Pit-1 Sites Research Articles

Design of Drainage System Coal Mine at PT. Tebo Agung International Site Project, Semambu Village, Sumay District, Tebo Regency, Jambi

Tebo Agung Internasional Ltd is one of the companies in coal mining that employs the open-pit mining method. This sort of method will create a large basin that can accumulate water inside the mining pit. The drainage system is applied at the Pit-1 Site Semambu is mine-dewatering carried out by draining the water into the sump so that it can be pumped out of the mining area and prevent the runoff from coming inside through an open drainage system (ditch). The result of data analysis demonstrated that referring to the daily rainfall in 2010 – 2019 through the distribution of Log Person Type III, the maximum rainfall being planned was 508.019 mm/day within the return period of 5 years. The intensity of rainfall at the research site was 82.5 mm/hour having a rain duration averagely of 3.1 hours/day. Pit-1 Site Semambu had a catchment area around 469,317.15 m2 and a water discharge totally 61,238.81 m3/day derived from rainwater discharge of 48,530.48 m3/day and runoff water discharge 12,708.33 m3/day, whereas the capacity of the temporary accommodating pond (Sump) was 39,539.55 m3. The pump at the research site could not be operated anymore (broken). Accordingly, the recommendation that can be given to the company is changing the broken pumps with 4 units of multi fall 420 pumps having operating speed 1,300 rpm and actual discharge yielded by the pump 871.64 m3/hour. The pump worked for 14 hours/day and the total water discharge that could be produced was 12,202.96 m3/day. Open channel (ditch) at Pit-1 Site Semambu was the only one with accommodated discharge 1.27 m3/second. Thus, the recommendation that could be delivered to optimally prevent the runoff water coming into the mining area is by adding 2 ditches having the capacities of 0.90 m3/second and 0.75 m3/second which have been adjusted to the planned water discharge.

Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

Fish growth-hormone genes: Evidence of functionality of paralogous genes in Levanidov’s charr Salvelinus levanidovi

The growth-hormone gene is presented as a single copy in the genome of most vertebrates. In contrast, in salmonids, one of the genome duplication events resulted in the multiplication of many genes, including the growth hormone. In salmonids, the growth-hormone gene exists as two independently inherited functional paralogues, gh1 and gh2. In this study, we performed a comparative analysis of gh1 and gh2 growth-hormone genes and their adjacent sequences in Levanidov’s charr Salvelinus levanidovi to determine their functionality and define the potential differences. We found that both genes have the same gene structure and are composed of six exons (I–VI) and five introns (A, B, C, D, E). However, the respective gene sequences differ in length. A comparison of exons showed that the size of each exon is identical in both paralogues. The overall length of genes differs due to the varying lengths of introns. Coding sequence of both genes contains an open reading frame for 210 amino acids. We identified regulatory elements in the promoter region of both genes: TATA box, A/T-rich regions that contain binding sites for pituitary-specific transcriptional activator Pit-1, and regions responsible for interaction with other transcriptional activators and initiators, in particular hormone receptors. Thus, the obtained data indicates that both gh1 and gh2 genes in Levanidov’s charr Salvelinus levanidovi genome are functional.

Fish growth-hormone genes: functionality evidence of paralogous genes in Levanidov's charr

In the genome of most vertebrates growth-hormone gene is presented in a single copy, while in salmonids after one of the duplication events many genes were multiplied, including growth hormone gene. In salmonids, the growth-hormone gene exists as two independently inherited functional paralogues, gh1 and gh2. In this study, we performed a comparative analysis of gh1 and gh2 growth-hormone genes and their adjacent sequences in Levanidov's charr Salvelinus levanidovi to determine their functionality and define the potential differences. We found that both genes have the same gene structure and are composed of six exons (I-VI) and five introns (A, B, C, D, E). However, the respective gene sequences differ in length. A comparison of exons showed that the size of each exon is identical in both paralogues. The overall length of genes differs due to the varying lengths of introns. Coding sequence of both genes contains an open reading frame for 210 amino acids. We identified regulatory elements in the promoter region of both genes: TATA box, A/T-rich regions that contain binding sites for pituitary-specific transcriptional activator Pit-1, and regions responsible for interaction with other transcriptional activators and initiators, in particular hormone receptors. The obtained data indicate that both genes are functional.

