Identification of porcine Lhx3 and SF1 as candidate genes for QTL affecting growth and reproduction traits in swine.

The distal portion of the long arm of porcine chromosome 1 has been shown to harbour several quantitative trait loci affecting growth and reproductive traits in swine. In order to identify potential candidate genes that might underlie these effects, a comparative mapping analysis was undertaken to define the extent of orthologous segments of human chromosome 9. A microsatellite associated with heat shock protein (HSP) A5 was used to define the proximal boundary of the quantitative trait loci (QTL) region, which suggests the human orthologue of the gene(s) responsible for the observed effects lies between HSPA5 and the q arm telomere of human chromosome 9. Examination of this region revealed two candidate genes with known roles in production of hormones essential to growth and reproductive function. The steroidogenic factor 1 and Lhx3 LIM homeodomain transcription factor genes were mapped to 123 and 155 cM, respectively, of the Sus scrofa chromosome 1 (SSC1) linkage group, placing both genes within the confidence interval for the observed QTL. To further evaluate Lhx3, we examined the expression profile during porcine embryonic development. Low levels were detected at early embryonic stages, when development of the nervous system is proceeding. A transient increase in expression level is observed during the time of pituitary organogenesis and again at the time of differentiation of anterior pituitary cells, with relatively high levels of expression persisting in the adult pituitary gland. This ontology is consistent with Lhx3 being a candidate gene for the QTL.

Biochemical and genetic characterization of the porcine Prophet of Pit-1 pituitary transcription factor.

Prophet of Pit-1 (Prop-1) is a paired class homeodomain transcription factor that is specifically expressed in the pituitary gland. Mutations in the Prop-1 gene cause compound pituitary diseases in mouse models and human patients. We have cloned and analyzed the porcine ortholog of Prop-1. Analysis of cDNAs revealed that the porcine Prop-1 sequence is similar to the mouse and human proteins within the homeodomain and carboxyl terminus, but the amino terminus is poorly conserved. The Prop-1 gene consists of three exons and two introns and spans 3.8 kilobases of genomic DNA. In addition, we mapped Prop-1 to the q arm of pig chromosome two. During development, Prop-1 is expressed at the time of pituitary organogenesis. In the adult, expression was observed at low levels only in the pituitary gland. The porcine Prop-1 protein displays similar biochemical, DNA binding, and transcriptional activities to human PROP-1. We conclude that, although the structural divergence between the porcine and human PROP-1 molecules may indicate some distinct functions, the porcine Prop-1 gene encodes a pituitary transcription factor with similar overall activities to the human ortholog.

Differential activation of pituitary hormone genes by human Lhx3 isoforms with distinct DNA binding properties.

Lhx3 is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. We identified two isoforms of human Lhx3, hLhx3a and hLhx3b, which differ in their ability to trans-activate pituitary gene targets. These factors are identical within the LIM domains and the homeodomain, but differ in their amino-terminal sequences preceding the LIM motifs. Both isoforms are localized to the nucleus and are expressed in the adult human pituitary, but gene activation studies demonstrate characteristic functional differences. Human Lhx3a trans-activated the alpha-glycoprotein subunit promoter and a reporter construct containing a high-affinity Lhx3 binding site more effectively than the hLhx3b isoform. In addition, hLhx3a synergized with the pituitary POU domain factor, Pit-1, to strongly induce transcription of the TSHbeta-subunit gene, while hLhx3b did not. We demonstrate that the differences in gene activation properties between hLhx3a and hLhx3b correlate with their DNA binding to sites within these genes. The short hLhx3b-specific amino-terminal domain inhibits DNA binding and gene activation functions of the molecule. These data suggest that isoforms of Lhx3 may play distinct roles during development of the mammalian pituitary gland and other neuroendocrine systems.

Characterization of the porcine Lhx3/LIM-3/P-Lim LIM homeodomain transcription factor.

Lhx3/LIM-3/P-Lim is a LIM homeodomain transcription factor which is essential in mice for the development of anterior and intermediate lobes of the pituitary gland. We report the cloning and characterization of porcine Lhx3. The porcine Lhx3 protein exhibits strong similarity to murine Lhx3 within the amino terminal LIM domains and the homeodomain, however, it is diverged in regions outside these motifs. Expression vectors for porcine Lhx3 activated murine and porcine alpha-glycoprotein reporter genes in transfection assays, and recombinant porcine Lhx3 protein specifically bound to a target site within the porcine alpha-glycoprotein gene upstream sequence. In addition, porcine Lhx3 synergistically induced transcription from prolactin enhancer/promoter reporter genes in cooperation with the Pit-1 pituitary transcription factor. Porcine Lhx3 protein interacted with Pit-1 protein in solution and also with the LIM domain-binding protein NLI/Lbd1/CLIM. Together, these data indicate that many aspects of Lhx3 function in the mammalian pituitary are conserved and that Lhx3 may be involved in the activation of trophic hormone genes during early and late stages of pituitary organogenesis. Divergence in the Lhx3 amino acid sequence between mammalian species may suggest distinct activities for this protein in some species and may help identify important functional domains of this key developmental transcription factor.