Regulation of monoamine oxidase A by the SRY gene on the Y chromosome

Abstract

Monoamine oxidase A (MAO A), encoded by the X chromosome, catalyzes the oxidative deamination of monoamine neurotransmitters, such as serotonin, and plays a critically important role in brain development and functions. Abnormal MAO A activity has been implicated in several neuropsychiatric disorders, such as depression, autism, and attention deficit hyperactivity disorder, which show sexual dimorphism. However, the molecular basis for these disease processes is unclear. Recently, we found that MAO A was a putative target gene directly regulated by a transcription factor encoded by the sex-determining region Y (SRY) gene located on the Y chromosome. We demonstrated that SRY activates both MAO A-promoter and catalytic activities in a human male neuroblastoma BE(2)C cell line. A functional SRY-binding site in the MAO A core promoter was identified and validated by electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses. Coimmunoprecipitation and ChIP assays showed that SRY and Sp1 form a transcriptional complex and synergistically activate MAO A transcription. This is the first study demonstrating that the Y-encoded transcription factor SRY is capable of regulating an X-located gene, suggesting a novel molecular mechanism for sexual dimorphism in neural development, brain functions, and initiation/progression of neural disorders associated with MAO A dysfunction.

Figure 1.

SRY activates both MAO A-promoter and catalytic activities in BE(2)C cells. A) Effect of SRY overexpression on MAO A mRNA expression. MAO A mRNA levels were determined by quantitative real-time PCR in BE(2)C cells overexpressing SRY. In control (ctrl) cells, pCMV vector carrying neomycin-resistant gene was stably transfected. GAPDH was used as an internal control. Data were analyzed by 2^−ΔΔCT method. MAO A mRNA expression level in control cells was arbitrarily set as 1. B) Effect of SRY overexpression on MAO A catalytic activity. MAO A catalytic activity was determined by MAO A catalytic activity assay in BE(2)C cells overexpressing SRY. MAO A catalytic activity in control cells was set as 100%. C) Knockdown of endogenous SRY in BE(2)C cells. SRY siRNA was transfected into BE(2)C cells; 48 h later, cells were harvested and analyzed by Western blot using anti-SRY antibody. β-Actin was used as loading control. Nonsense (NS) siRNA was transfected similarly in control cells. D) Effect of SRY knockdown on MAO A catalytic activity. BE(2)C cells were transfected with SRY siRNA; 48–72 h later, cells were harvested and analyzed by MAO A catalytic activity assay. MAO A catalytic activity in control cells was set as 100%. E) Effect of SRY on MAO A-promoter activity. MAO A 2-kb-luc was cotransfected with various amounts of FLAG-SRY expression construct into BE(2)C cells, 24–48 h later, cells were harvested, and the luciferase activity was determined. Activity of MAO A 2-kb-luc (expressing firefly luciferase) was normalized with cotransfected pRL-TK (expressing Renilla luciferase). pcDNA was added to keep the DNA amount of each transfection constant. Activity of MAO A 2-kb-luc without transfection of SRY was set as 1. F) Analysis of transfection efficiency of FLAG-SRY. Western blot was performed to document the FLAG-SRY expression in BE(2)C cells using anti-FLAG antibody. β-Actin was used as the loading control. G) MAO A 2- or 0.24-kb-luc was cotransfected with SRY siRNA into BE(2)C cells; 48 h later, cells were harvested, and the luciferase activity was determined. Activity of MAO A 2- or 0.24-kb-luc without transfection of SRY siRNA was set as 100%. All data are presented as the means ± sd from 3 independent experiments with triplicates for each experiment. Representative gels are shown. *P < 0.05, **P < 0.01.

Figure 2.

