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• W1994501148 abstract "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 thehGH-N promoter contains functional binding sites for multiple transcription factors, including Sp1, Zn-15, and Pit-1, predictable and developmentally appropriate expression ofhGH-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 linkedhGH-N transgene. Because the Pit-1 sites in thehGH-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. 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 thehGH-N promoter contains functional binding sites for multiple transcription factors, including Sp1, Zn-15, and Pit-1, predictable and developmentally appropriate expression ofhGH-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 linkedhGH-N transgene. Because the Pit-1 sites in thehGH-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. locus control region electrophoretic mobility shift assay human growth hormone murine growth hormone hypersensitive site(s) polymerase chain reaction base pair(s) kilobase pair(s) polymerase chain reaction monoclonal antibody Tissue-specific regulation of eukaryotic genes is mediated by the combined action of ubiquitous and tissue-restricted transcription factors. These factors are brought into functional concert by binding to multiple classes of gene regulatory determinants, leading to the appropriate induction of transcription (reviewed in Ref. 1Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar). One class of regulatory elements appears to function primarily at the level of chromatin structure. The role of these elements is to establish transcriptionally competent domains that are available for interactions with the appropriate trans-factors. These chromatin elements are most reliably detected and mapped based on their ability to support expression of linked transgenes independent of the site of integration in the host genome (2Grosveld F. Bloom B. van Assendelft D. Greaves R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1442) Google Scholar). When detected in the mammalian genome, these elements are termed locus control regions (LCRs1; reviewed in Ref. 3Kioussis D. Festenstein R. Curr. Opin. Genet. Dev. 1997; 7: 614-619Crossref PubMed Scopus (125) Google Scholar). Components of locus control regions, which may be situated at significant distances from their target genes, can be identified by their ability to establish DNase I hypersensitive sites (HS) in the chromatin of expressing tissues. The mechanism for LCR function is inferred to be biphasic, involving the initial alteration of chromatin structure and subsequent transcriptional activation (reviewed in Ref.4Kamakaka R.T. Trends Biochem. Sci. 1997; 22: 124-128Abstract Full Text PDF PubMed Scopus (27) Google Scholar). The human growth hormone gene, hGH-N, is the 5′-most member of a five-gene cluster on chromosome 17. These genes share greater than 95% sequence identity and encode structurally similar proteins (5Seeburg P.H. DNA. 1982; 1: 239-249Crossref PubMed Scopus (213) Google Scholar) (Fig. 1). Despite this structural similarity, the hGH-N gene is expressed exclusively in the somatotropes and lactosomatotropes of the anterior pituitary, while the remaining genes of the cluster (hCS-L, hCS-A, hGH-V, andhCS-B) are expressed exclusively in the syncytiotrophoblasts of the placental villi (6MacLeod J.N. Lee A.K. Liebhaber S.A. Cooke N.E. J. Biol. Chem. 1992; 267: 14219-14226Abstract Full Text PDF PubMed Google Scholar, 7McWilliams D. Boime I. Endocrinology. 1980; 107: 761-765Crossref PubMed Scopus (34) Google Scholar, 8Liebhaber S.A. Urbanek M. Ray J. Tuan R.S. Cooke N.E. J. Clin. Invest. 