Gene expression, function, and diversity of Nkx2-4 in the rainbow trout, Oncorhynchus mykiss

Gen Comp Endocrinol. 2014 Sep 15;206:193-202. doi: 10.1016/j.ygcen.2014.07.007. Epub 2014 Jul 19.

Abstract

Nkx2 homeodomain transcription factors are involved in various developmental processes and cell specification: e.g. in mammals, NKX2-1 is essential for thyroid-specific gene expression and thyroid morphogenesis. Among Nkx2 proteins, information is still very limited for Nkx2-4. In the present study, we have identified three distinct cDNAs encoding Nkx2-4 isoforms (Nkx2-4a, -b, and -c) from the rainbow trout thyroid tissue, and characterized their transcriptional properties. The trout Nkx2-4 proteins were all predicted to conserve three characteristic domains: the tinman-like amino terminal decapeptide, the NK2 homeodomain, and the NK2-specific domain, and also share 75-89% amino acid similarity. It was shown by dual luciferase assay that Nkx2-4a and Nkx2-4b, but not Nkx2-4c, significantly activated transcription from a cotransfected rat thyroglobulin (TG) promoter. An electrophoretic mobility shift assay indicated that all the Nkx2-4 isoforms could bind to the TG promoter, implying that the faint transcriptional activity of Nkx2-4c might result from some critical amino acid substitution(s) outside the homeodomain. RT-PCR analysis revealed similar tissue distribution patterns for Nkx2-4a and Nkx2-4b mRNAs. Both mRNAs were expressed abundantly in the thyroid, and weakly in the testis. On the other hand, Nkx2-4c mRNA was detected in the ovary as well as in the thyroid. The expression sites of Nkx2-4c mRNA were localized, by in situ hybridization histochemistry, to the ovarian granulosa cells and to the thyroid follicular cells. The results suggest that in the rainbow trout, Nkx2-4a and Nkx2-4b might play a major role in TG gene transcription whereas Nkx2-4c might have some functions in the ovary as well as the thyroid.

Keywords: Nkx2-1; Nkx2-4; Ovary; Rainbow trout; TTF-1; Thyroid transcription factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation*
  • In Situ Hybridization
  • Molecular Sequence Data
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism*
  • Oncorhynchus mykiss / metabolism
  • Phylogeny
  • Protein Isoforms / metabolism
  • RNA, Messenger / metabolism
  • Sequence Homology, Amino Acid
  • Thyroglobulin / metabolism
  • Thyroid Nuclear Factor 1
  • Tissue Distribution
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism*
  • Transcriptional Activation

Substances

  • DNA, Complementary
  • Nuclear Proteins
  • Protein Isoforms
  • RNA, Messenger
  • Thyroid Nuclear Factor 1
  • Transcription Factors
  • Thyroglobulin