Identification and characterization of four splicing variants of ovine POU1F1 gene.
Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p<0.05). Concerning the GH promoter, the beta variant presented a trans-activation capacity 10% higher than Wt. Wt and beta variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants gamma and delta showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly (p<0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.
Bastos
Estela
E
Centro de Genética e Biotecnologia (CGB-UTAD), Universidade de Trás-os-Montes e Alto Douro, Apdo. 1013, 5001-801 Vila Real, Portugal. ebastos@utad.pt
Avila
Sílvia
S
Cravador
Alfredo
A
Renaville
Robert
R
Guedes-Pinto
Henrique
H
Castrillo
José Luis
JL
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D014644:Genetic Variation; D013006:Growth Hormone / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D010902:Pituitary Gland / Q000378:metabolism; D011388:Prolactin / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D012756:Sheep / Q000235:genetics*; D050817:Transcription Factor Pit-1 / Q000235:genetics*; D015533:Transcriptional Activation
D011994:Recombinant Proteins; D050817:Transcription Factor Pit-1; D011388:Prolactin; D013006:Growth Hormone; D004247:DNA