Paired-like homeodomain transcription factors 1 and 2 regulate follicle-stimulating hormone beta-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.

Figure 1

PITX proteins regulate basal murine and human Fshb/FSHB gene transcription through a PITX/bicoid-like binding element in the proximal promoter. A, Western blots for endogenous PITX1 and PITX2 in whole-cell lysates from heterologous HepG2 cells and homologous LβT2 cells. B, HepG2 cells seeded in 24-well plates were transfected with the indicated murine Fshb-luc reporters along with empty, PITX1, or PITX2C expression vectors. Data are presented as fold change in reporter activity in the presence of PITX expression vector relative to empty vector transfected cells with the same reporter. Data represent the mean (±sem) of three experiments performed in triplicate. * and #, Statistically significant differences from the empty pGL3-Basic vector for PITX1 and PITX2C, respectively (Scheffé post hoc test of significant interaction). C, Alignment of proximal Fshb/FSHB promoters in several species. Bases that differ from the mouse are shaded, and gaps are shown by a colon (:). The conserved cis-element, AAATCC, is boxed. D, HepG2 cells were cotransfected with WT or PBE mutant forms of −126/+7 hFSHB-luc along with PITX1 or PITX2C. Data from five replicate experiments are presented as in B. Lowercase (PITX1) and uppercase (PITX2C) letters indicate significant differences from empty vector. Bars with different letters also differed from one another.

Figure 2

PITX proteins bind to a proximal cis-element. A, Gel shift analyses were performed with a radiolabeled probe corresponding to −61/−40 of the murine Fshb promoter. The probe was incubated with nuclear extracts from CHO cells transfected with empty (pcDNA3.0) (lanes 1 and 2), PITX1 (lanes 3–8), or PITX2C (lanes 9–17) expression vectors. Complexes associated with PITX1 and PITX2C proteins are labeled on the left (b, c, g, and f) and right (a and e) sides, respectively. Complex d corresponds to an unknown endogenous CHO cell protein complex that specifically interacts with the probe. Lanes 15–17 were run on a separate gel and the gel exposed to film for a longer time to more clearly show that the PITX2 antibody (which is of low titer) could supershift PITX2C bound to the probe. Free (unbound) probe is not pictured, and the lanes are numbered at the bottom. B, CHO cells were cotransfected with Flag-PITX1 and myc-PITX1 alone or in combination. Whole-cell lysates were immunoprecipitated (IP) using anti-Flag M2 affinity gel. Bound proteins were eluted, separated by SDS-PAGE, and immunoblotted with the indicated antibodies. C, Southwestern blot (SWB) analysis performed with CHO cell nuclear extracts used in A: pcDNA3.0 (lane 1), PITX1 (lane 2), and PITX2C (lane 3). The top two panels represent immunoblots (IB) with the indicated antibodies. The bottom two panels are autoradiograms from blots probed with radiolabeled probes corresponding to the indicated base pairs in the murine Fshb promoter. D, DNA precipitation assay with WT −61/−40 (WT) and −61/−40 PBE mutant (Mut) probes. Immunoblots were performed with the indicated antibodies on LβT2 whole-cell extract (WCE) (lane 1), supernatants (Sup.) from extracts incubated with WT and Mut probes bound to magnetic beads (lanes 2 and 3), and proteins eluted from the beads (lanes 4 and 5, Precip.). E, ChIP was performed using LβT2 cells. After cross-linking, IP was performed by incubating with no antibody, IgG, or antibodies against PITX1 or acetylated histone H3 (Lys 9). Input and precipitated DNA were analyzed by PCR using the primers for murine Fshb promoter regions indicated on the right side of the panels. Note that the PCR products in the upper and lower panels were run on separate agarose gels with different sized combs. F, ChIP was performed using LβT2 cells transfected with −1990/+1 mFshb-luc WT or PBEmut together with Flag-PITX1 expression vector. IP was performed by incubating fractions of the same samples with IgG or anti-Flag antibodies. Precipitated DNAs in each condition were quantified using the real-time PCR with primers in the promoter (−119/−98 forward) and the vector (pGL3 reverse). Data are presented as a percentage of binding observed with the WT promoter set to 100%.

