Pit-1 Binding Research Articles

PiT1/Slc20a1 Is Required for Endoplasmic Reticulum Homeostasis, Chondrocyte Survival, and Skeletal Development.

During skeletal mineralization, the sodium-phosphate co-transporter PiT1Slc20a1 is assumed to meet the phosphate requirements of bone-forming cells, although evidence is missing. Here, we used a conditional gene deletion approach to determine the role of PiT1 in growth plate chondrocytes. We show that PiT1 ablation shortly after birth generates a rapid and massive cell death in the center of the growth plate, together with an uncompensated endoplasmic reticulum (ER) stress, characterized by morphological changes and increased Chop, Atf4, and Bip expression. PiT1 expression in chondrocytes was not found at the cell membrane but co-localized with the ER marker ERp46, and was upregulated by the unfolded protein response cascade. In addition, we identified the protein disulfide isomerase (Pdi) ER chaperone as a PiT1 binding partner and showed that PiT1 ablation impaired Pdi reductase activity. The ER stress induced by PiT1 deficiency in chondrocytes was associated with intracellular retention of aggrecan and vascular endothelial growth factor A (Vegf-A), which was rescued by overexpressing a phosphate transport-deficient mutant of PiT1. Our data thus reveal a novel, Pi-transport independent function of PiT1, as a critical modulator of ER homeostasis and chondrocyte survival during endochondral ossification. © 2018 American Society for Bone and Mineral Research.

Disruption of the Phosphate Transporter Pit1 in Hepatocytes Improves Glucose Metabolism and Insulin Signaling by Modulating the USP7/IRS1 Interaction

The liver plays a central role in whole-body lipid andglucose homeostasis. Increasing dietary fat intake results in increased hepatic fat deposition, which is associated with a risk for development ofinsulin resistance and type 2 diabetes. In this study, we demonstrate a role for the phosphate inorganic transporter 1 (PiT1/SLC20A1) in regulating metabolism. Specific knockout of Pit1 in hepatocytes significantly improved glucose tolerance and insulin sensitivity, enhanced insulin signaling, and decreased hepatic lipogenesis. We identified USP7 as a PiT1 binding partner and demonstrated that Pit1 deletion inhibited USP7/IRS1 dissociation upon insulin stimulation. This prevented IRS1 ubiquitination and its subsequent proteasomal degradation. As a consequence, delayed insulin negative feedback loop and sustained insulin signaling were observed. Moreover, PiT1-deficient mice were protected against high-fat-diet-induced obesity and diabetes. Our findings indicate that PiT1 has potential as a therapeutic target in the context of metabolic syndrome, obesity, and diabetes.

A Pit-1 Binding Site Adjacent to E-box133 in the Rat PRL Promoter is Necessary for Pulsatile Gene Expression Activity.

Recent evidence reveals that prolactin gene expression (PRL-GE) in mammotropes occurs in pulses, but the molecular process(es) underlying this phenomenon remains unclear. Earlier, we have identified an E-box (E-box133) in the rat PRL promoter that binds several circadian elements and is critical for this dynamic process. Preliminary analysis revealed a Pit-1 binding site (P2) located immediately adjacent to this E-box133 raising the possibility that some type of functional relationship may exist between these two promoter regions. In this study, using serum shocked GH3 cell culture system to synchronize PRL-GE activity, we determined that Pit-1 gene expression occurred in pulses with time phases similar to that for PRL. Interestingly, EMSA analysis not only confirmed Pit-1 binding to the P2 site, but also revealed an interaction with factor(s) binding to the adjacent E-box133 promoter element. Additionally, down-regulation of Pit-1 by siRNA reduced PRL levels during pulse periods. Thus, using multiple evidences, our results demonstrate clearly that the Pit-1 P2 site is necessary for PRL-GE elaboration. Furthermore, the proximity of this critical Pit-1 binding site (P2) and the E-box133 element coupled with the evidences of a site-to-site protein interactions suggest that the process of PRL-GE pulse activity might involve more dynamic and intricate cross-talks between promoter elements that may span some, or all, of the proximal region of the PRL promoter in driving its pulsatile expression.

