GH Promoter Research Articles

OXR1A, a Coactivator of PRMT5 Regulating Histone Arginine Methylation

Oxidation resistance gene 1 (OXR1) protects cells against oxidative stress. We find that male mice with brain-specific isoform A knockout (Oxr1A-/-) develop fatty liver. RNA sequencing of male Oxr1A-/- liver indicates decreased growth hormone (GH) signaling, which is known to affect liver metabolism. Indeed, Gh expression is reduced in male mice Oxr1A-/- pituitary gland and in rat Oxr1A-/- pituitary adenoma cell-line GH3. Oxr1A-/- male mice show reduced fasting-blood GH levels. Pull-down and proximity ligation assays reveal that OXR1A is associated with arginine methyl transferase PRMT5. OXR1A-depleted GH3 cells show reduced symmetrical dimethylation of histone H3 arginine 2 (H3R2me2s), a product of PRMT5 catalyzed methylation, and chromatin immunoprecipitation (ChIP) of H3R2me2s shows reduced Gh promoter enrichment. Finally, we demonstrate with purified proteins that OXR1A stimulates PRMT5/MEP50-catalyzed H3R2me2s. Our data suggest that OXR1A is a coactivator of PRMT5, regulating histone arginine methylation and thereby GH production within the pituitary gland.

Decreased Activity of the Ghrhr and Gh Promoters Causes Dominantly Inherited GH Deficiency in Humanized GH1 Mouse Models.

Isolated growth hormone deficiency type II (IGHD2) is mainly caused by heterozygous splice-site mutations in intron 3 of the GH1 gene. A dominant-negative effect of the mutant GH lacking exon 3 on wild-type GH secretion has been proposed; however, the molecular mechanisms involved are elusive. To uncover the molecular systems underlying GH deficiency in IGHD2, we established IGHD2 model mice, which carry both wild-type and mutant copies of the human GH1 gene, replacing each of the endogenous mouse Gh loci. Our IGHD2 model mice exhibited growth retardation along with intact cellular architecture and mildly activated endoplasmic reticulum stress in the pituitary gland, caused by decreased GH-releasing hormone receptor (Ghrhr) and Gh gene promoter activities. Decreased Ghrhr and Gh promoter activities were likely caused by reduced levels of nuclear CREB3L2, which was demonstrated to stimulate Ghrhr and Gh promoter activity. To our knowledge, this is the first in vivo study to reveal a novel molecular mechanism of GH deficiency in IGHD2, representing a new paradigm that differs from widely accepted models.

Non-secreting pituitary tumours characterised by enhanced expression of YAP/TAZ.

Tumours of the anterior pituitary can manifest from all endocrine cell types but the mechanisms for determining their specification are not known. The Hippo kinase cascade is a crucial signalling pathway regulating growth and cell fate in numerous organs. There is mounting evidence implicating this in tumour formation, where it is emerging as an anti-cancer target. We previously demonstrated activity of the Hippo kinase cascade in the mouse pituitary and nuclear association of its effectors YAP/TAZ with SOX2-expressing pituitary stem cells. Here, we sought to investigate whether these components are expressed in the human pituitary and if they are deregulated in human pituitary tumours. Analysis of pathway components by immunofluorescence reveals pathway activity during normal human pituitary development and in the adult gland. Poorly differentiated pituitary tumours (null-cell adenomas, adamantinomatous craniopharyngiomas (ACPs) and papillary craniopharyngiomas (PCPs)), displayed enhanced expression of pathway effectors YAP/TAZ. In contrast, differentiated adenomas displayed lower or absent levels. Knockdown of the kinase-encoding Lats1 in GH3 rat mammosomatotropinoma cells suppressed Prl and Gh promoter activity following an increase in YAP/TAZ levels. In conclusion, we have demonstrated activity of the Hippo kinase cascade in the human pituitary and association of high YAP/TAZ with repression of the differentiated state both in vitro and in vivo. Characterisation of this pathway in pituitary tumours is of potential prognostic value, opening up putative avenues for treatments.

Contribution of LHX4 Mutations to Pituitary Deficits in a Cohort of 417 Unrelated Patients.

