Twin of I-POU: a two amino acid difference in the I-POU homeodomain distinguishes an activator from an inhibitor of transcription.

I-POU, a POU domain nuclear protein that lacks two conserved basic amino acids of the POU homeodomain is coexpressed in the developing Drosophila nervous system with a second POU domain transcription factor, Cf1-a. I-POU does not bind to DNA but forms a POU domain-mediated, high affinity heterodimer with Cf1-a, inhibiting its ability to bind and activate the dopa decarboxylase gene. The I-POU/Cf1-a dimerization interface encompasses only the N-terminal basic region and helices 1 and 2 of the POU homeodomains with precise amino acid and alpha-helical requirements. twin of I-POU, an alternatively spliced transcript of the I-POU gene, encodes a protein containing the two basic amino acid residues absent in I-POU. Twin of I-POU is incapable of dimerizing with Cf1-a, but can act as a positive transcription factor on targets distinct from those regulated by Cf1-a. These findings suggest that the I-POU genomic locus simultaneously generates both a specific activator and inhibitor of gene transcription, capable of modulating two distinct regulatory programs during neural development.

I-POU: a POU-domain protein that inhibits neuron-specific gene activation.

A novel, structurally distinct POU-domain protein has been identified that inhibits activation by another positive POU-domain regulator of neuron-specific transcription units. Two Drosophila POU-domain proteins, I-POU and Cf1-a, are coexpressed in overlapping subsets of neurons during development. Because I-POU lacks two basic residues in the N terminus of its homeodomain, it cannot bind DNA, but it does form a stable heterodimeric complex with Cf1-a, preventing Cf1-a from binding to DNA recognition elements and from transactivating the dopa-decarboxylase gene. The inhibition by I-POU provides a potential strategy by which the activation of genes in development is controlled by a homeodomain-containing protein that does not bind DNA.

Functional glucocorticoid inducible enhancer activity in the 5'-flanking sequences of the rat growth hormone gene.

Glucocorticoid regulation of rat growth hormone (rGH) gene expression has been investigated in a series of gene transfer studies into cells in culture. It has been established that sequences (-12 to -523) immediately flanking the start site for rGH gene transcription behave as a functional glucocorticoid inducible enhancer when associated with a heterologous promoter (RSV), displaying independence of orientation and position in mediating the glucocorticoid effect. The induction of chloramphenicol acetyl transferase (CAT) gene expression in these constructs by dexamethasone was established at the enzyme and mRNA levels and was inhibited in the presence of the antiglucocorticoid, RU 38486. The glucocorticoid inducible enhancer activity was not restricted to pituitary cells. The constructs containing the rGH-5'-flanking sequences, associated with the RSV promoter, also mediated glucocorticoid induction of CAT gene expression when transiently transfected into MH1C1 cells, a hepatoma cell line. The effect was similarly demonstrable on co-transfection of these constructs with a glucocorticoid receptor expression vector into receptor deficient COS cells. Two elements within these rGH sequences (-97 to -111 and -250 to -264) display partial homology with a consensus sequence computed for a group of glucocorticoid regulatory elements. Mutation of both of these elements or of the more proximal element alone (-97/-111) led to a complete loss of ability to mediate glucocorticoid induction of gene expression. However, the rGH sequences still mediated glucocorticoid induction of gene expression when the distal GRE-like element was mutated or deleted. Thus, the proximal rGH GRE-like element is absolutely required to mediate this glucocorticoid inducible enhancer activity.

Autoregulation of pit-1 gene expression mediated by two cis-active promoter elements.

The pit-1 gene is a member of a large family of genes that encode proteins which are involved in development and which contain a highly homologous region, referred to as the POU domain. Pit-1, a pituitary-specific transcription factor, can activate the transcription of the growth hormone and prolactin promoters. It is expressed in mature thyrotroph, somatotroph and lactotroph cell types of the anterior pituitary which arise sequentially during development; somatotrophs and lactotrophs, which secrete growth hormone and prolactin, respectively, are the last to arise. Intriguingly, during ontogeny, pit-1 transcripts are observed in the rat neural tube and neural plate (embryonic day 10-11) and disappear thereafter (day 13), only to reappear exclusively in the anterior lobe of the pituitary gland (day 15) just before activation of prolactin and growth hormone. This biphasic pattern suggests a complex mechanism of initial activation of pit-1 gene expression. Transcription and transfection analyses in vitro using wild-type and mutated promoters indicate that Pit-1 can positively autoregulate the expression of the pit-1 promoter as a consequence of binding to two Pit-1-binding elements. Mutation of the 5' Pit-1-binding site abolished positive autoregulation, whereas mutation of the element located immediately 3' of the cap site markedly increased expression of the pit-1 promoter. These data are consistent with a positive, attenuated autoregulatory loop that seems to function in maintaining pit-1 gene expression.

A two-base change in a POU factor-binding site switches pituitary-specific to lymphoid-specific gene expression.

