Novel forms of Paired-like homeodomain transcription factor 2 (PITX2): generation by alternative translation initiation and mRNA splicing

Abstract

Background: Members of the Paired-like homeodomain transcription factor (PITX) gene family, particularly PITX1 and PITX2, play important roles in normal development and in differentiated cell functions. Three major isoforms of PITX2 were previously reported to be produced through both alternative mRNA splicing (PITX2A and PITX2B) and alternative promoter usage (PITX2C). The proteins derived from these mRNAs contain identical homeodomain and carboxyl termini. Differences in the amino-termini of the proteins may confer functional differences in some contexts.

Results: Here, we report the identification of two novel PITX2 isoforms. First, we demonstrate that the Pitx2c mRNA generates two protein products, PITX2Calpha and PITX2Cbeta, via alternative translation initiation. Second, we identified a novel mRNA splice variant, Pitx2b2, which uses the same 5' splice donor in intron 2 as Pitx2b (hereafter referred to as Pitx2b1), but employs an alternative 3' splice acceptor, leading to an in-frame deletion of 39 base pairs relative to Pitx2b1. Pitx2b2 mRNA is expressed in both murine and human pituitary. The data show that in a murine gonadotrope cell line and adult murine pituitary what was previously thought to be PITX2B1 is actually PITX2Cbeta, or perhaps PITX2B2. PITX2B1 is expressed at lower levels than previously thought. PITX2Cbeta and PITX2B2 activate gonadotrope-specific gene promoter-reporters similarly to known PITX2 isoforms.

Conclusion: We have identified and characterized two novel isoforms of PITX2, generated by alternative translation initiation (PITX2Cbeta) and alternative mRNA splicing (PITX2B2). These proteins show similar DNA binding and trans-activation functions as other PITX2 isoforms in vitro, though their conservation across species suggests that they may play distinct, as yet unidentified, roles in vivo.

Figure 1

Schematic representation of the major isoforms of PITX2 in mouse and human. The PITX2 gene consists of 6 exons, which are depicted as boxes. The introns and flanking sequences are represented as lines connecting the boxes. The unfilled and filled boxes represent the non-coding and coding sequences, respectively. The homeodomain is indicated as a black region spanning exons 5 and 6. The solid line joins the exons present in PITX2A (top) and PITX2B (bottom) mRNAs; the broken line connects the exons present in the PITX2C mRNA. PITX2D is not pictured.

Figure 2

Alternative translation initiation sites are present in PITX2C mRNA. A) Comparison of the nucleotides flanking the start codon (Met1) and two other in-frame candidate start codons (Met4 and Met35) in the Pitx2c/PITX2C mRNAs from various mammalian species. The Kozak consensus sequence is shown at the top to facilitate the comparison. [GenBank accession nos. for the human: NM_000325, mouse: NM_001042502, rat: AJ222971, cow: NM_001097991, and dog: XM_846277]. B) Schematic representation of the expression constructs designed to study the two PITX2C isoforms. The large and small arrows represent strong and weak AUG^START codons in each cDNA sequence. C) Immunoblot analysis showing the PITX2C protein isoforms translated from the expression constructs in panel B when transfected into CHO cells. Lysates were run on a 10% Nupage^® Novex Bis-Tris gel with MOPS buffer.

Figure 3

A Novel PITX2 mRNA splice variant. A) Full-length Pitx2a, Pitx2b1, and Pitx2b2 cDNAs amplified by RT-PCR from LβT2 mRNA. B) Schematic representation of the splicing events giving rise to Pitx2b1 (top) or Pitx2b2 (bottom). The 3' end of exon 2 (donor) and 5' end of exon 3 are indicated in white type with black shading. Upper and lower case letters are used for exon and intron nucleotides, respectively. The components of the lariat for the 3' alternative acceptor site are shown, with a branch point (circled, with invariant A shown as bold and italicized), followed by a poly-pyrimidine tract (underlined) and a terminal AG (boxed) at the extreme 3' end. C) RT-PCR analysis of Pitx2a/PITX2A, Pitx2b1/PITX2B1 and Pitx2b2/PITX2B2 mRNAs in LβT2 cells, and murine and human pituitary. D) Immunoblot analysis of PITX2 isoforms expressed in LβT2 cells (left) and combined nuclear extracts from CHO cells (right) transfected with different PITX2 isoforms run on 13.5% Tis-glycine SDS-PAGE gels. A, PITX2A; B1, PITX2B1; B2, PITX2B2; Cα, PITX2Cα; Cβ, PITX2Cβ.

Figure 4

The major PITX2 isoforms in gonadotrope cells, pituitary and skeletal muscle. A) Immunoblot analysis of PITX2 protein expression in CHO cells transfected with different PITX2 isoforms, LβT2 cells, and murine pituitary run on a 12% Nupage^® Novex Bis-Tris gel with MOPS buffer. Abbreviations: pcDNA3.0, control; A, PITX2A; B1, PITX2B1; C, PITX2C; Cα, PITX2Cα; Cβ, PITX2Cβ; A+C, PITX2A and PITX2C; nuc, nuclear extract; WCE, whole cell extract. B) PITX2 immunoblot of murine skeletal muscle (Sk. mus.) WCE (lane 1), and LβT2 and αT3-1 nuclear extracts (lane 2 and 3) run on a Nupage^® Novex 7% Tris-acetate gel. C) RT-PCR analysis of Pitx2a, Pitx2b1 and Pitx2b2 (top) and Pitx2c (middle) mRNA in LβT2 cells and murine skeletal muscle (hindlimb). Rpl19 (bottom) was used as a loading control.

Figure 5

PITX2A, PITX2B1 and PITX2B2 isoforms trans-activate gonadotrope-specific genes. A, C and D) CV-1 cells seeded in 12-well plates were co-transfected with the indicated promoter-reporters (0.9 μg/well) along with empty (pcDNA3.0) or PITX2 expression vectors (0.3 μg/well). B) The Lhb promoter-reporter (0.9 μg/well) was co-transfected with PITX2 expression vectors plus or minus SF-1 (both expression vectors at 0.3 μg/well; DNA in all wells was balanced with pcDNA3.0). After approximately 30 h, lysates were collected for luciferase assays. Data were derived from three to five experiments performed in triplicate. Bars with different symbols differ significantly. E) Immunoblot showing relative PITX2A, B1 and B2 expression in CV-1 cells, whole cell extracts run on a 4–12% Nupage^® Novex Bis-Tris gel with MOPS buffer.

Figure 6

Trans-activation of gonadotrope-specific genes by PITX2C isoforms. CV-1 cells were transfected as in Figure 5, except with PITX2C expression vectors [PITX2C, expressing both forms; PITX2Cα (M35L/T-3A); PITX2Cβ (L5stop)]. Immunoblot in panel E shows relative PITX2Cα and β expression in the transfected CV-1 cells (gel conditions as in Fig. 5E).

Figure 7

All PITX2 isoforms bind the Fshb promoter. A) Immunoblot showing PITX2 isoforms over-expressed in CHO cell extracts run on 10% Nupage^® Novex Bis-Tris gel with MOPS buffer. B) Gel shift assay performed with a radio-labeled probe corresponding to -61/-40 of the murine Fshb promoter with the same nuclear extracts used in panel A. The free probe is not pictured and lanes are numbered at the bottom.