using chromatin immunoprecipitation assays. Diencephalic regions of fetal brains at embryonic day 16 were analyzed, which revealed ARP-1 recruitment to the aro-AII site. To analyze the effects of ARP-1 on transcriptional regulation of the brain-specific aromatase promoter, a luciferase reporter plasmid driven by the brain-specific promoter was transfected into CV-1 cells together with a plasmid expressing ARP-1 protein. These analyses revealed that ARP-1 induced promoter activity in a dose-dependent manner. Furthermore, to determine whether ARP-1 is required for aromatase expression in neurons, ARP-1 knockdown was conducted in neuronal cell primary culture. Knockdown of ARP-1 significantly suppressed the increase in aromatase mRNA observed in cultured neurons. These results indicate that ARP-1 is involved in the transcriptional regulation of the brain-specific promoter of the aromatase gene.

Keywords: aromatase; chicken ovalbumin upstream promoter transcription factor; estrogen; nuclear receptor; sexual differentiation; steroid hormone.

Figure 1

Brain-specific exon 1 and its promoter region in the mouse aromatase gene. The number +1 corresponds to a potential transcription start site. A TATA box is shown in the shadowed box. The open boxes indicate the aro-AII and aro-B sites found in previous studies (27). The two primers used in the chromatin immunoprecipitation assay are also indicated in the figure by the arrows.

Figure 2

Identification of ARP-1 as a candidate binding protein of the aro-AII site. (A) The aro-AII-binding protein recognized the sequence “TTGGCCCCT,” which is shown inside the square. The wild-type and mutant oligonucleotide probes, AII, AIIM1, and AIIM3, are shown. Mutations introduced into the aro-AII oligonucleotide are shown by outlined characters. The nucleotide sequence of the ARP-1 binding site in the promoter region of the human and mouse ApoAI gene is also shown in the figure. Tandem repeats of human and mouse ApoAI oligonucleotides are shown in the oligonucleotide by arrows. The box indicates the portion of nucleotides essential for ARP-1 binding as described in our previous report. The putative tandem repeats in the AII probe are shown by broken arrows. (B) A gel shift assay was performed using 5 μg of nuclear protein. A 200-fold molar excess of unlabeled probe was used in the competition assay. The specific signals are indicated by the arrow on left side. The bracket shows the non-specific signals (N.S.). (C) For supershift assays, nuclear protein was incubated with 2 μg of the indicated antibody on ice for 30 min before the addition of a radiolabeled probe. The probe/nuclear protein complex and supershift signals are indicated by the arrow and arrowhead, respectively, on the left side.

Figure 3

ARP-1 protein binds to the aro-AII sequence in vivo. (A) A gel shift assay was performed using 5 μg of nuclear protein or an aliquot of in vitro synthesized ARP-1 protein. For supershift assays, nuclear protein was incubated with 2 μg of the indicated antibody on ice for 30 min before the addition of a radiolabeled probe. The probe/nuclear protein complex and supershift signals are indicated by the arrow and arrowhead, respectively, on the left side. The bracket shows the non-specific signals (N.S.). A 200-fold molar excess of unlabeled probe was used in the competition assay. The probe/ARP-1 complex and supershift signals are indicated by the hatched arrow and arrowhead, respectively, on the right side. (B) A chromatin immunoprecipitation assay confirmed that ARP-1 could associate with the aro-AII site in the fetal mouse brain in vivo. Fresh diencephalic regions of E16 mouse brains were treated with 1% formaldehyde. The fixed tissues were dissolved, and the DNA was sheared and immunoprecipitated with anti-ARP-1 antibody or preimmune IgG. The recovered genomic DNA was subjected to PCR with primers specific for the mouse aromatase gene as shown in Figure 1.

Figure 4

ARP-1 increases brain-specific promoter activity of the mouse aromatase gene. (A) ARP-1 increases the promoter activity in a dose-dependent manner. The reporter plasmid (500 ng), pGL3aroBr, was co-transfected with the indicated amounts of pFLAG-ARP-1 plasmid (50, 100, 250, and 500 ng) and 50 ng of the internal control plasmid into CV-1 cells. The total amount of expression plasmid was adjusted to 500 ng with an empty plasmid (p3XFLAG-myc-CMV-26). The cells were harvested after 48 h, and a Dual-Luciferase Reporter Assay was carried out as described in the Materials and Methods section. (B) Effects of ARP-1 on the activity of aromatase and apolipoprotein AI promoters in HepG2 cells. A Dual-Luciferase Reporter Assay was conducted as described in (A). The mean ± SEM of at least three independent experiments is shown in the figure. One-way ANOVA showed a significantly different distribution (p < 0.0001 for (A,B). The p-value of the Tukey–Kramer test is indicated with the symbols as follows. Asterisks indicate statistically significant differences in relative promoter activity between the empty plasmid alone and that after co-transfection with the ARP-1 expression plasmid (p < 0.05). Pound signs indicate statistically significant differences in comparisons between indicated pairs (p < 0.05).

Figure 5

Effect of ARP-1-targeting siRNA on expression of the endogenous aromatase gene in primary cultured neural cells. (A) Diencephalic neurons were transfected with ARP-1-targeting siRNA and cultured for 48 h, and then the lysates were subjected to western blotting with an anti-ARP-1 antibody and an anti-β-actin antibody (top panel). The amount of ARP-1 protein is quantitated and expressed as the relative ARP-1/β-actin value (lower panel). The mean ± SEM of three independent experiments is shown in the figure. One-way ANOVA showed a significantly different distribution (p < 0.0002). The p-value of the Tukey–Kramer test is indicated with the symbol as follows. Asterisks indicate statistically significant differences in ARP-1 protein levels between the ARP-1 siRNA and negative control siRNA groups (p < 0.05). (B) Effect of ARP-1 knockdown on the aromatase mRNA level in neural cells. Diencephalic neurons were prepared as in (A), and total RNA was extracted from the cultured cells. The total RNA was analyzed by RT-qPCR to determine the amount of aromatase mRNA as described in the Materials and Methods section. The results are presented as the mean ± SEM of three independent experiments. One-way ANOVA showed a significantly different distribution (p < 0.0002). The p-value of the Tukey–Kramer test is indicated with the symbol as follows. Asterisks indicate statistically significant differences in aromatase mRNA levels between the ARP-1 siRNA and the negative control siRNA groups (p < 0.05).