Ras-dva Is a Novel Pit-1- and Glucocorticoid-Regulated Gene in the Embryonic Anterior Pituitary Gland

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

A Pit-1 Threonine 220 Phosphomimic Reduces Binding to Monomeric DNA Sites to Inhibit Ras and Estrogen Stimulation of the Prolactin Gene Promoter

Pit-1 is a POU-homeodomain transcription factor that dictates the ontogeny of pituitary somatotrophs, lactotrophs, and thyrotrophs through regulation of their respective protein hormone genes: GH, prolactin (PRL), and TSHbeta. Although Pit-1 threonine 220 (T220) and serine 115 are protein kinase phospho-acceptor sites, the transcriptional role of Pit-1 phosphorylation remains unclear. In the rat PRL promoter (rPRL), Ras-stimulated transcription is mediated by binding of Ets-1 and Pit-1 at a composite site (FPIV). Ets-1 and Pit-1 physically interact, and Pit-1 T220 is a major Ets-1 contact point. T220 was mutated to aspartic acid (D, to mimic phosphorylation) or a neutral alanine (A), and DNA binding and transcriptional activity were tested. The Pit-1 T220D mutation reduced binding at monomeric Pit-1 sites (FPIV, PRL-1d), but not dimeric Pit-1 sites (FPI). Pit-1 T220A bound all sites with wild-type (WT) affinity. In transfections of HeLa cells, each Pit-1 mutant transcriptionally activated the -425rPRL promoter and cooperated with Ets-1 to WT levels. In contrast, Pit-1-mediated Ras activation of the -425 rPRL promoter was significantly inhibited by T220D. Finally, Pit-1 synergistic activation of the 2500-bp rPRL promoter with estrogen receptor was reduced by T220D compared with T220A and WT Pit-1. Thus, phosphorylation of Pit-1 T220 reduces binding to monomeric sites blunting Ras and estrogen/estrogen receptor stimulation of the rPRL gene promoter. Consequently, T220 phosphorylation of Pit-1 by protein kinase A, protein kinase C, or cell cycle-dependent kinases appears to serve as a regulatory switch, inhibiting Ras and estrogen/estrogen receptor regulatory pathways, while enhancing the cAMP/protein kinase A response, thus allowing a more precise integration of pituitary responses to distinct signaling stimuli.

Genomic structure and transcriptional regulation of grass carp calmodulin gene

A fish calmodulin (CaM) gene was characterized for the first time in grass carp. The CaM gene is about 12-Kb in size with identical intron/exon organization as that of mammalian CaM genes. When compared to mammalian counterparts, the 5′-promoter region of grass carp CaM gene contains a TATA box and has a much lower GC content and CpG dinucleotide frequency. Interestingly, the 5′-promoter of carp CaM gene is AT-rich with multiple IRS elements and putative binding sites for Pit-1, Sp1/Sp3 and AP1. Using luciferase reporter assay, a potent silencer region was identified in the distal region of grass carp CaM promoter. Besides, the CaM promoter activity could be upregulated by IGF but suppressed by PACAP, forskolin and over-expression of Sp1 and Sp3. These findings, taken together, indicate that grass carp CaM gene does not exhibit the typical features of housekeeping genes and its expression is under the control of hormone factors, presumably by coupling with the appropriate signaling pathways/transcription factors.

Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim, a phenyl-furan-benzimidazole dication DB293 binds AT-rich sites as a monomer and 5′-ATGA sequence as a stacked dimer, both in the minor groove. Here, we used a protein/DNA array approach to evaluate the ability of DB293 to specifically inhibit transcription factors DNA-binding in a single-step, competitive mode. DB293 inhibits two POU-domain transcription factors Pit-1 and Brn-3 but not IRF-1, despite the presence of an ATGA and AT-rich sites within all three consensus sequences. EMSA, DNase I footprinting and surface-plasmon-resonance experiments determined the precise binding site, affinity and stoichiometry of DB293 interaction to the consensus targets. Binding of DB293 occurred as a cooperative dimer on the ATGA part of Brn-3 site but as two monomers on AT-rich sites of IRF-1 sequence. For Pit-1 site, ATGA or AT-rich mutated sequences identified the contribution of both sites for DB293 recognition. In conclusion, DB293 is a strong inhibitor of two POU-domain transcription factors through a cooperative binding to ATGA. These findings are the first to show that heterocyclic dications can inhibit major groove transcription factors and they open the door to the control of transcription factors activity by those compounds.

