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Department of Medicine and Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908
Address all correspondence and requests for reprints to: Richard Day, University of Virginia Health Science Center, Department of Medicine and Cell Biology, Box 800578 HSC, 300 Jefferson Park Avenue, MSB Room 7121, Charlottesville, Virginia 22908-0578. E-mail: . rnd2v{at}virginia.edu
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Introduction |
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The abnormal pituitary gland development in patients with ARS, coupled with the demonstration that loss of Pitx1 function resulted in changes in the transcription of multiple pituitary genes (4), implicated the Pitx proteins in pituitary gland development. This was substantiated by the observation that both Pitx1 and Pitx2 appear early in the development of the oral plate ectoderm from which Rathke’s pouch, the primordium of the anterior pituitary, is derived. Further, the phenotype of Pitx2-/- knockout mice recapitulated the multiple developmental defects of ARS, including the abnormal pituitary gland development. The pituitaries in the Pitx2-/- mice failed to progress beyond embryonic d 10.5, when the ectoderm cells would normally begin to proliferate and populate the pituitary gland (2, 5). Recently, this observation was confirmed and extended by using a mouse model for reduced Pitx2 function (Pitx2neo, Ref. 1). The Pitx2neo mice survive much longer than the knockout mice, which allowed the analysis of reduced Pitx2 levels on various aspects of pituitary development. The results revealed a functional overlap between Pitx1 and Pitx2, and indicated that haploinsufficiency, where functional Pitx2 is reduced below a critical level, could prevent the proliferation of the somatotroph, thyrotroph, and gonadotroph cell lineages (6). This positioned Pitx2 early in the cascade of transcription factors that determine endocrine cell fate in the anterior pituitary. Do the Pitx proteins have functions beyond development, in the adult pituitary cells?
Pitx1 and Pitx2 are both expressed uniformly throughout the cells of the anterior pituitary (7, 8). The homeodomains of Pitx1 and Pitx2, the major determinant of DNA binding specificity in vitro, are nearly identical (2). Therefore, it is not surprising that both Pitx proteins recognize the same consensus TAA(T/G)CC DNA element. This motif is common to the promoters of most of the pituitary hormone-coding genes, which are expressed only in specific subpopulations of pituitary cells. Moreover, it appears that both Pitx1 and Pitx2 have redundant roles in the transcriptional regulation of these cell-type restricted genes (9, 10). That the Pitx proteins are present in all adult anterior pituitary cell types and recognize DNA elements that are common to many pituitary genes is paradoxical. How is it that the Pitx proteins function to regulate the cell type-restricted expression of pituitary hormone genes? Lessons learned from the Hox family of proteins in flies provide some insight into this paradox. Hox genes encode a conserved family of homeodomain proteins that control morphogenesis along that anteroposterior axis in Drosophila. Yet, most Hox family proteins recognize the same DNA binding site in vitro. The specificity for the regulation of target genes in vivo results from the partnerships formed between the Hox proteins and other homeodomain proteins (11, 12). In this regard, the homeobox factors act as the allosteric regulators proposed by Lefstin and Yamamoto (13) that adopt different conformations in different promoter contexts, and this guides the selective recruitment of promoter specific cofactors. Thus, like the Hox proteins, the Pitx proteins may occupy binding sites common to many genes, but they achieve differential transcriptional regulation through the partnerships that they form with other proteins.
Indeed, the interaction between the Pitx proteins and transcription factors restricted to particular pituitary cell lineages has been demonstrated. For example, Lamonerie et al. (14) identified Pitx1 by its binding to a promoter element called CE3 that directs the corticotroph-restricted expression of proopiomelanocortin (POMC). Work from the Drouin laboratory (15) showed that the basic helix-loop-helix protein NeuroD1 and the Brachyury/ T box family protein Tpit (16) are obligate partners for Pitx1 activation of POMC transcription. Further, Pitx1 was identified in screens for proteins interacting with the POU (Pit-1, oct-1, and Caenorhabditas elegans unc-86) homeodomain transcription factor, Pit-1 (17). Pit-1 functions to establish the somatolactotroph and thyrotroph cell lineages, and through its combinatorial interactions with other nuclear proteins acts to control the expression of several pituitary-specific genes, including the prolactin (PRL) gene. Both Pitx1 and Pitx2 were found to act synergistically with Pit-1 to induce PRL gene expression (17, 18). Structural studies of Pit-1 demonstrated flexibility in the conformations it adopts when interacting with a variety of DNA response elements (19). Similar to the mechanisms described above for the Hox proteins, it appears the combinatorial interactions between Pit-1 and its protein partners are determined by the conformations that Pit-1 adopts on different DNA elements (20). Thus, the interaction surfaces that Pit-1 and the Pitx proteins display in the context of the PRL promoter, for instance, may function to direct the assembly of a multiprotein transcription complex (21) that determines the temporal and cell-restricted pattern of PRL expression. How point mutations affect these interaction surfaces can potentially provide insight into the molecular mechanisms that mediate Pitx2 functions in both the developing and adult pituitary gland.