1989; 83: 1985-1991Crossref PubMed Scopus (82) Google Scholar). This tissue specificity does not appear to be attributable to proximal transcriptional control elementsper se, as evidenced by the similar expression ofhGH-N and the placenta-specific genes in transfected pituitary cells (9Nickel B.E. Kardami E. Cattini P.A. J. Biol. Chem. 1990; 267: 653-658Google Scholar). Furthermore, retention of as much as 7.5 kb of 5′-flanking sequence in cis to the hGH-N gene is insufficient to recapitulate appropriate levels and specificity of expression in transgenic mice (10Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (152) Google Scholar). These data suggest the participation of distal regulatory element(s) in the developmental activation of hGH-N gene expression. A set of DNase I HS located between 14.5 and 30 kb upstream of the hGH gene cluster has been identified exclusively in pituitary and placental chromatin (HS I–V; Fig. 1). The tissue-specific nature of these sites implies that they play a role in regulation of the hGH gene cluster. Human GH-N transgenes with extensive 5′-flanking regions encompassing these remote HS are expressed at high levels and in a position-independent and copy number-dependent manner in the mouse pituitary. These data indicate that these DNase I HS constitute an LCR for the hGH gene cluster and that the transgenic mouse is an appropriate model system in which to study its function (10Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (152) Google Scholar). The somatotrope-specific determinants of the LCR map to the region marked by two closely linked HS approximately 14.5 kb 5′ to thehGH-N gene (HS I,II; Fig. 1) (10Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (152) Google Scholar). HS I,II activity was initially isolated on a 1.6-kb fragment and was subsequently sublocalized to a 404-bp segment (the F14 segment) (11Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (55) Google Scholar). This F14 segment is sufficient to confer high level, somatotrope-specific, and position-independent expression of a linked hGH-N gene in transgenic mouse pituitary. Furthermore, the F14 segment mediates appropriately timed induction of the linked hGH-N transgene during mouse embryonic development, paralleling induction of the endogenous mGH gene (11Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (55) Google Scholar). In the present study, we identified multiple binding sites for the POU domain transcription factor Pit-1 within the F14 segment of the HS I,II region and have established that these sites are essential components of LCR-mediated gene activation in the pituitary in vivo. The previously described F14 segment contains the 3′-terminal 404 bp of the 1602-bp HS I,II sequence (GenBankTM accession no.AF039413). DNA fragments used as probes in electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays were generated by PCR using a plasmid (pHSI, II-11) containing the 1602-bp HS I,II region as a template. PCRs consisted of 10 mm Tris (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 0.2 mm each dNTP, 0.5 μm each primer, 10 ng of template DNA, and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer) in a total of 100 μl. Primers for fragments F14.1, F14.2, F14.3, F14.4, and F14.5 were designed to add EcoRI sites to both ends of the resulting PCR product to allow 3′-end labeling (Table I). The end points of these fragments relative to the 1602-bp HS I,II region are as follows: F14.1, 1164–1279; F14.2, 1275–1359; F14.3, 1356–1455; F14.4, 1435–1524; F14.5, 1520–1602. The F14 region DNase I footprinting probe containing nucleotides 1164–1525 was amplified with primers designed to add anEcoRI site at one end of the amplicon to allow the sense strand relative to hGH-N to be 3′-end-labeled. The 3′-ends of probes digested with EcoRI were radiolabeled using [α-32P]dATP and Klenow DNA polymerase. Fragments F14.3.1, F14.3.2, F14.3.3, and F14.3.4 were generated by annealing complementary synthetic oligonucleotides and 5′-end labeling with [γ-32P]ATP and T4 polynucleotide kinase. All radiolabeled probes and unlabeled competitor fragments were gel-purified on 5% polyacrylamide. Wild type and mutant duplex Pit-1 oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).Table