Temporal regulation of a paired-like homeodomain repressor/TLE corepressor complex and a related activator is required for pituitary organogenesis.

Understanding the functional significance of the coordinate expression of specific corepressors and DNA-binding transcription factors remains a critical question in mammalian development. During the development of the pituitary gland, two highly related paired-like homeodomain factors, a repressor, Hesx1/Rpx and an activator, Prop-1, are expressed in sequential, overlapping temporal patterns. Here we show that while the repressive actions of Hesx1/Rpx may be required for initial pituitary organ commitment, progression beyond the appearance of the first pituitary (POMC) lineage requires both loss of Hesx1 expression and the actions of Prop-1. Although Hesx1 recruits both the Groucho-related corepressor TLE1 and the N-CoR/Sin3/HDAC complex on distinct domains, the repressor functions of Hesx1 in vivo prove to require the specific recruitment of TLE1, which exhibits a spatial and temporal pattern of coexpression during pituitary organogenesis. Furthermore, Hesx1-mediated repression coordinates a negative feedback loop with FGF8/FGF10 signaling in the ventral diencephalon, required to prevent induction of multiple pituitary glands from oral ectoderm. Our data suggest that the opposing actions of two structurally-related DNA-binding paired-like homeodomain transcription factors, binding to similar cognate elements, coordinate pituitary organogenesis by reciprocally repressing and activating target genes in a temporally specific fashion, on the basis of the actions of a critical, coexpressed TLE corepressor.

Signaling and transcriptional mechanisms in pituitary development.

During the development of the pituitary gland, distinct hormone-producing cell types arise from a common population of ectodermal progenitors, providing an instructive model system for elucidating the molecular mechanisms of patterning and cell type specification in mammalian organogenesis. Recent studies have established that the development of the pituitary occurs through multiple sequential steps, allowing the coordinate control of the commitment, early patterning, proliferation, and positional determination of pituitary cell lineages in response to extrinsic and intrinsic signals. The early phases of pituitary development appear to be mediated through the activities of multiple signaling gradients emanating from key organizing centers that give rise to temporally and spatially distinct patterns of transcription factor expression. The induction of these transcriptional mediators in turn acts to positionally organize specific pituitary cell lineages within an apparently uniform field of ectodermal progenitors. Ultimately, pituitary cell types have proven to be both specified and maintained through the combinatorial interactions of a series of cell-type-restricted transcription factors that dictate the cell autonomous programs of differentiation in response to the transient signaling events.

Signaling mechanisms in pituitary morphogenesis and cell fate determination.

The development of the pituitary gland has provided an instructive model system for exploring the mechanisms by which differentiated cell types arise from a common primordium in response to extrinsic and intrinsic signals. Recent studies have established that organ commitment, early patterning, proliferation and positional determination of cell types in the developing pituitary are mediated through the integral actions of multiple signaling gradients acting on an initially uniform ectodermal cell population. Studies of the cell-autonomous transcriptional mediators of the transient signaling events have also provided insight into the molecular mechanisms by which overlapping patterns of transcription factor expression can positionally specify pituitary cell lineages. There is emerging evidence for a morphogenetic code for the development of the pituitary gland based on the cooperative and opposing actions of multiple signaling gradients, mediated by corresponding expression patterns of temporally and spatially induced transcription factors.

Combinatorial codes in signaling and synergy: lessons from pituitary development.

The development of the hormone-secreting cell types in the pituitary gland provides an excellent model system in which to explore the complex transcriptional mechanisms underlying the specification and maintenance of differentiated cell types in mammalian organogenesis. Pituitary development is orchestrated through the combinatorial actions of a repertoire of signaling-gradient-induced transcription factors which, on the basis of their distinct and overlapping expression patterns, and functional interactions, ultimately has led to the generation of functionally distinct cell phenotypes from a common ectodermal primordium.

Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types.

The mechanisms by which transient gradients of signaling molecules lead to emergence of specific cell types remain a central question in mammalian organogenesis. Here, we demonstrate that the appearance of four ventral pituitary cell types is mediated via the reciprocal interactions of two transcription factors, Pit1 and GATA2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as the molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA binding-independent function of Pit1, suppressing the ventral GATA2-dependent gonadotrope program by inhibiting GATA2 binding to gonadotrope- but not thyrotrope-specific genes, indicating that both DNA binding-dependent and -independent actions of abundant determining factors contribute to generate distinct cell phenotypes.

