Characterization of a Novel POU1F1 Mutation Identified on Screening 160 Growth Hormone Deficiency Patients.

The objective of the study is the functional characterization of a novel POU1F1 c.605delC mutation in combined pituitary hormone deficiency (CPHD) and to report the clinical and genetic details of 160 growth hormone deficiency patients. Screening of GH1, GHRHR, POU1F1, PROP1, and HESX1 genes by Sanger sequencing was carried out in 160 trios and 100 controls followed by characterization of the POU1F1 c.605delC mutation by expression studies including site directed mutagenesis, co-transfection, protein degradation, and luciferase assays to compare the wild type and mutant POU1F1. In vitro studies showed that the POU1F1 c.605delC mutation codes for a truncated protein with reduced transactivation capacity on its downstream effectors, viz., growth hormone (GH) and prolactin (PRL) causing severe CPHD. Experiments using different protease inhibitors reveal rescue of the protein upon blockage of the lysosomal pathway that might be useful in novel drug designing using targeted approach thereby maintaining the milieu and preventing/delaying the disease. The study provides an insight into the disease causing mechanism of POU1F1 c.605delC mutation identified in a CPHD child with severe short stature and failure to thrive. It also shows mutation effect on the expression, function and turnover of protein and highlights mechanistic details by which these potent regulators may operate.

Evolution of the POU1F1 transcription factor in mammals: Rapid change of the alternatively-spliced ß-domain.

The POU1F1 (Pit-1) transcription factor is important in regulating expression of growth hormone, prolactin and TSH ß-subunit, and controlling development of the anterior pituitary cells in which these hormones are produced. POU1F1 is a conserved protein comprising three main domains, an N-terminal transcription activation domain (TAD), a POU-specific domain and a C-terminal homeodomain. Within the TAD, a ß-domain can be inserted by alternative splicing, giving an extended 'ß-variant' with altered properties. Here sequence data from over 100 species were used to assess the variability of POU1F1 in mammals. This showed that the POU-specific domain and homeodomain are very strongly conserved, and that the TAD is somewhat less conserved, as are linker and hinge regions between these main domains. On the other hand, the ß-domain is very variable, apparently evolving at a rate not significantly different from that expected for unconstrained, neutral evolution. In several species stop and/or frameshift mutations within the ß-domain would prevent expression of the ß-variant as a functional protein. In most species expression of the ß-variant is low (<5% of total POU1F1 expression). The rate of evolution of POU1F1 in mammals shows little variation, though the lineage leading to dog does show an episode of accelerated change. This comparative genomics study suggests that in most mammalian species POU1F1 variants produced by alternative splicing may have little physiological significance.

Biochemical and structural characterization of a novel cooperative binding mode by Pit-1 with CATT repeats in the macrophage migration inhibitory factor promoter.

Overexpression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is linked to a number of autoimmune diseases and cancer. MIF production has been correlated to the number of CATT repeats in a microsatellite region upstream of the MIF gene. We have characterized the interaction of pituitary-specific positive transcription factor 1 (Pit-1) with a portion of the MIF promoter region flanking a microsatellite polymorphism (-794 CATT5-8). Using fluorescence anisotropy, we quantified tight complex formation between Pit-1 and an oligonucleotide consisting of eight consecutive CATT repeats (8xCATT) with an apparent Kd of 35 nM. Using competition experiments we found a 23 base pair oligonucleotide with 4xCATT repeats to be the minimum DNA sequence necessary for high affinity interaction with Pit-1. The stoichiometry of the Pit-1 DNA interaction was determined to be 2:1 and binding is cooperative in nature. We subsequently structurally characterized the complex and discovered a completely novel binding mode for Pit-1 in contrast to previously described Pit-1 complex structures. The affinity of Pit-1 for the CATT target sequence was found to be highly dependent on cooperativity. This work lays the groundwork for understanding transcriptional regulation of MIF and pursuing Pit-1 as a therapeutic target to treat MIF-mediated inflammatory disorders.

Differential abilities of chicken Pit1 isoforms to regulate the GH promoter: evidence for synergistic activation.

