The Role Of Dlx3 In Gene Regulation In The Mouse Placenta
Distal-less 3 (Dlx3) is a homeodomain containing transcription factor that is required for the normal development of the mouse placenta. Moreover in human trophoblasts, DLX3 appears to be a necessary transcriptional regulator of the glycoprotein hormone [alpha] subunit gene, a protein subunit of placental-derived chorionic gonadotropin. The aim of my studies described here was to determine the role of Dlx3 in gene regulation within the placenta. My studies initially sought to determine if Dlx3 could interact physically with other transcriptional regulators in placental trophoblast cells and how these proteinprotein interactions might influence Dlx3-dependent gene expression. Yeast twohybrid screens provide evidence that mothers against decapentaplegic homolog 6 (SMAD6) was a binding partner for DLX3. SMAD6 was found to be expressed and nuclear localized in placental trophoblasts and interacted directly with DLX3. Structure-function analysis revealed that this interaction occurred within a DLX3 domain that overlapped the homeobox, a key domain necessary for DLX3 DNA binding. SMAD6 was found to repress DLX3 transcriptional activity through inhibition of DLX3-DNA interactions at target genes. To develop a more comprehensive understanding of the gene profile regulated by Dlx3, microarray studies were used to determine differences in gene expression within the placenta of wild type and Dlx3 null mice at embryonic day 9.5. Loss of Dlx3 resulted in a marked reduction in gene expression of placental growth factor (Pgf). To investigate the mechanism(s) of regulation of Pgf gene expression, 5.2 kb of the mouse Pgf gene promoter was cloned and used in reporter gene assays. Overexpression of Dlx3 increased the Pgf promoter activity in choriocarcinoma cells, suggesting that Dlx3 may have a direct effect on transcriptional activation of the Pgf gene promoter. To determine the functional importance of Dlx3 in the regulation of Pgf gene transcription, two Dlx3 binding sites in the mouse Pgf 5.2 kb promoter were identified and mutated by site-directed mutagenesis. The mutations effectively abolished the ability of Dlx3 to bind these sites. Pgf promoter activity was found to be reduced by the combined mutation of both Dlx3 binding sites. Thus, Dlx3 appears to be an important determinant for Pgf gene promoter activity and ultimately Pgf secretion in placental trophoblast cells. Since PGF is an important marker for risk of preeclampsia in women, studies in a mouse model of preeclampsia were undertaken to determine if Dlx3 and Pgf were mis-regulated during gestation in this model. Indeed, both Dlx3 and Pgf transcript levels were greatly reduced in early gestation in BPH/5 mice suggesting that Dlx3 and Dlx3 target genes may underlie genetic mechanisms associated with preeclampsia prior to the onset of maternal disease. To determine if similar mis-regulation of the DLX3 gene network might occur in placentas from women with preeclampsia, we next examined DLX3 and PGF transcript abundance in a preliminary cohort of patients. This preliminary study supported our findings in the BPH/5 mouse model. Together, these data suggest that Dlx3 may be an important factor during the development of mouse placenta by regulating genes such as Pgf. Further, misregulation of this gene network may direct early changes in placental function leading to symptoms characteristic of preeclampsia.
dissertation or thesis