The structurally related POU homeo domain proteins Pit-1 and Oct-2 activate pituitary- and lymphoid-specific transcription, respectively, by binding to similar AT-rich motifs in their target genes. In this study we identify bases critical for recognition and activation by Pit-1 and examine how small differences in Pit-1 and Oct-2-binding sites can impart differential transcriptional responses in pituitary and B-lymphoid cells. Scanning mutagenesis of Pit-1 response elements in both the rat prolactin and growth hormone genes reveals a critical binding motif recognized in an identical manner by the native Pit-1 protein and cloned Pit-1 gene product. This motif, ATTATTCCAT, differs by only two bases from the octamer element, ATTTGCAT, required for Oct-2-dependent activation of immunoglobulin genes. Cross recognition of Pit-1 and Oct-2 sites by both factors can be demonstrated in competitive binding assays, in which an oligometric Pit-1 site from the prolactin gene is converted to an Oct-2 site by a double point mutation. In contrast to the binding data, no cross activation of transcription is detectable in cultured cell lines. When inserted immediately 5' to a prolactin TATA box, the wild-type prolactin element enhances transcription strongly in pituitary cells but is inactive in B cells, whereas the octamer variant of the prolactin site activates expression in B cells but is silent in pituitary lines. Both elements are nonfunctional in heterologous cell lines that lack Pit-1 and Oct-2.(ABSTRACT TRUNCATED AT 250 WORDS)

A family of POU-domain and Pit-1 tissue-specific transcription factors in pituitary and neuroendocrine development.

The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types, within an organ, a central question in development. The rat prolactin and growth hormone genes are selectively expressed in distinct cell types (lactotrophs and somatotrophs) of the anterior pituitary gland, which reflect differential mechanisms of gene activation or restriction because of interactions of multiple factors binding to these genes. We find that the pituitary-specific 33,000 dalton transcription factor, Pit-1, normally expressed in somatotrophs, lactotrophs, and thyrotrophs, can bind to and activate both growth hormone and prolactin promoters in vitro at levels even tenfold lower than those normally present in pituitary cells. In the case of the prolactin gene, high levels of expression in transgenic animals required two cis-active regions; a distal enhancer (-1.8 to -1.5 kb) and a proximal region (-422 to +33 bp). Each of these regions alone can direct low levels of fusion gene expression to prolactin-producing cell types in transgenic mice, but a synergistic interaction between these regions is necessary for high levels of expression. The initial appearance of the prolactin transgene expression closely follows the appearance of high levels of Pit-1, but later increases in expression coincident with appearance of mature lactotrophs suggest the operation of additional, critical positive factor(s). Unexpectedly, transgenes containing the distal enhancer removed from its normal context are expressed in both the prolactin-producing lactotrophs and the TSH-producing thyrotrophs, thereby suggesting that sequences flanking this enhancer are necessary to restrict expression to the correct cell type within the pituitary. These data indicate that distinct processes of gene activation and restriction are necessary for the fidelity of cell-type specific expression within an organ. Consistent with this model, we find that lactotroph cell lines that cannot express the growth hormone gene contain high levels of functional Pit-1. We suggest a large, highly related POU-domain gene family, potentially exceeding 100 members, has been conserved and expanded in evolution to meet the increasing requirements for more intricate patterns of cell phenotypes. The POU-domain subgroup of the homeodomain gene family, in concert with other homeodomain proteins and with other classes of transcription factors, is likely to contribute to the establishment of the mammalian neuroendocrine system.

Expression of a large family of POU-domain regulatory genes in mammalian brain development.

A novel region referred to as the POU-domain is present in two tissue-specific transcription factors, Pit-1 and Oct-2, that activate expression of genes specifying pituitary and lymphocyte phenotypes. We report the identification of multiple new members of a large family of POU-domain genes expressed in adult brain, and document that all the known mammalian POU-domain genes, including Pit-1 and Oct-2, are expressed widely in the developing nervous system.

Tissue specific trans-acting factor interaction with proximal rat prolactin gene promoter sequences.

Using an exonuclease III protection assay, strong, reversible and tissue-specific binding of GH3 cell nuclear factors to proximal regions of the rat prolactin (rPrl) promoter (-31 to -77) has been detected. A second less prominent region of factor binding, that may have a correlate in HeLa cell extracts, was detected in the region (-155 to -180). The binding is eliminated in the presence of excess unlabelled rPrl promoter sequences (-423 to +38), excess unlabelled distal rPrl 5'-flanking sequences (-1960 to -1260) and SV40-enhancer/promoter sequences; it is largely unaffected by growth hormone (rGH) promoter and RSV-LTR sequences. A plasmid containing the proximal rPrl promoter sequences (-75 to +38) was also shown to be an avid inhibitor, at low concentrations, of rPrl promoter driven chloramphenicol acetyl transferase (CAT) gene expression in transient cotransfection competition studies; under these assay conditions distal rPrl 5'-flanking sequences and RSV and rGH promoter plasmids do not compete. The results emphasize the critical importance of proximal rPrl promoter sequences for prolactin gene expression in GH3 cells but recognize the related functional potential of more distal sequences.