Paired-Like Homeodomain Transcription Factors 1 and 2 Regulate Follicle-Stimulating Hormone β-Subunit Transcription through a Conserved cis-Element

Abstract

Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone-encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine FSH beta-subunit (Fshb) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LbetaT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human Fshb/FSHB promoter-reporter activities, and in both cases, mutation of the critical cis-element abrogated these effects. In homologous LbetaT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C or knocking down PITX1 or -2 expression by RNA interference in LbetaT2 cells inhibited murine Fshb transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in coprecipitation experiments, and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated Fshb transcription. In summary, both PITX1 and PITX2C regulate murine and human Fshb/FSHB transcription through a conserved cis-element in the proximal promoter. Furthermore, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.

Pitx3 directly regulates Foxe3 during early lens development

“…No conserved bicoid site for putative Pitx3 binding was observed in the upstream region of the Tube1 gene. Nevertheless, we considered two bicoid-like motifs (TAAGCC), known to bind Pitx family members in vitro albeit showing less affinity than consensus bicoid sites (Lamba et al, 2008); starting -1,293 bp and -1,856 bp upstream (E10.5, E11.5, E12.5, and E14.5) (-3,377 / -3,415) containing the bicoid site TAATCC. The specificity of this binding was confirmed by a supershift assay using an anti-Pitx3 antibody and competition assay with unlabelled DNA containing the same bicoid site (Fig.…”

Conservation of mechanisms mediating gonadotrophin-releasing hormone 1 stimulation of human luteinizing hormone β subunit transcription

“…Murine PITX1, Flag-PITX1, myc-PITX1 and PITX2 expression vector were described earlier (Lamba et al, 2008a, b). To make Flag-tagged EGR1 and SF1, the parental constructs were sub-cloned using strategies described earlier for Flag-PITX1 (Lamba et al, 2008b). Constitutively active MKK6 was a gift from Dr David Engelberg (Hebrew University, Jerusalem, Israel), and Raf-CAAX was from Dr Linda Van Aelst (Cold Spring Harbor Lab).…”

“…CHO and CV1 cells were provided by Dr Patricia Morris (Population Council, New York, NY). All cells were cultured and transfected as described previously (Lamba et al, 2008b;Wang et al, 2008). Briefly, LbT2 cells were transfected overnight with 450 ng reporter/well and the indicated amounts of plasmid DNA or siRNA.…”

“…Following the addition of 0.05 pmol of 32 P-labeled double-stranded probe and incubation for 15 min at room temperature, protein:DNA complexes were resolved on 5% native polyacrylamide gels at 48C. DNAP assays using streptavidin-coupled Dynabeadsw M-280 (Dynal, Invitrogen) were performed as previously described (Lamba et al, 2006(Lamba et al, , 2008b using the biotinylated probes. Following elution from the beads, proteins were resolved on 10% SDS -PAGE gels as described previously (Bernard, 2004).…”

Pin1 Facilitates the Phosphorylation-Dependent Ubiquitination of SF-1 To Regulate Gonadotropin β-Subunit Gene Transcription

“…Also crucial in LH␤ gene expression is the early growth factor 1 (Egr-1), whose expression and phosphorylation are induced by GnRH, allowing its synergistic interaction with SF-1 and Pitx1 and mediation of the GnRH effect (10,15,19,21,39,59). The molecular mechanisms of the basal and GnRH-stimulated effects on the FSH␤ gene promoter have been less well elucidated, but they appear to involve SF-1 and various Pitx proteins, including Pitx1, as well as the activator protein-1 (AP-1) transcriptional complex, comprising c-Jun and c-Fos, both of which are transcriptionally regulated and phosphorylated by GnRH (7,23,37,39,62,70). SF-1 and Pitx1 act synergistically on both the LH␤ and Mullerian-inhibiting-substance gene promoters, and this was shown to be a result of Pitx1 binding directly to SF-1.…”