SRY binds to a functional SRY-binding site in MAO A core promoter both in vitro and in vivo. A) Serial deletion analysis of MAO A-promoter activation by SRY. Various deletions of MAO A-promoter-luc were cotransfected with SRY into BE(2)C cells; 24 to 48 h later, cells were harvested, and the luciferase activity was determined. Promoterless pGL2-basic vector was used as control. Fold of activation of MAO A-promoter-luc by SRY is indicated. Activity of MAO A 2-kb-luc without transfection of SRY was set as 1. B) Demonstration of SRY binding to MAO A 0.24-kb core promoter in vivo. Schematic representation of MAO A 2-kb (−2072/−64) and 0.24-kb (−303/−64) promoter structure is not proportional to the real promoter length. A in the start codon was set as +1. BE(2)C cells were transiently transfected with FLAG-SRY; 48 h later, cells were subjected to ChIP assay using anti-FLAG antibody and PCR with primers specific for MAO A core-promoter region (−360/−17). IgG was used as a negative control for IP. BE(2)C genomic DNA (gDNA) and ddH₂O were used as positive and negative controls, respectively, for PCR. C) Sequence of canonical SRY-binding site (top panel), a potential SRY-binding site (−117/−111) in MAO A 0.24-kb promoter (middle panel), and the introduced point mutations (italic) used to inactivate the potential SRY-binding site (bottom panel). D) Effect of SRY on wild-type (wt) and mutated (mut) MAO A 0.24-kb-promoter-luc construct in BE(2)C cells. Fold of activation of wt MAO A 0.24-kb-luc by SRY was set as 100%. E) EMSA of in vitro translated SRY protein with MAO A 0.24-kb-promoter-derived, ³²P-labeled oligonucleotide containing wt SRY-binding site. In vitro translation product using pcDNA vector as a template was used as mock. All data are presented as means ± sd from 3 independent experiments with triplicates for each experiment. Representative gels are shown. *P < 0.05.

Figure 3.

Sp1 synergistically enhances, but is not essential, for the SRY activation of MAO A promoter. A) Effect of Sp1 on MAO A-promoter activation by SRY. MAO A 0.24-kb-luc was cotransfected with various amounts of HA-Sp1 in the presence or absence of SRY into BE(2)C cells; 24 to 48 h later, cells were assayed for luciferase activity. Activity of MAO A 0.24-kb-luc transfected alone was set as 1. B) Summary of increases of MAO A-promoter activity obtained in A. C) Western blot analysis of transfected cells correlating the transfection efficiency of HA-Sp1 used in A. D) Knockdown of endogenous Sp1 in BE(2)C cells by siRNA interference technology. E) Effect of Sp1 knockdown on the SRY activation of MAO A promoter. MAO A 0.24-kb-luc was cotransfected with SRY together with or without Sp1 siRNA into BE(2)C cells; 48 h later, cells were assayed for luciferase activity. Activity of MAO A 0.24-luc transfected alone was set as 1. All data are presented as means ± sd from 3 independent experiments with triplicates for each experiment. *P < 0.05, **P < 0.01.

Figure 4.

Sp1 enhances SRY binding to MAO A promoter by interacting and forming a complex with SRY at the MAO A core promoter in vivo. A) Coimmunoprecipitation and Western blot analysis of interaction between SRY and Sp1 in BE(2)C cells. Rabbit polyclonal anti-Sp1 antibody was used for IP, and mouse monoclonal anti-SRY antibody was used for Western blot. IgG was used as a negative control for IP. 10% input was loaded as control. B) ChIP/re-ChIP analysis demonstrating the simultaneous presence of Sp1 and SRY in natural MAO A core promoter. BE(2)C cells were transiently transfected with FLAG-SRY; 48 h later, cells were subjected to ChIP assay using anti-FLAG antibody. Anti-FLAG immunoprecipitates were subjected to re-ChIP assay using anti-Sp1 antibody. IgG was used as a negative control for IP. PCR was performed using primers targeting MAO A core promoter region. ddH₂O was used as a negative control (NTC) for PCR. C) Overexpression of Sp1 enhanced SRY binding to MAO A core promoter in vivo. BE(2)C cells were transiently transfected with FLAG-SRY together with or without Sp1; 48 h later, cells were subjected to ChIP assay using anti-FLAG antibody. PCR was performed using primers targeting MAO A core-promoter region. Intensity of DNA bands was quantified by Labworks analysis software (UVP). Representative gels are shown.

Figure 5.

Schematic diagram of MAO A transcriptional activation cooperatively regulated by SRY and Sp1.