IPrimers used to generate probes for binding experimentsProbePrimersF14.15′-GGAATTCCCAAGCCTTTCCCAGTTATAC5′-GGAATTCCTGGGAGTCTCATGGTTTAGGF14.25′-GGAATTCCCCAGATTTTGCCCCACTCCC5′-GGAATTCAGGTCAGCTTGAGGCCCATGGF14.35′-GGAATTCACCTCAGGTGATGTTTATATT5′-GGAATTCTGTTTCCAGATGTTCCAAATGF14.45′-GGAATTCCATTTGGAACATCTGGAAACA5′-GGAATTCTCAGGGGCAGGAGGGTTAAGGF14.55′-GGAATTCCTCCTGCCCCTGACTTCCGTG5′-GGAATTCGATCTTGGCCTAGGCCTCGGAF14 footprinting5′-CCAAGCCTTTCCCAGTTATAC5′-GGAATTCTCAGGGGCAGGAGGGTTAAGG Open table in a new tab GH3 (12Tansey W.P. Catanzaro D.F. J. Biol. Chem. 1991; 266: 9805-9813Abstract Full Text PDF PubMed Google Scholar), GHFT1 (13Lew D. Brady H. Klausing K. Yaginuma K. Theill L.E. Stauber C. Karin M. Mellon P.L. Genes Dev. 1993; 7: 683-693Crossref PubMed Scopus (117) Google Scholar), and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Nuclear extracts of NIH/3T3, GHFT1, GH3, and primary mouse pituitary cells were prepared as described (14Dyer R.B. Herzog N.K. BioTechniques. 1995; 19: 192-195PubMed Google Scholar). Pituitaries excised from multiple CD-1 mice were placed in ice-cold phosphate-buffered saline and collected by centrifugation at 1000 × g at 4 °C for 10 min. After removing the supernatant, the pituitaries were incubated in dissociation buffer (Life Technologies, Inc.) for 1 min at room temperature and then disaggregated by repeated pipetting, and the larger material was allowed to sediment. The buffer and dissociated cells were removed to a tube containing 40 ml of cold Dulbecco's modified Eagle's medium with 10% fetal bovine serum. This process was repeated until the pituitary tissue was completely dissociated. Protein-DNA complexes were detected in vitro on the basis of changes in the electrophoretic mobility of a radiolabeled DNA probe (15Fried M. Crothers D.M. Nucleic Acids Res. 1981; 9: 6505-6525Crossref PubMed Scopus (1687) Google Scholar). Binding reactions (25 μl) contained 5 ng of an end-labeled, double-stranded DNA fragment in buffer composed of 10 mm HEPES (pH 7.9), 1 mm EDTA, 1 mm dithiothreitol, 32 mm KCl, 10% glycerol, 0.08 g/liter poly(dI-dC), and 0.3 g/liter bovine serum albumin. Nuclear extract (4 μg) was added last, and the mixture was incubated for 20 min at room temperature. Samples were resolved on a 5% nondenaturing polyacrylamide (25:1 acrylamide:bisacrylamide) gel in 2× Tris-glycine buffer at 150 V for 2–3 h. When necessary, unlabeled competitor DNA was added prior to the addition of nuclear extract. For supershift experiments, 2 μg of polyclonal Pit-1 (Santa Cruz Biotechnology) or monoclonal Pit-1 (Transduction Laboratories) antibody was added at the end of the initial 20-min binding reaction, and the reactions were incubated for an additional 30 min prior to gel loading. Regions of the HS I,II F14 region protected by bound proteins were detected by cleavage with DNase I (16Galas D.J. Schmitz A. Nucleic Acids Res. 1978; 5: 3157-3170Crossref PubMed Scopus (1341) Google Scholar). Binding reactions (80 μl) contained 5 ng of a 3′ end-labeled, double-stranded DNA fragment, 12 mm HEPES (pH 7.6), 48 mm KCl, 4 mm MgCl2, 0.8 mm EDTA, 10% glycerol, 0.2 g/liter poly(dI-dC), and 80 μg of GH3 cell nuclear extract. Control reactions without nuclear extract were supplemented with an equal volume of nuclear extract diluent. Samples of unbound DNA (no added protein) were digested with 1 μl of 10 ng/μl DNase I (Sigma) for 60 s at room temperature, and samples of bound DNA (with nuclear extract) were digested with 1 μl of 100 ng/μl DNase I for 180 s at room temperature. Reactions were stopped by adding 2 μl of 0.5 m EDTA. DNA was purified from the binding reactions by phenol/chloroform extraction and ethanol precipitation in the presence of 20 μg of yeast tRNA and resolved on a denaturing 6% polyacrylamide (20:1 acrylamide:bisacrylamide), 7 m urea gel in 0.5× TBE at 2000 V for 1–2 h. Chemical sequencing reactions were performed by the method of Maxam and Gilbert (17Maxam A. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9014) Google Scholar). DNA subfragments of the HS I,II region were generated by PCR as described above using a plasmid (pHSI, II-11) containing the 1602-bp HS I,II region as a template. Primers were designed to add an XhoI restriction site to the 5′-end and a HindIII restriction site to the 3′-end of the amplicons (relative to the transcriptional orientation of the hGH-N gene) for subsequent cloning (Table II). The F14, F14Δ.3, F14mut5′Pit-1, F14mut3′Pit-1, and F14mut5′,3′Pit-1 fragments all included nucleotides 1198–1602 of the HS I,II region. In the F14Δ.3 deletion mutant, nucleotides 1357–1456 were deleted. The F14.3 fragment extends from nucleotide 1357 to 1456 of the HS I,II region. Deletion and point mutations were introduced into the F14 fragment by SOE (splicing by overlap extension) PCR as described (18Vallejo A.N. Pogulis R.J. Pease L.R. Dieffenbach C.W. Dveksler G.S. PCR Primer. Cold Spring Harbor Press, Plainview, NY1995: 603-612Google Scholar). After digestion with XhoI andHindIII, PCR fragments were cloned into pBSIIKS-hGHN digested with XhoI and HindIII, upstream of thehGH-N gene. This plasmid contains the 2.6-kbEcoRI fragment R2 (19Chen E.Y. Liao Y.-C. Smith D.H. Barrera-Saldana H.A. Gelinas R.E. Seeburg P. Genomics. 1989; 4: 479-497Crossref PubMed Scopus (443) Google Scholar) cloned into the EcoRI site of pBSIIKS (Stratagene). The sequences of all constructs were verified by the Sequencing Core of the University of Pennsylvania School of Medicine.Table IIPrimers used to generate fragments for recombinant plasmidsConstruct fragmentPrimersF145′-GCTCGAGGGAGACACTAGCCCCAAAGTT5′-GGAAGCTTGATCTTGGCCTAGGCCTCGGAF14Δ.3Same as F14, plus mutagenic primers:5′-GTTCTTTCTCAGCTTGAGGCCCATGGGCCC5′-CTCAAGCTGAGAAAGAACATCTGGGGCTGCF14.35′-GCTCGAGACCTCAGGTGATGTTTATATT5′-GGAAGCTTTGTTTCCAGATGTTCCAAATGF14mut5′Pit-1Same as F14, plus mutagenic primers:5′-AACAGCTCATCCCTATCCCCATCACCTG5′-CAGGTGATGGGGATAGGGATGAGCTGTTF14mut3′Pit-1Same as F14, plus mutagenic primers:5′-ATGTTCCAAATTCCACCCCATGGCTCTGA5′-TCAGAGCCATGGGGTGGAATTTGGAACATF14mut5′,3′Pit-1Same as F14; same mutagenic primers as F14mut5′Pit-1 using the pBS-F14mut3′Pit-1-hGHN plasmid as a template. Open table in a new tab DNA constructs released from vector sequences by BssHII digestion were resolved on agarose gels, recovered with glass beads, purified by Elutip (Schleicher and Schuell), and diluted to 3 ng/μl in 10 mmTris-HCl (pH 7.6), 0.1 mm EDTA. One-picogram aliquots of DNA were microinjected into fertilized C57BL/6J × SJL oocytes. Twenty-two eggs were reimplanted into each pseudopregnant recipient, and the mothers were sacrificed 18.5 days postcoitus (embryonic day 18.5). Placental DNA was analyzed for transgene DNA by dot blotting. Relative transgene copy numbers for each embryo were estimated by comparing the hybridization signal from an hGH-N gene probe to that from an endogenous mouse ζ-globin gene probe on duplicate blots. Each embryo was bisected with a parasagittal incision, fixed in 10% phosphate-buffered formalin, and embedded in paraffin, and the pituitary was located by serial sectioning. A highly specific anti-hGH monoclonal antibody has been previously described (mAb 9) (11Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (55) Google Scholar). Anti-rat growth hormone, which cross-reacts with mGH, was obtained from the National Hormone and Pituitary Program. Sections of paraffin-embedded mouse embryos (4–5 μm) were stained with hematoxylin and eosin. Anti-rat growth hormone was used at a 1:2500 dilution, and mAb 9 was used at 1:1500; incubations were for 24 h at room temperature. The specificity of immunostaining was documented by nonimmune sera and by competition with purified hGH (Dako). All reactions were visualized with streptavidin-biotin-peroxidase (Autoprobe III detection system; Biomeda) and were revealed with the chromagen 3,3′-diaminobenzidine (11Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (55) Google