Figure 3

A single PITX binding element exists within −61/−40 of the Fshb promoter. A, Sequences of the probes used in the gel shift analysis in B; − indicates no change in sequence from the WT probe. B, Gel shift assay with radiolabeled −61/−40 probe and nuclear extracts from CHO cells overexpressing PITX1. Complexes were competed with the indicated unlabeled probes (500-fold molar excess). Free probe is not pictured.

Figure 4

PITX proteins do not bind to the putative PITX2C-binding region. Gel shift analyses were performed with a radiolabeled probe corresponding to −232/−200 of the murine Fshb promoter. The probe was incubated with CHO cell nuclear extracts as in Fig. 2. A 500-fold molar excess of unlabeled −232/−200 (lanes 6 and 12), −61/−40 (lanes 4 and 10), and −61/−40 PBE mutant (Mut) (lanes 5 and 11) murine Fshb was used as competitor.

Figure 5

The proximal PBE is required for activin A-stimulated transcription. A, LβT2 cells seeded in 24-well plates were transfected with −1990/+1 mFshb-luc (WT) or a similar construct containing PBEmut. The next day, cells were treated with 1 nm activin A or control medium and lysates collected 24 h later. Data from three experiments are presented as fold change in the reporter activity, normalized relative to the untreated WT reporter set to 1. B, As in A, LβT2 cells were transfected with −1028/+7 hFSHB-luc (WT), with the −1028/+7 reporter containing PBEmut, or with empty pGL3-Basic and treated with activin A. Data are derived from three experiments performed in triplicate. Statistical differences are indicated with different letters (Tukey test of significant interaction).

Figure 6

Dominant-negative PITX proteins and Pitx siRNAs inhibit Fshb transcription. A, LβT2 cells were transfected with −1990/+1 mFshb-luc along with 200 ng/well of PITX2C WT or K50 mutants (either K141A or K141E). The following day, cells were treated with 1 nm activin A for 24 h. Data were normalized to untreated, pcDNA3.0-transfected control cells. Data from three experiments are presented. Bars with different letters differ significantly from each other (Bonferroni). B, LβT2 cells were transfected as in A except with WT and K139A forms of PITX1. C, LβT2 cells were transfected with a murine Fshb reporter and the indicated concentrations of control (Ctrl), Pitx1, or Pitx2 siRNAs either alone (10 nm) or together (5 nm each) and treated the next day with 1 nm activin A for 24 h. Data are presented as fold change in the reporter activity relative to the untreated, 10 nm Ctrl siRNA condition. Bars represent mean (±sem) data derived from four experiments performed in duplicate. Bars with different letters differed significantly (Bonferroni of significant siRNA main effect; note that the ligand × siRNA interaction was not significant). D, LβT2 cells were transfected with a murine Fshb-luc construct with PBEmut along with 10 nm of the indicated siRNAs and treated with 1 nm activin A for 24 h. Data are derived from three experiments and were analyzed as in C.

Figure 7

Physical and functional interaction between PITX and Smad proteins. A and B, CHO cells were cotransfected with PITX1 (A) or PITX2C (B) alone or in combination with Flag-Smad3 and were treated with activin A or control media for 1 h. Whole-cell lysates were immunoprecipitated (IP) using anti-Flag M2 affinity gel. Bound proteins were eluted and separated by SDS-PAGE and immunoblotted with the indicated antibodies. C, LβT2 cells were seeded in six-well plates and transfected with −1990/+1 mFshb-luc WT (WT) or PBEmut along with expression vectors for Smad2, -3, and -4 (black bars) or empty vector (white bars). The data are from three experiments. *, Smad2/3/4 stimulation of the WT promoter differed from all other groups, which did not differ from one another (Bonferroni).