Constitutive Somatostatin Receptor Subtype-3 Signaling Suppresses Growth Hormone Synthesis

Somatostatin signals through somatostatin receptor subtypes (SSTR) 2 and 5 to attenuate GH secretion. Although expressed in normal pituitary glands and in GH-secreting pituitary tumors, SSTR3 function was unclear, and we have now determined the role of SSTR3 in somatotroph function. Stable rat pituitary tumor cell (GC) transfectants of human SSTR3 (GpSSTR3WT) showed suppression of rat (r) GH promoter activity, GH mRNA expression, and secreted GH concordant with suppressed cAMP/protein kinase A (PKA) signaling. In contrast, cAMP levels and GH expression were unchanged in cells expressing a mutant SSTR3 DRY motif (GpSSTR3R141A). GH expression was rescued by treatment of GpSSTR3WT with forskolin and 8-bromo-cAMP. GpSSTR3WT exhibited activation of glycogen synthase kinase3-β (GSK3-β), a PKA substrate, which was also reversed by 8-Bromo-cAMP treatment. Moreover, SSTR3-dependent GH transcriptional inhibition was rescued by inhibition of GSK3-β. GpSSTR3WT exhibited elevated Pit-1 serine phosphorylation and decreased Pit-1 occupancy of the rGH promoter with sustained Pit-1 expression. GSK3-β and Pit-1 physically interacted with each other, indicating that Pit-1 may be a GSK3-β phosphorylation substrate. In conclusion, constitutive SSTR3 activity mediates transcriptional repression of GH through cAMP/PKA, leading to subsequent activation of GSK3-β and increased Pit-1 phosphorylation and ultimately attenuating Pit-1 binding to the rGH promoter.

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.

Enhancer-Blocking Activity Is Associated with Hypersensitive Site V Sequences in the Human Growth Hormone Locus Control Region

Activation of the human growth hormone gene (hGH-N) is linked to a locus control region (LCR) containing four (I-III, V) hypersensitive sites (HS). Pit-1 binding to HS I/II is required for efficient pituitary expression. However, inclusion of HS III and V, located about 28 and 32 kb upstream of the hGH-N gene, respectively, is also required for consistent hGH-N expression levels in vivo. HS V is referred to as a boundary for the hGH LCR, but no specific enhancer blocking or barrier function is reported. We examined a 547 bp fragment containing HS V sequences (nucleotides -32,718/-32,172 relative to hGH-N) for enhancer-blocking activity using a well-established transient gene transfer system and assessed these sequences for CCCTC binding factor (CTCF), which is linked to enhancer-blocking activity. The 547 bp HS V fragment decreased enhancer activity with a reverse-orientation preference when inserted between HS III enhancer sequences and a minimal thymidine kinase promoter (TKp). These sequences are associated with CTCF in human pituitary and nonpituitary chromatin. Enhancer-blocking activity with an orientation preference was further localized to a 45 bp sub-fragment, with evidence of CTCF and upstream binding factor 1 (USF1) binding; USF1 is linked more closely with barrier function. The presence of yin and yang 1 (Yy1) that cooperates with CTCF in the regulation of X-chromosome inactivation was also seen. A decrease in CTCF and Yy1 RNA levels was associated with a significant reduction in enhancer-blocking activity. Assessment of CpG-dinucleotides in the TKp indicates that the presence of HS V sequences are associated with an increased incidence of CpG-dinucleotide methylation of the GC box region. These data support association of CTCF and enhancer-blocking activity with HS V that is consistent with a role as a (LCR) boundary element and also implicates Yy1 in this process.

Identification and functional analysis of novel human growth hormone-releasing hormone receptor (GHRHR) gene mutations in Japanese subjects with short stature

Growth hormone-releasing hormone receptor (GHRHR) gene mutations have been identified in patients of different ethnic origins with isolated GH deficiency (IGHD) type IB. However, the prevalence of these mutations in the Japanese population has yet to be fully determined. This study aimed to evaluate the contributions of GHRHR mutations to the molecular mechanism underlying short stature in Japanese subjects. The GHRHR gene was sequenced in 127 unrelated Japanese patients with either IGHD (n = 14) or idiopathic short stature (ISS; n = 113). Sequence variants were evaluated in family members and 188 controls, and then examined in functional studies. A novel homozygous E382E (c.1146G>A) synonymous variant, at the last base of exon 12, was identified in an IGHD family with two affected sisters. In vitro splicing studies showed this mutation to result in skipping of exon 12. In one ISS patient, a heterozygous ATG-166T>C variant was found in the distal Pit-1 P2 binding element of the GHRHR promoter. In two control subjects, a close but distinct variant, ATG-164T>C, was detected. Functional studies showed that both promoter variants diminish promoter activity by altering Pit-1 binding ability. Four missense variants were also found in both patient and control groups but had no detectable functional consequences. The homozygous GHRHR mutation was rare, being detected in only one Japanese IGHD family. Future research is needed to clarify the genetic contributions of heterozygous functional promoter variants to GHD, ISS and normal-stature variations.