Essential Role of GATA2 in the Negative Regulation of Thyrotropin β Gene by Thyroid Hormone and Its Receptors

Previously we reported that the negative regulation of the TSHbeta gene by T(3) and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15-30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.

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.

Modular changes of cis‐regulatory elements from two functional Pit1 genes in the duplicated genome of Cyprinus carpio

The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth hormone (GH). In the ectotherm teleost Cyprinus carpio, Pit1 plays an important role in regulation of the adaptive response to seasonal environmental changes. Two Pit1 genes exist in carp, a tetraploid vertebrate and transcripts of both genes were detected by RT-PCR analysis. Powerful comparative analyses of the 5'-flanking regions revealed copy specific changes comprising modular functional units in the naturally evolved promoters. These include the precise replacement of four nucleotides around the transcription start site embedded in completely conserved regions extending upstream of the TATA-box, an additional transcription factor binding site in the 5'-UTR of gene-I and, instead, duplication of a 9 bp element in gene-II. Binding of nuclear factors was assessed by electro mobility shift assays using extracts from rat pituitary cells and carp pituitary. Binding was confirmed at one conserved Pit1, one conserved CREB and one consensus MTF1. Interestingly, two functional Pit1 sites and one putative MTF1 binding site are unique to the Pit1 gene-I. In situ hybridization experiments revealed that the expression of gene-I in winter carp was significantly stronger than that of gene-II. Our data suggest that the specific control elements identified in the proximal regulatory region are physiologically relevant for the function of the duplicated Pit1 genes in carp and highlight modular changes in the architecture of two Pit1 genes that evolved for at least 12 MYA in the same organism.

A variation in a Pit-1 site in the growth hormone gene (GH1) promoter induces a differential transcriptional activity

The proximal promoter of the human growth hormone gene (GH1) is highly polymorphic. We tested if promoter haplotypes differing at possibly functional sites, namely −278T/G (in the NF1 binding site), −75A/G (in the proximal Pit-1 binding site) and −57G/T (in the VDR binding site), induced a different luciferase activity when transfected in a rat pituitary cell line. The presence of a G instead of an A at position −75 induced a more than two-fold reduced activity (p<0.0001). In accordance with this findings the electrophoretic mobility shift assay demonstrated a reduced affinity of the −75G for the pituitary transcription factor Pit-1. Despite the strong effect of this polymorphism in vitro, the −75G variation was not associated to an impairment of the GH secretion in vivo.

Role of specificity protein-1, PPARγ, and pituitary protein transcription factor-1 in transcriptional regulation of the murine CORS-26 promoter

The collagenous repeat-containing sequence of 26-kDa protein (CORS-26) was recently described as a new gene that is induced during adipocyte differentiation. Since the transcription factors specificity protein-1 (SP-1) and PPARγ have been demonstrated to modulate transcriptional activation of adipocytic genes, we investigated the putative role of SP-1 and PPARγ in the regulation of the murine CORS-26 promoter. Computer-based sequence analysis revealed two putative SP-1 binding sites and binding sites for PPARγ and Pit-1 within the TATA-box containing promoter. Electrophoretic mobility shift assays (EMSA) with nuclear extracts from 3T3-L1 adipocytes and appropriate promoter fragments demonstrated that SP-1 binds specifically to both SP-1 binding sites. Specificity was demonstrated by (i) the appearance of supershift bands, (ii) competition experiments and, (iii) by using oligonucleotides carrying mutated SP-1 binding sites. Functional promoter activity was analyzed by Luciferase reporter gene assays and SP-1 was shown to exert inhibitory effects on the transcriptional activation of the murine CORS-26 gene. Additionally, specific binding activity of PPARγ and Pit-1 to the CORS-26 promoter was demonstrated. Taken together, the present data demonstrate the functionality of the proximal murine CORS-26 promoter, which is regulated specifically by two SP-1 binding sites via SP-3-independent repressive effects of SP-1 on transcriptional activation. Pit-1 and PPARγ can bind specifically to the promoter and might play an additive functional role in gene regulation of murine CORS-26.

Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSHβ gene

Previous studies showed that Pit-1 functionally cooperates with GATA-2 to stimulate transcription of the TSHβ gene. Pit-1 and GATA-2 are uniquely coexpressed in pituitary thyrotropes and activate transcription by binding to a composite promoter element. To define the domains of Pit-1 important for functional cooperativity with GATA-2, we cotransfected a set of Pit-1 deletions with an mTSHβ-luciferase reporter. Plasmids were titrated to express equivalent amounts of protein. A mutant containing a deletion of the hinge region between the POU and homeodomains retained the ability to fully synergize with GATA-2. In contrast, mutants containing deletions of amino acids 2–80 or 72–125 demonstrated 56 or 34% of the synergy found with the full-length protein, suggesting that these regions contributed to cooperativity. Mutants with deletions of the POU-specific or homeodomain further reduced the effect signifying the requirement for DNA binding. GST interaction studies demonstrated that only the homeodomain of Pit-1 interacted with GATA-2. Finally, several mutations between the Pit-1 and GATA-2 sites on the TSHβ promoter reduced binding for each factor and greatly reduced ternary complex formation. Thus multiple domains of Pit-1 are required for full synergy with GATA-2 and sequences between the two binding sites contribute to co-occupancy with both factors on the proximal TSHβ promoter.

Specification of unique Pit-1 activity in the hGH locus control region.

The human GH (hGH) gene cluster is regulated by a remote 5' locus control region (LCR). HSI, an LCR component located 14.5 kb 5' to the hGH-N promoter, constitutes the primary determinant of high-level hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast, the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary.

A composite hormone response element regulates transcription of the rat GHRH receptor gene.

To further elucidate the molecular mechanisms underlying the transcriptional regulation of the GHRH receptor (GHRH-R) gene, hormonal regulation of the promoter activity of this gene was examined. An approximately 3-kb genomic fragment spanning the promoter region of the gene was sequenced and the transcription start site was determined by RT-PCR and RNase protection assay. A major start site was localized at -105 (relative to the translation initiation codon, ATG), and a pit-1 binding sequence characteristic of pituitary specific genes was found at -155 to -146. Deletion and mutation studies demonstrated this site to be functional. In the presence of dexamethasone, the GHRH-R promoter (from -2935 to -11) directed luciferase expression in MtT-S cells, a somatotropic cell line, but not in the PC12 cells that normally do not express GHRH-R. While T(3), all trans-RA, and 9cis-RA alone weakly enhanced the reporter gene expression, each of these substances was found to act as a synergistic enhancer in the presence of dexamethasone. Additional deletion and mutation analyses demonstrated a functional RA response element at -1090 to -1074. Two functional glucocorticoid response elements and a T(3) response element were found in an 80-bp 5'-flanking sequence of the pit-1 site. Interestingly, it is suggested that the 6-bp half-site AGGACA (from -209 to -204) functions as a 3'-half-site of T(3) response element as well as a 5'-half-site of one of the glucocorticoid response elements.

Trout GH promoter analysis reveals a modular pattern of regulation consistent with the diversification of GH gene control and function in vertebrates