Pit1, pituitary-specific transcription factor 1, regulates differentiation of cells of the Pit1 lineage in the anterior pituitary and the synthesis of peptide hormones by these cell types, including GH. Pit1 is characterized by an N-terminal transactivation domain and a C-terminal POU domain. Alternative forms of Pit1, differing from each other in the N-terminal domain, have been reported in several species, but the functional implication of having multiple isoforms is not known. Several PIT1 mRNA transcripts exist in chickens that have not been characterized. This study was conducted to determine which, if any, of the chicken Pit1 isoforms regulate the chicken GH (cGH) promoter. During the course of this work, Pit1ß2, a novel isoform of chicken Pit1, was discovered. Effects of known and novel isoforms (Pit1α, Pit1ß1, Pit1ß2, and Pit1γ) on cGH promoter activity were characterized in chicken Leghorn male hepatoma cells. Three of the isoforms, Pit1α, Pit1ß1, and Pit1ß2, activated the cGH promoter, whereas Pit1γ did not. Results from gel-shift assays indicated that Pit1γ does not bind to the proximal Pit1-bindng site of the cGH promoter, suggesting a possible mechanism underlying its inactivity. We found a functional advantage for having multiple isoforms expressed. When Pit1ß1 was coexpressed with Pit1α or Pit1ß2, significantly greater activation of the cGH promoter occurred than with any one isoform alone, with synergistic activation occurring when Pit1α and Pit1ß1 were coexpressed. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Identification of isoforms with specific functions will facilitate identification of their respective interacting partners that are essential for GH gene expression.

Involvement of Prop1 homeobox gene in the early development of fish pituitary gland.

When mutated in mammals, paired-like homeobox Prop1 gene produces highly variable pituitary phenotypes with impaired regulation of Pit1 and eventually defective synthesis of Pit1-regulated pituitary hormones. Here we have identified fish prop1 orthologs, confirmed their pituitary-specific expression, and blocked the splicing of zebrafish prop1 transcripts using morpholino oligonucleotides. Very early steps of the gland formation seemed unaffected based on morphology and expression of early placodal marker pitx. Prop1 knock-down reduced the expression of pit1, prl (prolactin) and gh (growth hormone), as expected if the function of Prop1 is conserved throughout vertebrates. Less expectedly, lim3 was down regulated. This gene is expressed from early stages of vertebrate pituitary development but is not known to be Prop1-dependent. In situ hybridizations on prop1 morphants using probes for the pan pituitary gene pitx3 and for the hormone gene markers prl, gh and tshß, revealed abnormal shape, growth and cellular organization of the developed adenohypophysis. Strikingly, the effects of prop1 knock-down on adenohypophysis morphology and gene expression were gradually reversed during late development, despite persistent splice-blocking of transcripts. Therefore, prop1 function appears to be conserved between mammals and fish, at least for the mediation of hormonal cell type differentiation via pit1, but the existence of other fish-specific pathways downstream of prop1 are suggested by our observations.

Folding of the Pit1 homeodomain near the speed limit.

Current questions in protein folding mechanisms include how fast can a protein fold and are there energy barriers for the folding and unfolding of ultrafast folding proteins? The small 3-helical engrailed homeodomain protein folds in 1.7 µs to form a well-characterized intermediate, which rearranges in 17 µs to native structure. We found that the homologous pituitary-specific transcription factor homeodomain (Pit1) folded in a similar manner, but in two better separated kinetic phases of 2.3 and 46 µs. The greater separation and better fluorescence changes facilitated a detailed kinetic analysis for the ultrafast phase for formation of the intermediate. Its folding rate constant changed little with denaturant concentration or mutation but unfolding was very sensitive to denaturant and energy changes on mutation. The folding rate constant of 3 × 10(5) s(-1) in water decreased with increasing viscosity, and was extrapolated to 4.4 × 10(5) s(-1) at zero viscosity. Thus, the formation of the intermediate was partly rate limited by chain diffusion and partly by an energy barrier to give a very diffuse transition state, which was followed by the formation of structure. Conversely, the unfolding reaction required the near complete disruption of the tertiary structure of the intermediate in a highly cooperative manner, being exquisitely sensitive to individual mutations. The folding is approaching, but has not reached, the downhill-folding scenario of energy landscape theory. Under folding conditions, there is a small energy barrier between the denatured and transition states but a larger barrier between native and transition states.

