A point mutation in the LIM domain of Lhx3 reduces activation of the glycoprotein hormone alpha-subunit promoter.
Lhx3, a member of the LIM homeodomain family of transcription factors, is required for development of the pituitary in mice. A recent report has described a point mutation in the human LHX3 gene that is associated with a combined pituitary hormone disorder. The mutation is predicted to lead to the replacement of a tyrosine residue with a cysteine in the second LIM domain of LHX3. We have characterized the effects of this point mutation (Y114C) when analyzed in the context of the mouse Lhx3 coding sequence. Mobility shift assays demonstrated that the Lhx3 Y114C mutant is capable of binding DNA, although a decrease in the formation of a specific complex was observed. Transfection assays using an expression vector for either full-length Lhx3 or a GAL4-Lhx3 LIM domain fusion provided evidence that the Lhx3 Y114C mutant has a decreased ability to stimulate transcription. In particular, a GAL4-Lhx3 Y114C LIM mutant was unable to support Ras responsiveness of a modified glycoprotein hormone alpha-subunit reporter gene. Protein interaction studies suggest that the Y114C mutation may modestly reduce binding to the POU transcription factor, Pit-1. Interestingly, the Y114C mutation essentially abrogated binding to the putative co-activator/adapter, selective LIM-binding protein. The findings provide insights into the mechanisms mediating transcriptional activation by Lhx3 and suggest that the observed phenotype of the human mutation probably involves reduced transcriptional activity of the mutant LHX3.
Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
P W
PW
Howard
R A
RA
Maurer
D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D060850:LIM-Homeodomain Proteins; D009154:Mutation; D010641:Phenotype; D017354:Point Mutation; D011233:Precipitin Tests; D011388:Prolactin / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C094898:Lhx3 protein; C494187:POU1F1 protein, human; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D014443:Tyrosine; D011388:Prolactin; D004247:DNA; D005982:Glutathione Transferase; D003545:Cysteine