Appearance of the pituitary factor Pit-1 increases chromatin remodeling at hypersensitive site III in the human GH locus

Expression of pituitary and placental members of the human GH and chorionic somatomammotropin (CS) gene family is directed by an upstream remote locus control region (LCR). Pituitary-specific expression of GH requires direct binding of Pit-1 (listed as POU1F1 in the HUGO database) to sequences marked by a hypersensitive site (HS) region (HS I/II) 14.6 kb upstream of the GH-N gene (listed as GH1 in the HUGO database). We used human embryonic kidney 293 (HEK293) cells overexpressing wild-type and mutant Pit-1 proteins as a model system to gain insight into the mechanism by which Pit-1 gains access to the GH LCR. Addition of Pit-1 to these cells increased DNA accessibility at HS III, located 28 kb upstream of the human GH-N gene, in a POU homeodomain-dependent manner, as reflected by effects on histone hyperacetylation and RNA polymerase II activity. Direct binding of Pit-1 to HS III sequences is not supported. However, the potential for binding of ETS family members to this region has been demonstrated, and Pit-1 association with this ETS element in HS III sequences requires the POU homeodomain. Also, both ETS1 and ELK1 co-precipitate from human pituitary extracts using two independent sources of Pit-1 antibodies. Finally, overexpression of ELK1 or Pit-1 expression in HEK293 cells increased GH-N RNA levels. However, while ELK1 overexpression also stimulated placental CS RNA levels, the effect of Pit-1 appeared to correlate with ETS factor levels and target GH-N preferentially. These data are consistent with recruitment and an early role for Pit-1 in remodeling of the GH LCR at the constitutively open HS III through protein-protein interaction.

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.

Expression of Pit-1 in Nonsomatotrope Cell Lines Induces Human Growth Hormone Locus Control Region Histone Modification and hGH-N Transcription

The POU domain transcription factor Pit-1 is expressed in somatotropes, lactotropes, and thyrotropes of the anterior pituitary. Pit-1 is essential for the establishment of these lineages during development and regulates the expression of genes encoding the peptide hormones secreted by each cell type, including the growth hormone gene expressed in somatotropes. In contrast to rodent growth hormone loci, the human growth hormone ( hGH) locus is regulated by a distal locus control region (LCR), which is required in cis for the proper expression of the hGH gene cluster in transgenic mice. The hGH LCR mediates a domain of histone acetylation targeted to the hGH locus that is associated with distal hGH-N activation, and the discrete determinants of this activity coincide with DNaseI hypersensitive site (HS) I of the LCR. The identification of three in vitro Pit-1 binding sites within the HS-I region suggested a model in which Pit-1 binding at HS-I initiates the chromatin modification mechanism associated with hGH LCR activity. To test this hypothesis directly and to determine whether Pit-1 expression is sufficient to confer hGH locus histone acetylation and activate hGH-N transcription from an inactive locus, we expressed Pit-1 in nonpituitary cell types. We show that Pit-1 expression established a domain of histone hyperacetylation at the LCR and hGH-N promoter in these cells similar to that observed in pituitary chromatin. This was accompanied by the activation of hGH-N transcription and an increase in intergenic and CD79b transcripts proximal to HS-I. These effects were coincident with Pit-1 occupancy at HS-I and the hGH-N promoter and were observed irrespective of the basal histone modification status of HS-I in the heterologous cell line. These findings are consistent with a role for Pit-1 as an initiating factor in hGH locus activation during somatotrope ontogeny, acting through binding sites at HS-I of the hGH LCR.

Involvement of mPOU (Brn-5), a class VI POU protein, in the gene expression of Pit-1 as well as PRL

PRL is mainly expressed in the pituitary and its gene expression is regulated by a variety of transcription factors including Pit-1. Brn-5 is a transcription factor that binds to Pit-1 binding elements and stimulates PRL reporter gene expression. In this study, the role of Brn-5 was examined. RNA interference (RNAi) against Brn-5 leaded to reduction in PRL content of GH3 cells, indicating endogenous Brn-5 may play a role in PRL gene expression. Furthermore Brn-5 RNAi decreased Pit-1 mRNA. Transfection of expression vectors for mPOU (human ortholog of Brn-5) modestly but significantly stimulated activities of PRL-Luc and Pit-1-Luc reporter genes in GH3 and HEK 293 cells. In addition, mPOU showed synergistic action with Pit-1 and CBP on PRL-Luc expression. mPOU-FL, a splicing variant of mPOU, showed weaker activity than mPOU. Chip assay suggested binding of mPOU to PRL and Pit-1 promoters of genomic DNA. Taken together, these results suggest that mPOU (Brn-5) enhances PRL gene expression directly in associate with Pit-1 and CBP, and indirectly via the activation of Pit-1 gene expression.

