Single-stranded DNA-binding proteins regulate the abundance and function of the LIM-homeodomain transcription factor LHX2 in pituitary cells

Abstract

A family of single-stranded DNA-binding proteins (or SSBPs) has been shown to augment the function of LIM-homeodomain (LIM-HD) transcription factors in embryogenesis by interaction with LIM domain-binding protein-1 (LDB1). No DNA-binding complex has been described, however, containing a LIM-HD protein, LDB1, and SSBP, and the mechanism by which SSBPs affect LIM-HD function had not been elucidated. Through use of electrophoretic mobility shift, antibody supershift, and ChIP analyses, we show that an Lhx2-Ldb1-Ssbp3 complex binds a specific element in the Lhx2 target gene Cga (encoding the alpha subunit of glycoprotein hormones) in the alphaT3-1 pituitary cell line. Using overexpression and knockdown approaches, we demonstrate that SSBP3 inhibits Lhx2 and Ldb1 turnover, stimulates assembly of this DNA-binding complex, promotes its recruitment to the Cga promoter, and enhances Cga transcription. These studies provide novel insights into the regulation of pituitary gene expression and LIM-HD function more generally.

Fig. 1

Ssbp3 is an integral component of a pituitary Lhx2- and Ldb1-containing transcriptional complex. EMSA and supershift analysis of nuclear extracts from non-transduced (A) and SSBP2-transduced αT3-1 cells (B). Ssbp3-, Ldb1-, and Lhx2-containing DNA-binding complex is marked with arrowhead. Complexes supershifted by Lhx2, Ldb1 and Ssbp3 antibodies are marked with open circles. Non-specific complexes are marked with filled circles. (C) ChIP analysis of Ssbp3, Ldb1, and Lhx2 occupancy of the Cga promoter (upper panel) and its 3′ UTR (lower panel). Transient transfection analysis with a Cga promoter-luciferase reporter plasmid in αT3-1 cells transduced with an Ssbp3 or Ssbp2 cDNA or parental vector (D) or with shRNAs to Ssbp3 (Ssbp3 shRNA, left), EGFP (EGFP shRNA, middle) and Ssbp2 (Ssbp2 shRNA, right) (E). (F) Reporter analysis in αT3-1 cells transduced with indicated combinations of pIRES-Ldb1, pIRES-Ldb1(Δ214-223), and pIRES-Ssbp3.

Fig. 2

Ssbp3 regulates assembly of the Lhx2-Ldb1-Ssbp3 complex, its occupancy of the Cga promoter, and Cga gene expression in αT3-1 cells. (A) RT-PCR analysis of S16, Ssbp2, Ssbp3, Ssbp4, and Cga mRNAs in short-term puromycin-selected αT3-1 cells transduced with shRNAs for Ssbp2, Ssbp3, or an irrelevant RNA (EGFP). (B) Western blot analysis of Ssbp3 and Ssbp2 abundance in nuclear extracts from αT3-1 cells transduced with the indicated shRNA. (C) EMSA and supershift analysis of nuclear extracts from αT3-1 cells transduced with the indicated vectors. The Lhx2-Ldb1-Ssbp3 complex is marked with arrowhead and two nonspecific DNA-binding complexes used in normalization of binding activity are marked with open circles. The fold change in DNA-binding activity was determined from densitometry. (D) Quantitative ChIP analysis of factor occupancy on the Cga promoter in short-term puromycin-selected αT3-1 cells transduced with expression vectors for Ssbp3 or EGFP shRNAs.

Fig. 3

Ssbp3 regulates Ldb1 and Lhx2 protein turnover in αT3-1 cells. (A, B) Western blot analysis of Ssbp3, Ssbp2, Ldb1, Lhx2, and Hdac2 abundance in nuclear extracts from transduced αT3-1 cells. (A) Results from cells transduced with full-length SSBP2 or SSBP3 cDNAs. (B) Results from cells transduced with EGFP, Ssbp2, or Ssbp3 shRNAs. (C) Protein turnover analysis in Ssbp3 knockdown and control αT3-1 cells treated with CHX (100 μM) for the indicated times. Lhx2 (upper) and Ldb1 (lower) were detected by Western blot analysis and their levels quantified by densitometry of X-ray films. (D) Percent Ldb1 and Lhx2 remaining is shown as a function of time following CHX addition. (E) Results of Western blot analysis of Ldb1, Ssbp3, and α-actin abundance in vector- and Ssbp3-transfected cells treated with 1 μm MG132 or vehicle for 6 h.

Fig. 4

Ssbp3 does not regulate Lhx2 DNA-binding affinity. (A) The affinity of the Lhx2-Ldb1-Ssbp3 complex for DNA was assessed in pIRES-SSBP3- and vector-transduced αT3-1 cells by EMSA using increasing ratios of unlabeled to labeled PGBE probe. The Lhx2-containing complex is marked with arrowhead. Non-specific complexes are marked with filled circles. (B) Quantification of Lhx2 DNA-binding data from (A). Open circles denote SSBP3-overexpressing αT3-1 cells and filled triangles vector-transduced cells.