Molecular cloning and functional characterization of two forms of Pax8 in the rainbow trout, Oncorhynchus mykiss

Gen Comp Endocrinol. 2014 Mar 1;198:22-31. doi: 10.1016/j.ygcen.2013.12.009. Epub 2013 Dec 28.

Abstract

We have identified two distinct Pax8 (a and b) mRNAs from the thyroid gland of the rainbow trout (Oncorhynchus mykiss), which seemed to be generated by alternative splicing. Both Pax8a and Pax8b proteins were predicted to possess the paired domain, octapeptide, and partial homeodomain, while Pax8b lacked the carboxy-terminal portion due to an insertion in the coding region of the mRNA. RT-PCR analysis showed each of Pax8a and Pax8b mRNAs to be abundantly expressed in the thyroid and kidney. In situ hybridization histochemistry further detected the expression of Pax8 mRNA in the epithelial cells of the thyroid follicles of the adult trout and in the thyroid primordial cells of the embryo. The functional properties of Pax8a and Pax8b were investigated by dual luciferase assay. The transcriptional regulation by the rat thyroid peroxidase (TPO) promoter was found to be increased by Pax8a, but not by Pax8b. Pax8a further showed synergistic transcriptional activity with rat Nkx2-1 for the human TPO upstream region including the enhancer and promoter. On the other hand, Pax8b decreased the synergistic activity of Pax8a and Nkx2-1. Electrophoretic mobility shift assay additionally indicated that not only Pax8a but also Pax8b can bind to the TPO promoter and enhancer, implying that the inhibitory effect of Pax8b might result from the lack of the functional carboxy-terminal portion. Collectively, the results suggest that for the trout thyroid gland, Pax8a may directly increase TPO gene expression in cooperation with Nkx2-1 while Pax8b may work as a non-activating competitor for the TPO transcription.

Keywords: Nkx2-1; Pax8; Rainbow trout; TTF1; Thyroid gland; Thyroid transcription factor 1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Amino Acid Sequence
  • Animals
  • Autoantigens / genetics*
  • Autoantigens / metabolism
  • Base Sequence
  • Cloning, Molecular
  • Electrophoretic Mobility Shift Assay
  • Fish Proteins / genetics
  • Fish Proteins / metabolism*
  • Gene Expression Regulation / physiology*
  • In Situ Hybridization
  • Iodide Peroxidase / genetics*
  • Iodide Peroxidase / metabolism
  • Iron-Binding Proteins / genetics*
  • Iron-Binding Proteins / metabolism
  • Membrane Proteins / genetics
  • Molecular Sequence Data
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Oncorhynchus mykiss / genetics*
  • Oncorhynchus mykiss / growth & development
  • Paired Box Transcription Factors / genetics*
  • Phylogeny
  • Promoter Regions, Genetic / genetics
  • Protein Isoforms
  • RNA, Messenger / genetics
  • Rats
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Thyroid Gland / cytology
  • Thyroid Gland / metabolism*
  • Thyroid Nuclear Factor 1
  • Tissue Distribution
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcriptional Activation

Substances

  • Autoantigens
  • Fish Proteins
  • Iron-Binding Proteins
  • Membrane Proteins
  • NKX2-1 protein, human
  • Nkx2-1 protein, rat
  • Nuclear Proteins
  • Paired Box Transcription Factors
  • Protein Isoforms
  • RNA, Messenger
  • Serinc5 protein, rat
  • Thyroid Nuclear Factor 1
  • Transcription Factors
  • TPO protein, human
  • Iodide Peroxidase