Down-regulation of goldfish ( Carassius auratus) prolactin gene expression by dopamine and thyrotropin releasing hormone

Prolactin (PRL) is the most versatile hormone identified with multiple functions from osmoregulation in euryhaline fish to lactation in mammals. However, little is known about the basic physiological control of PRL in stenohaline freshwater fish. In this study, goldfish is used as a model for the study of PRL gene expression in stenohaline fish. We report herein the identification of the goldfish PRL (gfPRL) genomic sequence which possesses five exons with its 5′-flanking gene promoter region characterized in vitro using GH3 and GH4 cell-lines. Dopamine and thyrotropin releasing hormone were found to down-regulate the transcription of this gfPRL gene promoter in vitro. This was further confirmed by the decrease of PRL mRNA levels in vitro (in goldfish pituitary primary cells) and in vivo (intra-peritoneal injection) following the administrations of dopamine and thyrotropin releasing hormone. The Pit-1 binding sites of gfPRL are highly conserved with a consensus DNA sequence of 5′TATNCAT-3′, as confirmed with the electrophoretic mobility shift assay using nuclear extract from the GH3 cell-line, may be responsible for the control of gfPRL gene promoter.

Cellular Expression Levels of the Vitamin D Receptor Are Critical to Its Transcriptional Regulation by the Pituitary Transcription Factor Pit-1

The biological role of 1,25-dihydroxyvitamin D(3) has generally been related to calcium homeostasis, but this hormone also has fundamental effects on processes of cellular proliferation and differentiation. The genomic actions of 1,25-dihydroxyvitamin D(3) are mediated by the vitamin D receptor (VDR) present in target cells. However, VDR transcriptional regulation is not well understood, probably attributable to the complexity of the VDR gene and its promoter. In the present study, it is demonstrated that administration of the pituitary transcription factor Pit-1 (originally found in the pituitary gland but also present in other nonpituitary cell types and tissues) to the MCF-7 (human breast adenocarcinoma) cell line induces a significant increase in VDR mRNA and protein levels. Conversely, Pit-1-targeted small interference RNA markedly reduced expression of VDR in MCF-7 cells. Reporter gene assays demonstrated that the effect of Pit-1 is mediated by its binding to a region located between -254 and -246 bp from the VDR transcription start site. Selective mutations of this site completely abolished VDR transcription. Chromatin immunoprecipitation analysis showed that binding of Pit-1 to the VDR promoter leads additionally to recruitment of cAMP response element-binding protein binding protein, acetylated histone H4, and RNA polymerase II. Surprisingly, Pit-1 binding also recruits VDR protein to the VDR promoter. Using several cell lines with different levels of VDR expression, it was demonstrated that up-regulation of VDR transcription by Pit-1 is dependent on the presence of VDR protein, suggesting that transcriptional expression of VDR in a given cell type is dependent on, among other factors, its own expression levels.

Regulation of growth hormone expression by Delta-like protein 1 (Dlk1)

Delta-like protein 1 (Dlk1) is a transmembrane protein characterized by epidermal growth factor (EGF)-like repeats. Dlk1, which is also known as preadipocyte factor 1 (pref-1) because of its ability to inhibit preadipocyte differentiation, regulates the differentiation of several other cell types through unknown mechanisms. To elucidate Dlk1 functions, identification of Dlk1-regulated target genes is critical. The observation that Dlk1 is expressed in many endocrine tissues suggests that Dlk1 may have endocrine-related functions. Because Dlk1 is expressed in GH producing cells, we hypothesize that one function of Dlk1 is to regulate GH expression. We found that GH mRNA, protein, and secretion were significantly decreased in GH3 pituitary cell clones that stably express Dlk1. In contrast, Dlk1 expression was unable to alter prolactin expression. Co-transfection of GH3 cells with a GH promoter-regulated reporter gene showed that Dlk1 repressed GH promoter activity. Deletion and mutation analysis of the GH promoter indicated that Pit-1 binding sites in the GH promoter are required for Dlk1-mediated repression. Furthermore, Dlk1 expression represses Pit-1-mediated transcription when both proteins are co-expressed in MCF-7 cells. Deletion analysis of Dlk1 revealed that the ability of Dlk1 to regulate GH promoter activity is independent of both its EGF-like repeats and its ability to modulate MAP kinase activity. The observation that Dlk1 regulates GH expression identifies the first endocrine function of Dlk1, establishes GH as a Dlk1-regulated target gene, and provides a model system to facilitate studies of Dlk1-mediated signaling.

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.

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.

