Regulation of LH-Beta Transcription by WT1 and DAX1 Proteins in the Pituitary Gonadotrope

Luteinizing hormone (LH) is secreted throughout the reproductive cycle from the gonadotrope cells of the anterior pituitary, and is required for steroidogenesis and ovulation. LH contains an α-subunit common with FSH, and a unique LHβ subunit that defines biological activity. Basal LHβ transcription is low and stimulated by the hypothalamic hormone GnRH. GnRH induces synthesis of Egr1 (early growth response protein-1) and stimulates the cyclic binding of transcription factors Egr1 and SF1 (steroidogenic factor-1) on the LHβ promoter. By blocking proteasomal degradation using the inhibitor MG132, our lab previously demonstrated that proteasomal inhibition hampered the cyclic binding of Egr1 and SF1 on the LHβ promoter, and we hypothesized that there could be a DNA-bound or transcription factor-bound inhibitory protein that hindered this cyclic association. These inhibitory proteins might require removal by proteasomal degradation to recruit transcriptional activators. Our candidate for the DNA binding repressor protein was WT1 (Wilms tumour1) and a potential candidate for the transcription factor-bound inhibitory protein was DAX1. WT1 (Wilms tumor protein1) is a zinc finger transcription factor with an essential role in urogenital system development. It regulates several reproductive genes via interactions with SF1 or binding to GC-rich elements such as Egr1 binding sites. DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1), regulates several reproductive and steroidogenic genes by interacting with SF1 and has been suggested to have both co-activator and co-repressor properties. In this thesis we investigated the potential roles for WT1 and DAX1 in LHβ transcription using clonal mouse gonadotrope LβT2 cells. We demonstrate the presence of WT1 in LβT2 and mouse pituitary cells, and that the protein bound to the endogenous LHβ promoter. The mRNAs for WT1(+KTS), which contains a three amino-acid insertion between the 3rd and 4th zinc-fingers, and the WT1 (-KTS) variant were both expressed at significant levels. WT1 mRNAs and protein were decreased approximately 50% by GnRH treatment, under conditions where Egr1 mRNA and protein, and LHβ transcription, were stimulated. Decreasing expression of mRNA for WT1(-KTS) decreased stimulation of LHβ and Egr1 by GnRH, whereas decreasing both WT1(-KTS) and (+KTS) increased endogenous LHβ transcription, and prevented LHβ but not Egr1 stimulation by GnRH, suggesting differing biological activities for the WT1 isoforms. Overexpression of WT1 showed that WT1(-KTS) enhanced LHβ promoter GnRH stimulation 2-to-3-fold and required only the 3’Egr1 site. WT1(+KTS) repressed both basal and GnRH-stimulated LHβ promoter activity by approximately 70%, and required both Egr1 and SF1 sites. Our data suggest that WT1 can modulate LHβ transcription, with differential roles for the two WT1 variants; WT1 (-KTS) enhances and WT1 (+KTS) suppresses transcription.

We investigated the role of DAX1 in LHβ transcription and the effect of proteasomal degradation in the cyclic binding of the co-regulatory proteins on the LHβ promoter. Chromatin immunoprecipiation showed that GnRH stimulates binding of DAX1 to the LHβ promoter, suggesting it acts on the endogenous gene. Inhibition of proteasomal activity prevents association of the SF1 co-activators SRC1 and GCN5, and regulatory proteins WT1 and DAX1, but sustained binding of the co-repressor SMRT to the LHβ promoter. DAX1 overexpression increased GnRH-stimulated LHβ promoter activity in a dose dependent manner. Decreasing endogenous DAX1 levels by siRNA decreased LHβ mRNA primary transcripts. Thus, DAX1 appears to be an endogenous activator of LHβ expression. To better understand DAX1 functions, we used full length and truncated forms of the LHβ promoter and found that the proximal GnRH response region is sufficient for DAX1 activity. We found that mutation of either SF1 site in the proximal region eliminated DAX1 enhancement of GnRH-stimulated promoter activity. However, subsequent experiments using siRNA against SF1 and measurement of DAX1 association with the LHβ promoter, or DAX stimulation of promoter activity, suggests that DAX1 can still bind and stimulate the LHβ promoter in the absence of SF1.

Overall our data shows that WT1 (+KTS) could serve as the DNA-bound inhibitory protein as per our hypothesis, and that proteasome activity regulates cyclic binding of coregulators to the LHβ promoter, suggesting exchange of negative and positive regulators is required for cyclic transcription factor association and transcription. In the process, we also found that DAX1 is a dose dependent, positive or negative co-regulator of GnRH stimulated LHβ transcription, and that the WT1 (–KTS) stimulates LHβ transcription.