LHX4 encodes a LIM-homeodomain transcription factor that is implicated in early pituitary development. In humans, only 13 heterozygous LHX4 mutations have been associated with congenital hypopituitarism. The aims of this study were to evaluate the prevalence of LHX4 mutations in patients with hypopituitarism, to define the associated phenotypes, and to characterize the functional impact of the identified variants and the respective role of the 2 LIM domains of LHX4. We screened 417 unrelated patients with isolated growth hormone deficiency or combined pituitary hormone deficiency associated with ectopic posterior pituitary and/or sella turcica anomalies for LHX4 mutations (Sanger sequencing). In vitro studies were performed to assess the functional consequences of the identified variants. We identified 7 heterozygous variations, including p.(Tyr131*), p.(Arg48Thrfs*104), c.606+1G>T, p.Arg65Val, p.Thr163Pro, p.Arg221Gln, and p.Arg235Gln), that were associated with variable expressivity; 5 of the 7 were also associated with incomplete penetrance. The p.(Tyr131*), p.(Arg48Thrfs*104), p.Ala65Val, p.Thr163Pro, and p.Arg221Gln LHX4 variants are unable to transactivate the POU1F1 and GH promoters. As suggested by transactivation, subcellular localization, and protein-protein interaction studies, p.Arg235Gln is probably a rare polymorphism. Coimmunoprecipitation studies identified LHX3 as a potential protein partner of LHX4. As revealed by functional studies of LIM-defective recombinant LHX4 proteins, the LIM1 and LIM2 domains are not redundant. This study, performed in the largest cohort of patients screened so far for LHX4 mutations, describes 6 disease-causing mutations that are responsible for congenital hypopituitarism. LHX4 mutations were found to be associated with variable expressivity, and most of them with incomplete penetrance; their contribution to pituitary deficits that are associated with an ectopic posterior pituitary and/or a sella turcica defect is ∼1.4% in the 417 probands tested.

STAT3 upregulation in pituitary somatotroph adenomas induces growth hormone hypersecretion.

Pituitary somatotroph adenomas result in dysregulated growth hormone (GH) hypersecretion and acromegaly; however, regulatory mechanisms that promote GH hypersecretion remain elusive. Here, we provide evidence that STAT3 directly induces somatotroph tumor cell GH. Evaluation of pituitary tumors revealed that STAT3 expression was enhanced in human GH-secreting adenomas compared with that in nonsecreting pituitary tumors. Moreover, STAT3 and GH expression were concordant in a somatotroph adenoma tissue array. Promoter and expression analysis in a GH-secreting rat cell line (GH3) revealed that STAT3 specifically binds the Gh promoter and induces transcription. Stable expression of STAT3 in GH3 cells induced expression of endogenous GH, and expression of a constitutively active STAT3 further enhanced GH production. Conversely, expression of dominant-negative STAT3 abrogated GH expression. In primary human somatotroph adenoma-derived cell cultures, STAT3 suppression with the specific inhibitor S3I-201 attenuated GH transcription and reduced GH secretion in the majority of derivative cultures. In addition, S3I-201 attenuated somatotroph tumor growth and GH secretion in a rat xenograft model. GH induced STAT3 phosphorylation and nuclear translocation, indicating a positive feedback loop between STAT3 and GH in somatotroph tumor cells. Together, these results indicate that adenoma GH hypersecretion is the result of STAT3-dependent GH induction, which in turn promotes STAT3 expression, and suggest STAT3 as a potential therapeutic target for pituitary somatotroph adenomas.

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.

Constitutive Somatostatin Receptor Subtype 2 Activity Attenuates GH Synthesis

Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2(WT) cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2(DRY) stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR(2DRY) did not exhibit an enhanced octreotide response as did GpSSTR2(WT) cells; however, both SSTR2(WT)-enhanced yellow fluorescent protein (eYFP) and SSTR2(DRY)-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2(WT) cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2(DRY) cells. In vivo SAHA also increased GH release from GpSSTR2(WT) but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2(WT) compared with that of either GpSSTR(2DRY) or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.

Hypoxia and cytoplasmic alkalinization upregulate growth hormone expression in lymphocytes

We report here that culture of lymphoid cells under hypoxic conditions showed an increase in both luciferase expression from a GH-promoter luciferase construct and the levels of lymphocyte GH. The effect was mimicked by treatment of cells with cobalt chloride consistent with a specific oxygen-sensing mechanism. We identified a putative hypoxia response element (HRE) in the GH promoter at the region −176bp to −172bp that contains a copy of the hypoxia-inducible factor-1 (Hif-1) binding motif (5′-ACGTG-3′). The results also showed that culture of primary rat spleen cells with different doses of TMA induced a dose-dependent increase in lymphocyte GH by Western blot analysis. Greater levels of GH are induced in T cell-enriched populations compared to B cell-enriched populations after treatment with CoCl2 or TMA. Our results suggest that the stressful cellular conditions likely to occur at sites of inflammation or tumor growth may induce the synthesis of lymphocyte GH.

Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression

Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α- and ERβ-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERβ mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERβ in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

Differential Abilities of Chicken Pit1 Isoforms to Regulate the GH Promoter: Evidence for Synergistic Activation

Pit1, pituitary-specific transcription factor 1, regulates differentiation of cells of the Pit1 lineage in the anterior pituitary and the synthesis of peptide hormones by these cell types, including GH. Pit1 is characterized by an N-terminal transactivation domain and a C-terminal POU domain. Alternative forms of Pit1, differing from each other in the N-terminal domain, have been reported in several species, but the functional implication of having multiple isoforms is not known. Several PIT1 mRNA transcripts exist in chickens that have not been characterized. This study was conducted to determine which, if any, of the chicken Pit1 isoforms regulate the chicken GH (cGH) promoter. During the course of this work, Pit1β2, a novel isoform of chicken Pit1, was discovered. Effects of known and novel isoforms (Pit1α, Pit1β1, Pit1β2, and Pit1γ) on cGH promoter activity were characterized in chicken Leghorn male hepatoma cells. Three of the isoforms, Pit1α, Pit1β1, and Pit1β2, activated the cGH promoter, whereas Pit1γ did not. Results from gel-shift assays indicated that Pit1γ does not bind to the proximal Pit1-bindng site of the cGH promoter, suggesting a possible mechanism underlying its inactivity. We found a functional advantage for having multiple isoforms expressed. When Pit1β1 was coexpressed with Pit1α or Pit1β2, significantly greater activation of the cGH promoter occurred than with any one isoform alone, with synergistic activation occurring when Pit1α and Pit1β1 were coexpressed. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Identification of isoforms with specific functions will facilitate identification of their respective interacting partners that are essential for GH gene expression.

The Adult Pituitary Shows Stem/Progenitor Cell Activation in Response to Injury and Is Capable of Regeneration

The pituitary gland constitutes, together with the hypothalamus, the regulatory core of the endocrine system. Whether the gland is capable of cell regeneration after injury, in particular when suffered at adult age, is unknown. To investigate the adult pituitary's regenerative capacity and the response of its stem/progenitor cell compartment to damage, we constructed a transgenic mouse model to conditionally destroy pituitary cells. GHCre/iDTR mice express diphtheria toxin (DT) receptor after transcriptional activation by Cre recombinase, which is driven by the GH promoter. Treatment with DT for 3 d leads to gradual GH(+) (somatotrope) cell obliteration with a final ablation grade of 80-90% 1 wk later. The stem/progenitor cell-clustering side population promptly expands after injury, concordant with the immediate increase in Sox2(+) stem/progenitor cells. In addition, folliculo-stellate cells, previously designated as pituitary stem/progenitor cells and significantly overlapping with Sox2(+) cells, also increase in abundance. In situ examination reveals expansion of the Sox2(+) marginal-zone niche and appearance of remarkable Sox2(+) cells that contain GH. When mice are left after the DT-provoked lesion, GH(+) cells considerably regenerate during the following months. Double Sox2(+)/GH(+) cells are observed throughout the regenerative period, suggesting recovery of somatotropes from stem/progenitor cells, as further supported by 5-ethynyl-2'-deoxyuridine (EdU) pulse-chase lineage tracing. In conclusion, our study demonstrates that the adult pituitary gland holds regenerative competence and that tissue repair follows prompt activation and plausible involvement of the stem/progenitor cells.

A Novel Dysfunctional LHX4 Mutation with High Phenotypical Variability in Patients with Hypopituitarism

LHX4 is a LIM homeodomain transcription factor involved in pituitary ontogenesis. Only a few heterozygous LHX4 mutations have been reported to be responsible for congenital pituitary hormone deficiency. A total of 136 patients with congenital hypopituitarism associated with malformations of brain structures, pituitary stalk, or posterior pituitary gland was screened for LHX4 mutations. Three novel allelic variants that cause predicted changes in the protein sequence of LHX4 (2.3%) were found (p.Thr99fs, p.Thr90Met, and p.Gly370Ser). On the basis of functional studies, p.Thr99fs mutation was responsible for the patients' phenotype, whereas p.Thr90Met and p.Gly370Ser were likely polymorphisms. Patients bearing the heterozygous p.Thr99fs mutation had variable phenotypes: two brothers presented somato-lactotroph and thyrotroph deficiencies, with pituitary hypoplasia and poorly developed sella turcica; the youngest brother (propositus) also had corpus callosum hypoplasia and ectopic neurohypophysis; their father only had somatotroph deficiency and delayed puberty with pituitary hyperplasia. Functional studies showed that the mutation induced a complete loss of transcriptional activity on POU1F1 promoter and a lack of DNA binding. Cotransfection of p.Thr99fs mutant and wild-type LHX4 failed to evidence any dominant negative effect, suggesting a mechanism of haploinsufficiency. We also identified prolactin and GH promoters as potential target genes of LHX4 and found that the p.Thr99fs mutant was also unable to transactivate these promoters. The present report describes three new exonic LHX4 allelic variants with at least one being responsible for congenital hypopituitarism. It also extends the phenotypical heterogeneity associated with LHX4 mutations, which includes variable anterior pituitary hormone deficits, as well as pituitary and extrapituitary abnormalities.