Factor-specific modulation of CREB-binding protein acetyltransferase activity.

CREB-binding proteins (CBP) and p300 are essential transcriptional coactivators for a large number of regulated DNA-binding transcription factors, including CREB, nuclear receptors, and STATs. CBP and p300 function in part by mediating the assembly of multiprotein complexes that contain additional cofactors such as p300/CBP interacting protein (p/CIP), a member of the p160/SRC family of coactivators, and the p300/CBP associated factor p/CAF. In addition to serving as molecular scaffolds, CBP and p300 each possess intrinsic acetyltransferase activities that are required for their function as coactivators. Here we report that the adenovirus E1A protein inhibits the acetyltransferase activity of CBP on binding to the C/H3 domain, whereas binding of CREB, or a CREB/E1A fusion protein to the KIX domain, fails to inhibit CBP acetyltransferase activity. Surprisingly, p/CIP can either inhibit or stimulate CBP acetyltransferase activity depending on the specific substrate evaluated and the functional domains present in the p/CIP protein. While the CBP interaction domain of p/CIP inhibits acetylation of histones H3, H4, or high mobility group by CBP, it enhances acetylation of other substrates, such as Pit-1. These observations suggest that the acetyltransferase activities of CBP/p300 and p/CAF can be differentially modulated by factors binding to distinct regions of CBP/p300. Because these interactions are likely to result in differential effects on the coactivator functions of CBP/p300 for different classes of transcription factors, regulation of CBP/p300 acetyltransferase activity may represent a mechanism for integration of diverse signaling pathways.

Signal-specific co-activator domain requirements for Pit-1 activation.

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Here we show that whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT, mSin3A/B and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, the histone acetyltransferase functions of CBP or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.

Mutations in PROP1 cause familial combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones. Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) is preserved. The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism. We have recently positionally cloned the putative Ames dwarf gene Prop1, which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression. In this report, we have identified four CPHD families with homozygosity or compound heterozygosity for inactivating mutations of PROP1. These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation. In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously. Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes.

Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism.

The gene apparently responsible for a heritable form of murine pituitary-dependent dwarfism (Ames dwarf, df) has been positionally cloned, identifying a novel, tissue-specific, paired-like homeodomain transcription factor, termed Prophet of Pit-1 (Prop-1). The df phenotype results from an apparent failure of initial determination of the Pit-1 lineage required for production of growth hormone, prolactin or thyroid-stimulating hormone, resulting in dysmorphogenesis and failure to activate Pit-1 gene expression. These results imply that a cascade of tissue-specific regulators is responsible for the determination and differentiation of specific cell lineages in pituitary organogenesis.

Phylogenetic footprinting of the human cytochrome c oxidase subunit VB promoter.

The human COX5B gene encodes subunit Vb of cytochrome c oxidase (COX). COX Vb is 1 of the 10 subunits of the mitochondrial COX complex encoded by a nuclear gene. We have defined a region in the human COX5B promoter essential for gene expression and shown by phylogenetic footprinting of 11 primate COX5B promoters that many cis-regulatory elements in this region are evolutionarily conserved. The transcription start site of human COX5B was mapped 58 bp upstream of the initiation Met codon by primer extension using a thermostable reverse transcriptase. A 475-bp region (-456 to +20) of the human COX5B gene was shown to function as a promoter for the chloramphenicol acetyl transferase (CAT) gene in expression vectors when transfected into HeLa cells. The human COX5B gene is located in a CpG island and contains several potential binding sites for the transcription factor Sp1, but no consensus TATA box element. Several sequence elements associated with the transcriptional regulation of respiratory genes were also found in the promoter and 5' flanking region, including a single NRF-1 site and two 9-bp direct repeats containing binding sites for ets-domain proteins, such as NRF-2/GABP. Many features of the human COX5B promoter are conserved in the COX5B promoters of primates, in particular, the presence of a single binding site for NRF-1 and multiple sites for Sp1 and NRF-2/GABP. Electrophoretic mobility shift assays demonstrate that the conserved NRF-1 site in primate COX5B promoters is specifically recognized by a factor present in HeLa nuclear extracts. Phylogenetic footprinting has identified additional conserved elements that may also function as binding sites for regulatory factors.