GATA4 Autoregulates Its Own Expression in Gonadal Cells via Its Distal 1b Promoter.

GATA4 is an essential transcription factor required for the development and function of multiple tissues derived from both mesoderm and endoderm. Among these tissues, GATA4 is highly expressed in the somatic cell lineages of the gonads in both sexes throughout development. Various knockout models in mice have demonstrated that GATA4 is essential for the onset of normal gonadal morphogenesis and is later recruited as a key regulator of numerous genes (e.g., Star, Cyp19a1, Inha) in response to hormonal cell signaling. Very recent evidence in the literature also supports a role for GATA4 in human gonadal development, at least in the male. In spite of its essential biological functions, the factors and mechanisms that control GATA4 expression have yet to be fully understood. We have shown that humans and rodents express the GATA4 gene as multiple transcript variants that differ solely in their untranslated first exons. The most frequently used first exons are exon 1a, which is closest to the ATG and driven by the proximal 1a promoter, and exon 1b whose usage is controlled by an alternative distal promoter (named 1b) that is approximately 30 kb further upstream. Since all GATA4 transcript variants produce the same GATA4 protein, our hypothesis is that differential promoter usage is an important mechanism for regulating spatiotemporal GATA4 expression in the gonads and possibly other tissues. Our previous work demonstrated that a conserved E-box motif in the proximal 1a promoter is essential for its activity both in vitro and in vivo. Using in situ hybridization, we now show that the Gata4 1b transcripts are also present in the somatic cell lineages of the developing testis and ovary. We also show, using quantitative real-time PCR (qPCR) that the Gata4 1b transcript is present in several gonadal cell lines (MSC-1, TM3, TM4, MA-10, DC3), albeit less abundant than the Gata4 1a transcript. Consistent with this observation, transient transfection studies revealed that the Gata4 1b regulatory region was less active than the Gata4 1a promoter in all cell lines tested. Progressive 5' deletion analysis of the Gata4 1b promoter showed that regulatory elements required for its activity are located in the first 171 bp. This region contains two conserved binding motifs for GATA family members (conserved at least in mouse, rat and human). Both motifs actively bind GATA4 protein as revealed by electrophoretic mobility shift assay (EMSA). The -171 bp Gata4 1b promoter was stimulated 3 to 7-fold by GATA4 in homologous and heterologous cells, respectively. Site-directed mutagenesis of both GATA motifs abolished GATA4-dependent activation of the 1b promoter. Finally, knockdown of GATA4 expression in MA-10 cells by siRNA also led to a marked drop in Gata4 1b promoter activity. Therefore, unlike the Gata4 1a promoter, which is regulated by ubiquitous factors binding to an E-box element, our present data suggest that GATA4 can also autoregulate its expression at the level of its distal 1b promoter. This represents a potentially key mechanism for regulating GATA4 expression in different cell types and at different timepoints during development. This research was supported by CIHR grant MOP-14796 to RSV. (poster)

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