Functional Sequestration of Transcription Factor Activity by Repetitive DNA

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) to the mouse major alpha-satellite repetitive DNA sequesters C/EBPalpha in the transcriptionally inert pericentromeric heterochromatin. We find that this sequestration reduces the transcriptional capacity of C/EBPalpha. Functional sequestration of C/EBPalpha was demonstrated by experimentally reducing C/EBPalpha binding to the major alpha-satellite DNA, which elevated the concentration of C/EBPalpha in the non-heterochromatic subcompartment of the cell nucleus. The reduction in C/EBPalpha binding to alpha-satellite DNA was induced by the co-expression of the transcription factor Pit-1, which removes C/EBPalpha from the heterochromatic compartment, and by the introduction of an altered-specificity mutation into C/EBPalpha that reduces binding to alpha-satellite DNA but permits normal binding to sites in some gene promoters. In both cases the loss of alpha-satellite DNA binding coincided with an elevation in the binding of C/EBPalpha to a promoter and an increased transcriptional output from that promoter. Thus, the binding of C/EBPalpha to this highly repetitive DNA reduced the amount of C/EBPalpha available for binding to and regulation of this promoter. The functional sequestration of some transcription factors through binding to repetitive DNAs may represent an underappreciated mechanism controlling transcription output.

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.

The NAD-dependent protein deacetylase Sirt1 is present in the human anterior pituitary gland where it plays a role in GH transcription

One of the best known experimental procedures that extend lifespan and resistance to aging-related diseases is caloric restriction. In yeast, C. elegans and Drosophila, Sir2 was shown to mediate the lifespan extending effect of caloric restriction. Sir2 and its mammalian homologue Sirt1 are NAD-dependent protein deacetylases that are sensitive to changes in cellular energy levels. Caloric restriction was shown to decrease plasma GH and IGF-I in rats, but the mechanism has never been understood. In the present study Sirt1 transcript was detected in the human anterior pituitary gland, and Sirt1 protein was found to be widely present in all endocrine cell types, as determined by immunohistochemistry. In order to investigate the effect of Sirt1 on GH expression, the rat mammosomatotrophinoma GH3 cells were transfected with a reporter vector bearing the GH promoter upstream the luciferase gene and Sirt1 was knocked down by RNA interference. Knocking down Sirt1 increased GH relative luciferase activity. GH transcription is under the control of the cAMP pathway. Sirt1 immunoprecipitated with Creb-binding protein (CBP), and was found to block CBP activity. It is possible that this is the mechanism mediating the inhibitory action of Sirt1 on GH transcription. These preliminary data show that Sirt1 is expressed in the anterior pituitary gland, where it can regulate GH synthesis in response to metabolic changes.

Conditional Overexpression of the Wild-Type Gsα as the gsp Oncogene Initiates Chronic Extracellularly Regulated Kinase 1/2 Activation and Hormone Hypersecretion in Pituitary Cell Lines

In pituitary cells, activation of the cAMP pathway by specific G protein-coupled receptors controls differentiative functions and proliferation. Constitutively active forms of the alpha subunit of the heterotrimeric G(s) protein resulting from mutations at codon 201 or 227 (gsp oncogene) were first identified in 30-40% of human GH-secreting pituitary adenomas. This rate of occurrence suggests that the gsp oncogene is not responsible for initiating the majority of these tumors. Moreover, there is a large overlap between the clinical phenotypes observed in patients with tumors bearing the gsp oncogene and those devoid of this oncogene. To explore the role of G(s)alpha in GH-secreting adenomas, we obtained somatolactotroph GH4C1 cell lines by performing doxycycline-dependent conditional overexpression of the wild-type G(s)alpha protein and expression of the gsp oncogene. Although the resulting adenylyl cyclase and cAMP levels were 10-fold lower in the wild-type G(s)alpha-overexpressing cell line, a sustained MAPK ERK1/2 activation was observed in both cell lines. Overexpression of the wild-type G(s)alpha protein as the gsp oncogene initiated chronic activation of endogenous prolactin synthesis and release, as well as chronic activation of ERK1/2-sensitive human prolactin and GH promoters.