In vertebrates, growth hormone (GH) gene expression requires the pituitary-specific transcription factor Pit-1/GHF1 but is differently regulated by a variety of factors in different vertebrate species. Here, we have studied the transcriptional activity of the trout GH (tGH) promoter, which is synergistically stimulated by cAMP and glucocorticoid. Gel shift assays indicated that Pit-1 binds as a dimer to three high affinity sites in the −226/+24 tGH region, and that recombinant cAMP response element (CRE)-binding protein (CREB) binds to a CRE situated between the two distal Pit-1 sites. Deletional and mutational transfection experiments, performed in pituitary Pit-1-expressing GC cells, showed that the different Pit-1 sites play distinct roles and are obligatory elements in the mechanisms mediating cAMP and glucocorticoid responses. Remarkably, the results suggest a hierarchical modular model of regulation of the tGH promoter, according to which a critical module, triggered by Pit-1 bound to the proximal Pit-1 site, is necessary and sufficient to turn on and drive basal levels of transcription. The latter may be stimulated synergistically by two Pit-1-dependent reciprocally non-cooperative auxiliary modules, activated by cAMP and glucocorticoid, respectively. Such modularity explains, in evolutionary terms, the crucial role played by Pit-1 in transcriptional activation and the emergence of the wide variety of mechanisms regulating transcriptional levels of GH, prolactin and other Pit-1-target genes in vertebrates.

Genomic Organization and Expression of the Mouse Equilibrative, Nitrobenzylthioinosine-Sensitive Nucleoside Transporter 1 (ENT1) Gene

We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C. About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3′-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5′-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5′-flanking region showed that the nucleotide sequence from −652 to −111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (−296/−303, −307/−313) and two MAZ binding sites (−353/−359, −522/−528) in this region. Luciferase assay results suggest that MAZ and Sp-1 transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.

Dual regulation of somatostatin receptor subtype 1 gene expression by pit-1 in anterior pituitary GH3 cells.

Somatostatin represents a major release inhibiting factor for hypophyseal hormones and mediates its action via five receptor subtypes, sst1-sst5, that are all present in the anterior pituitary. The pituitary specific transcription factor Pit-1 is essential for the pituitary development and pituitary-specific gene expression. Here the transcriptional regulation of the sst1 gene, which contains putative Pit-1-binding sites, was studied in anterior pituitary GH3 cells. We found that a fragment of 2 kb suffices to drive the expression of a reporter gene specifically in this cell line. Positive and negative cis-regulatory elements contributed to the promoter activity. Among these elements two functional binding sites for Pit-1 were identified. While the proximal site mediated transcriptional activation, the distal site attenuated transcription of reporter gene constructs. Mutations of the proximal Pit-1 site prevented expression of the reporter gene. Targeting Pit-1 mRNA by antisense oligonucleotides caused inhibition of transcription of reporter gene constructs containing the proximal Pit-1-binding site. Moreover, the expression of the endogenous sst1 gene in GH3 anterior pituitary cells was blocked. This resulted in reduced sst1 levels at the plasma membrane. Reduced sst1 levels were associated with a diminished antisecretory response to the sst1-specific agonist CH-275 and somatostatin. These results demonstrate the importance of Pit-1 for the expression of the sst1 gene, which hence is placed under common genetic control with the genes for hypophysiotropic hormones and the gene for the receptor of GH-releasing hormone.

Pit-1 Binding Sites at the Somatotrope-specific DNase I Hypersensitive Sites I, II of the Human Growth Hormone Locus Control Region Are Essential for in Vivo hGH-N Gene Activation

The human growth hormone gene cluster is composed of five closely related genes. The 5'-most gene in the cluster, hGH-N, is expressed exclusively in somatotropes and lactosomatotropes of the anterior pituitary. Although the hGH-N promoter contains functional binding sites for multiple transcription factors, including Sp1, Zn-15, and Pit-1, predictable and developmentally appropriate expression of hGH-N transgenes in the mouse pituitary requires the presence of a previously characterized locus control region (LCR) composed of multiple chromatin DNase I hypersensitive sites (HS). LCR determinant(s) necessary for hGH-N transgene activation are largely conferred by two closely spaced HS (HS I,II) located 14.5 kilobase pairs upstream of the hGH-N gene. The region sufficient to mediate this activity has recently been sublocalized to a 404-base pair segment of HS I,II (F14 segment). In the present study, we identified multiple binding sites for the pituitary POU domain transcription factor Pit-1 within this segment. Using a transgenic founder assay, these sites were shown to be required for high level, position-independent, and somatotrope-specific expression of a linked hGH-N transgene. Because the Pit-1 sites in the hGH-N gene promoter are insufficient for such gene activation in vivo, these data suggested a unique chromatin-mediated developmental role for Pit-1 in the hGH LCR.