Nuclear localization domain of thyroid transcription factor-1 in respiratory epithelial cells.
Thyroid transcription factor-1 (TITF-1) is a homeodomain containing transcription factor that binds to and selectively activates the expression of genes in thyroid and pulmonary epithelial cells. TITF-1 plays a critical role in gene expression and in organogenesis of lung and thyroid. In the present work, epitope-tagged TITF-1 proteins were used to identify the regions of the TITF-1 polypeptide that mediate nuclear localization and transcriptional activity in human lung adenocarcinoma cells. A series of TITF-1-flag deletion mutants was generated and transfected into H441 cells to determine amino acid sequences involved in translocation to the nucleus. Transfection of the TITF-1-flag mutants demonstrated that a nuclear localization signal (NLS) sequence, located at the N-terminus of the homeodomain, is critical for nuclear targeting. The NLS was essential but not sufficient for translocation of TITF-1 to the nucleus, since deletion of the homeodomain itself also blocked nuclear translocation in the presence of NLS. Deletion of the N-terminal transactivation domain of TITF-1 completely abolished its transcriptional activation on the human surfactant protein-B promoter, and deletion of the C-terminal domain partially reduced its stimulatory activity. Nuclear translocation of TITF-1 depends on both an NLS and the homeodomain of the polypeptide. Both C- and N-terminal regions of TITF-1 are involved in transactivation of surfactant protein B gene expression in pulmonary cells.
Ghaffari
M
M
Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, OH 45229-3039, USA.
Zeng
X
X
Whitsett
J A
JA
Yan
C
C
D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008168:Lung / Q000378:metabolism*; D016296:Mutagenesis; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics; D011510:Proteolipids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D017931:DNA Primers; C000614412:NKX2-1 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D009842:Oligopeptides; D010455:Peptides; D011498:Protein Precursors; D011510:Proteolipids; D011994:Recombinant Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; C105511:surfactant protein B propeptide; C085706:FLAG peptide