The role of pit-1 in the regulation of the rat growth hormone-releasing hormone receptor gene transcription by glucocorticoids

Glucocorticoids are involved in the regulation of the rat growth hormone-releasing hormone (GHRH) receptor gene expression, but they act only in the presence of the pituitary specific transcription factor, pit-1. In this study, the role of pit-1 in the glucocorticoid stimulation of the GHRH-receptor gene transcription was examined. The results suggest the presence of a silencer element in the promoter and it is postulated that pit-1 permits glucocorticoid action through suppressing the inhibitory effect of an as yet unknown factor that binds to this element. The present results also suggest that the synergistic activation of the rat GHRH-receptor gene transcription depends on the proper distance between the proximal glucocorticoid response element and the pit-1 binding site.

Hepatocyte Nuclear Factor-3α Binding at P Sequences of the Human Growth Hormone Locus Is Associated with Pituitary Repressor Function

The human GH family consists of five genes, including the placental chorionic somatomammotropins (CS), within a single locus on chromosome 17. Based on nuclease sensitivity, the entire GH/CS locus is accessible in pituitary chromatin, yet only GH-N is expressed. Previously, we reported a P sequence element (263P) capable of repressing placental CS-A promoter activity in transfected pituitary (GC) cells, and our data indicated a possible role for nuclear factor-1 (NF-1) and regulatory factor X1 in this repression. In this study we show the formation of two independent pituitary complexes in vitro: a repressor complex containing NF-1 and a nonfunctional complex containing regulatory factor X1. In vitro repressor function is stabilized by the presence of P sequence element C (PSE-C), downstream of the previously characterized PSE-A and PSE-B. Repressor function is also dependent on an intact Pit-1 binding site in the CS-A promoter. EMSAs with PSE-C reveal binding of the hepatocyte nuclear factor-3/forkhead (HNF-3/fkh) family of transcription factors in rat pituitary GC cells. This observation is extended to human pituitary tissue, where HNF-3alpha's association with P sequences is confirmed by chromatin immunoprecipitation. Furthermore, protein-protein interactions between HNF-3alpha and NF-1 family members are demonstrated. These results identify HNF-3alpha as an additional member of the pituitary P sequence regulatory complex, implicating it in tissue-specific expression of the human GH/CS family.

Characterization of the 5' flanking region of the growth hormone gene of the marine teleost, gilthead sea bream Sparus aurata: analysis of a polymorphic microsatellite in the proximal promoter

The 5′ flanking region (1712 bp) of the growth hormone (GH) gene of the gilthead sea bream Sparus aurata was cloned, sequenced and characterized. It contains a consensus sequence for TATA box and several Pit-1 binding site motifs. Consensus sequences related to cyclic AMP response element, glucocortiocoid response element, and several other transcription factors were identified by comparison to consensus sequences in fish or mammalian GH genes. The promoter contains two microsatellites (di- and tri-nucleotides repeats) with additional upstream microsatellites (tri-and tetranucleotides), 10-mer tandem repeat, and two long inverted repeats. Analysis of the proximal dinucleotide microsatellite by polymerase chain reaction revealed polymorphism among individuals from a hatchery population and an association of alleles 250 and 254 with growth performance. Segregation analysis of this marker in one family showed Mendelian inheritance. These results suggest that the microsatellite in the promoter region (termed saGHpCA) might be considered as a candidate genetic marker for broodstock management and growth selection programs of Sparus aurata.

The Zinc Finger Ikaros Transcription Factor Regulates Pituitary Growth Hormone and Prolactin Gene Expression through Distinct Effects on Chromatin Accessibility

The Ikaros transcription factors perform critical functions in the control of lymphohematopoiesis and immune regulation. Family members contain multiple zinc fingers that mediate DNA binding but have also been implicated as part of a complex chromatin-remodeling network. We show here that Ikaros is expressed in pituitary mammosomatotrophs where it regulates the GH and prolactin (PRL) genes. Ikaros was detected by Northern and Western blotting in GH4 pituitary mammosomatotroph cells. Wild-type Ikaros (Ik1) inhibits GH mRNA and protein expression but stimulates PRL mRNA and protein levels. Ikaros does not bind directly to the proximal GH promoter but abrogates the effect of the histone deacetylation inhibitor trichostatin A on this region. Ikaros selectively deacetylates histone 3 residues on the proximal transfected or endogenous GH promoter and limits access of the Pit1 activator. In contrast, Ikaros acetylates histone 3 on the proximal PRL promoter and facilitates Pit1 binding to this region in the same cells. These data provide evidence for Ikaros-mediated histone acetylation and chromatin remodeling in the selective regulation of pituitary GH and PRL hormone gene expression.