In this issue of Endocrinology, the paper by Quentien et al. (22) examines the effect of point mutations in the Pitx2 homeodomain on target gene expression in differentiated pituitary somatolactotroph cells. Five different Pitx2 mutants, each identified earlier in patients with developmental abnormalities, were selected for this study. Each mutant protein had a single amino acid alteration in the homeodomain, but importantly, none of these mutations interfered with specific binding to the CE3 DNA response element from the POMC gene. This showed that the mutant phenotypes did not result from disruption of DNA binding site recognition. Quentien et al. (22) characterize the effect of each point mutation on the function of Pitx2 in transactivation of PRL, GH, and Pit-1 gene promoters. Their findings revealed that all five of the Pitx2 mutant proteins had lost the transactivation function at these pituitary promoters, and none of the mutants exhibited the cooperative interaction with Pit-1. When expressed at subsaturating levels, four of the five mutant proteins also failed to affect wild-type Pitx2 induction of PRL promoter activity. Together, the results indicate that four of the tested ARS Pitx2 proteins behaved as loss-of-function mutants. These proteins likely act through a mechanism of haploinsufficiency in vivo, where a single functional allele is insufficient to achieve protein levels necessary for the regulation of target genes.
The fifth ARS mutant studied by Quentien et al. (22), Pitx2 R91P, behaved differently. When coexpressed with Pitx2, the R91P mutant acted as a dominant inhibitor, blocking wild-type Pitx2-dependent induction of PRL promoter activity and preventing its cooperative interactions with Pit-1. Consistent with this inhibitory activity, the R91P mutant also blocked PRL promoter activity in rat somatolactotroph cells. In this respect, the R91P mutant was similar to another Pitx2 homeodomain mutant, K88E, described by Saadi et al. (23). The lysine residue at position 88 is a defining feature of the Pitx subclass of homeodomain proteins, and is a major determinant of DNA-site recognition (2). In contrast to the R91P mutation, the K88E mutant failed to bind to a Pitx DNA response element. Interestingly, the dominant inhibitory activity of the K88E mutant appeared to dependent upon its interaction with Pit-1 bound to DNA (23). Recently, Priston et al. (24) reported another intriguing ARS Pitx2 protein mutated at position 83 in the homeodomain. In contrast to the dominant inhibitory Pitx2 mutants, this protein acted as a gain-of-function mutant exhibiting a 2-fold increase in transactivation activity over that of wild-type Pitx2 (24).
The activities of these different Pitx2 homeodomain mutants are providing important clues about the interactions of Pitx2 with other components of the transcription complex. The regulation of genes by the different classes of transcription factors is achieved through balanced interactions with both coactivator and corepressor protein complexes (20, 25). Subtle changes in protein conformation introduced by point mutations in DNA- or protein-interaction surfaces may dramatically alter the types of protein partnerships that form, and can potentially change the selection of cofactors that are recruited to a target gene promoter. Such a mechanism was proposed for one point mutation effecting the homeodomain of Pit-1. A mutation at position 254 of the Pit-1 homeodomain resulted in a dominant inhibitory protein that retained DNA binding specificity (25). The mechanism of action for this mutant protein appears to result from its increased affinity for the nuclear receptor corepressor complex, tipping the balance to inhibition. Thus, the dominant inhibitory action of Pitx2 homeodomain mutants could result from increased association with corepressor complexes, whereas the gain-of-function mutant may favor association with coactivator proteins.
Research over the past several years has provided a wealth of information regarding the structure and function of the coregulatory complexes recruited by different classes of transcription factors. However, the regulation of cell type- restricted gene expression, such as that observed in the anterior pituitary, requires the combinatorial assembly of many proteins into target-gene specific transcription complexes. Future studies that characterize mutations effecting selected surfaces of Pitx proteins, used in conjunction with targeted mutants of other essential factors, such as Pit-1, will ultimately provide important clues necessary to decipher this combinatorial code.
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Acknowledgments |
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Footnotes |
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Received May 17, 2002.
Accepted for publication May 22, 2002.
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