Thyroid transcription factor 1 rescues PAX8/p300 synergism impaired by a natural PAX8 paired domain mutation with dominant negative activity.
Mutations in the paired domain transcription factor PAX8 are a rare cause of congenital hypothyroidism due to thyroid dysgenesis. We identified a novel and unique PAX8 mutation segregating in seven affected members of a three-generations family. The mutation replaces an invariant serine residue within helix 2 of the paired DNA-binding domain for phenylalanine. The mutant protein (PAX8-S48F) does not induce the thyroglobulin promoter in nonthyroid cells, but displays almost half of wild-type PAX8 activity in thyroid cells. PAX8-S48F shows no defect in expression, nuclear targeting, or DNA binding and retains the ability to synergize with thyroid transcription factor 1 (TTF-1, NKX2.1). However, we found that in nonthyroid cells, the acetylation-independent synergism with the general transcriptional adaptor p300 is completely abrogated, suggesting that PAX8-S48F may be unable to efficiently recruit p300. Reconstitution experiments in nonthyroid cells reveal that TTF-1 can partially rescue PAX8-S48F/p300 synergism and thus reproduce the situation in thyroid cells. These functional characteristics result in a dominant negative effect of PAX8-S48F on coexpressed wild-type PAX8 activity, which is not observed in paired domain mutations with DNA binding defect. Our results describe the first dominant negative missense mutation in a paired domain and provide evidence for a crucial role of the p300 coactivator in mediating the functional synergism between PAX8 and TTF-1 in thyroid-specific gene expression.
Grasberger
Helmut
H
The University of Chicago, MC3090, 5841 South Maryland Avenue, Chicago, IL 60637, USA.
Ringkananont
Usanee
U
Lefrancois
Paule
P
Abramowicz
Marc
M
Vassart
Gilbert
G
Refetoff
Samuel
S
D000293:Adolescent; D000328:Adult; D000368:Aged; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002648:Child; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D006801:Humans; D007037:Hypothyroidism / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics; D013954:Thyroglobulin / Q000235:genetics; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism; D000074482:Thyroid Nuclear Factor 1; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
D004268:DNA-Binding Proteins; C000614412:NKX2-1 protein, human; D009687:Nuclear Proteins; D000072636:PAX8 Transcription Factor; C083325:PAX8 protein, human; D051761:Paired Box Transcription Factors; D000074482:Thyroid Nuclear Factor 1; D015534:Trans-Activators; D014157:Transcription Factors; D012694:Serine; D013954:Thyroglobulin