The 26-amino acid beta-motif of the Pit-1beta transcription factor is a dominant and independent repressor domain.

The POU-homeodomain transcription factor Pit-1 governs the pituitary cell-specific expression of Pit-1, GH, prolactin (PRL), and TSHbeta genes. Alternative splicing generates Pit-1beta, which contains a 26-amino acid beta-domain inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain (TAD). Pit-1beta represses GH, PRL, and TSHbeta promoters in a pituitary-specific manner, because Pit-1beta activates these same promoters in HeLa nonpituitary cells. Here we comprehensively analyze the role of beta-domain sequence, position, and context, to elucidate the mechanism of beta-dependent repression. Repositioning the beta-motif to the Pit-1 amino terminus, hinge, linker, and carboxyl terminus did not affect its ability to repress basal rat (r) PRL promoter activity in GH4 pituitary cells, but all lost the ability to repress Ras-induced rPRL promoter activity. To determine whether beta-domain repression is independent of Pit-1 protein and DNA binding sites, we generated Gal4-Pit-1TAD, Gal4-Pit-1betaTAD, and Gal4-beta-domain fusions and demonstrated that the beta-motif is sufficient to actively repress VP16-mediated transcription of a heterologous promoter. Moreover, beta-domain point mutants had the same effect whether fused to Gal4 or within the context of intact Pit-1beta. Surprisingly, Gal4-beta repression lost histone deacetylase sensitivity and pituitary specificity. Taken together, these results reveal that the beta-motif is a context-independent, modular, transferable, and dominant repressor domain, yet the beta-domain repressor activity within Pit-1beta contains cell type, promoter, and Pit-1 protein context dependence.

A single base difference between Pit-1 binding sites at the hGH promoter and locus control region specifies distinct Pit-1 conformations and functions.

Activation of the human growth hormone (hGH-N) gene in pituitary somatotropes is mediated by a locus control region (LCR). This LCR is composed of DNase I-hypersensitive sites (HS) located -14.5 kb to -32 kb relative to the hGH-N promoter. HSI, at -14.5 kb, is the dominant determinant of hGH-N expression and is essential for establishment of a 32-kb domain of histone acetylation that encompasses the active hGH locus. This activity is conferred by three binding sites for the POU domain transcription factor Pit-1. These Pit-1 elements are sufficient to activate hGH-N expression in the mouse pituitary. In contrast, Pit-1 sites at the hGH-N promoter are consistently unable to mediate similar activity. In the present study, we demonstrate that the functional difference between the promoter-proximal and the HSI Pit-1 binding sites can be attributed in part to a single base difference. This base affects the conformation of the Pit-1/DNA complex, and reciprocal exchange of the divergent bases between the two sets of Pit-1 elements results in a partial reversal of their transgenic activities. These data support a model in which the Pit-1 binding sites in the hGH LCR allosterically program the bound Pit-1 complex for chromatin activating functions.

Repression of the prolactin promoter: a functional consequence of the heterodimerization between Pit-1 and Pit-1 beta.

The POU-homeodomain transcription factor Pit-1 is required for the differentiation of the anterior pituitary cells and the expression of their hormone products. Pit-1beta, an alternate splicing isoform, has diametrically different outcomes when it is expressed in different cell types. Pit-1beta acts as a transcriptional repressor of prolactin (PRL) and growth hormone genes in pituitary cells, and as a transcriptional activator in non-pituitary cells. In order to explore these differences, we: (1) identified the transcriptional cofactors necessary for reconstitution of repression in non-pituitary cells; (2) tested the effect of the beta-domain on heterodimerization with Pit-1 and physical interaction with the co-activator CREB binding protein (CBP); and (3) determined the beta-domain sidechain chemistry requirements for repression. Co-expression of both Pit-1 isoforms reconstituted the repression of the PRL promoter in non-pituitary cells. The beta-domain allowed heterodimerization with Pit-1 but blocked physical interaction with CBP, and specific chemical properties of the beta-domain beyond hydrophobicity were dispensable. These data strongly suggest that Pit-1beta represses hormone gene expression by heterodimerizing with Pit-1 and interfering with the assembly of the Pit-1-CBP complex required for PRL promoter activity in pituitary cells.