The Human Growth Hormone Gene Contains a Silencer Embedded within an Alu Repeat in the 3′-Flanking Region

Alu family sequences are middle repetitive short interspersed elements (SINEs) dispersed throughout vertebrate genomes that can modulate gene transcription. The human (h) GH locus contains 44 complete and four partial Alu elements. An Sx Alu repeat lies in close proximity to the hGH-1 and hGH-2 genes in the 3'-flanking region. Deletion of the Sx Alu repeat in reporter constructs containing hGH-1 3'-flanking sequences increased reporter activity in transfected pituitary GC cells, suggesting this region contained a repressor element. Analysis of multiple deletion fragments from the 3'-flanking region of the hGH-1 gene revealed a strong orientation- and position-independent silencing activity mapping between nucleotides 2158 and 2572 encompassing the Sx Alu repeat. Refined mapping revealed that the silencer was a complex element comprising four discrete entities, including a core repressor domain (CRD), an antisilencer domain (ASE) that contains elements mediating the orientation-independent silencer activity, and two domains flanking the CRD/ASE that modulate silencer activity in a CRD-dependent manner. The upstream modulator domain is also required for orientation-independent silencer function. EMSA with DNA fragments representing all of the silencer domains yielded a complex pattern of DNA-protein interactions indicating that numerous GC cell nuclear proteins bind specifically to the CRD, ASE, and modulator domains. The silencer is GH promoter dependent and, in turn, its presence decreases the rate of promoter-associated histone acetylation resulting in a significant decrease of RNA polymerase II recruitment to the promoter. The silencer may provide for complex regulatory control of hGH gene expression in pituitary cells.

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.

Variable number of tandem repeats in the growth hormone gene of Sparus aurata: association with growth and effect on gene transcription

The GH gene of Sparus aurata(saGH) contains variable number of tandem repeats (VNTR). The hyper‐variable minisatellites in the first and third introns segregate in a Mendelian manner and exhibit numerous alleles. Analysis by PCR and sequencing of the two introns in several wild Sparidae species revealed comparable minisatellites with some variations. ‘Zoo blot’ with the first intron unit as a probe showed this sequence to be characteristic of several families from the Perciformes order. Unexpectedly, a similar minisatellite was found in the first intron of the GH gene in flounder, which belongs to a different order. Transfection of constructs containing a reporter gene and first intron of different length to four cell lines resulted in an inhibitory effect of the longer intron relative to the short intron. A (CA)n microsatellite (saGHpCA) is found in the GH promoter. A similar repeat at the same location is present in GH promoters of several other fish species. High variability (11 alleles) of the saGHpCA was found in a hatchery population. Full‐sib family genotyping showed a Mendelian inheritance of these alleles. A significant association was found between allele distribution and body mass in large and average size fishes from a hatchery population. The intron minisatellites may serve as markers for hybrid population and parental assignment. Its presence in families and orders of the higher teleosts may help solving classification uncertainties. Their conservation and inhibitory effect suggest a biological role. The saGHpCA is correlated with growth and may be a good candidate for predicting growth performance.

A novel cell type-specific mechanism for thyroid hormone-dependent negative regulation of the human type 1 deiodinase gene.

We have identified a cell type-specific, negative thyroid hormone-responsive element in the human type 1 iodothyronine deiodinase (hdio1) gene. This fragment, termed a JEG response element, bound tightly to a JEG-cell nuclear protein [JEG cell-specific transcription factor (JTF)] also present in placenta but not in COS-7, HeLa, or human embryonic kidney-293 cells. In JEG-3 cells, three copies of the JEG response element conferred a more than 40-fold transcriptional stimulation to the heterologous rat GH promoter which was further increased 2-fold by apo-thyroid hormone receptor (TR) and reduced 3-fold by T(3). Dimethyl sulfide footprinting showed overlapping contact sites for the high-affinity interaction of JTF and low-affinity binding of TR-retinoid X receptor. Expression of the same construct was unaffected by TR or T(3) in COS cells, indicating JTF was required for negative regulation by T(3)-TR. Mutations of the critical thyroid hormone responsive element binding P box amino acids EG to GS in TRalpha1 or TRbeta2 eliminated the apo-TR and T(3)-TR effects. These studies identify a novel mechanism for cell type-specific, promoter-independent negative regulation by T(3).