Scholar). Colocalization of hGH and mGH was determined by staining adjacent sections. Scoring of embryos was performed by an individual who was blind to transgene presence or identity. Because of the nonquantitative nature of the immunohistochemistry assay and the fact that HS I,II alone does not confer copy number dependence (10Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (152) Google Scholar), expression level per transgene copy was not addressed. Copy number was determined to control for the range of copy numbers carried by the embryos analyzed, such that only those carrying a parallel range of copy numbers of each construct were compared. It should be noted that although some of the hGH-positive embryos carried a relatively high transgene copy number, this did not bias the staining assay or expression data because F14Δ.3-GH embryos with comparable copy numbers were all negative for hGH-Nexpression. Formation of HS I,II of the hGH LCR is specific to nuclear chromatin from pituitary cells (Fig. 1) (10Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (152) Google Scholar). The determinants of HS I,II involved in the somatotrope-specific activation of the linkedhGH-N transgene have been localized to a 404-bp subsegment, F14 (nucleotides 1198–1602 in GenBankTM accession no.AF039413) (11Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (55) Google Scholar). Computer-assisted sequence analysis of this region identified multiple potential trans-factor binding sites, some of which are recognized by ubiquitous transcription factors and nuclear hormone receptors that could possibly contribute to its function. However, no motifs were identified that fit the pituitary-specific profile of F14 activity. To determine whether F14 could interact with one or more pituitary-specific factors, EMSAs were performed using nuclear extract from the mouse fibroblast cell line NIH3T3 and the mouse presomatotrope cell line GHFT1, as well as from the growth hormone-expressing rat somatotrope cell line GH3 and primary mouse pituitary cells. As an initial control to confirm the phenotypes of these four extracts, each was incubated with a probe containing a binding site for the pituitary-enriched POU domain transcription factor Pit-1 (Fig. 2 A). Consistent with the known phenotypes of each of the cells, strong binding to the Pit-1 probe was observed with extracts from the GHFT1, GH3, and mouse pituitary nuclei but not with NIH3T3 nuclear extracts (Fig. 2 A). The Pit-1 complexes formed by the three pituitary cell lines consisted of a strong doublet. An additional minor Pit-1 complex of higher mobility was formed by the two extracts originating from the mature somatotrope cells (GH3 and primary pituitary; Fig. 2 A). The presence of multiple Pit-1 complexes was attributed to the presence of multiple isoforms of Pit-1 (reviewed in Ref. 20Rosenfeld M.G. Bach I. Erkman L. Li P. Lin C. Lin S. McEvilly R. Ryan A. Rhodes S. Schonnemann M. Scully K. Rec. Prog. Horm. Res. 1996; 51: 217-238PubMed Google Scholar) and was not further explored. To screen the F14 segment of HS I,II for protein binding, the 404-bp segment was divided into five partially overlapping probes: F14.1, F14.2, F14.3, F14.4, and F14.5 (Fig. 2 B). Each of these probes was initially screened by incubation with GHFT1 and fibroblast (NIH/3T3) extracts. Whereas all five subfragments formed a variety of weak complexes that were largely observed in both the fibroblast and the GHFT1 extracts, a distinct and strong GHFT1-specific doublet complex was assembled on the F14.3 probe (Fig. 2 B;lane 9). Incubation of this F14.3 probe with the full set of nuclear extracts demonstrated formation of the strong doublet by proteins from each of the somatotrope-related cell lines (Fig. 2 C, lanes 3–5). The same doublet was also assembled on the F14.1 probe, although this complex was much less intense (Fig. 2 B, lane 3). The doublet complex could be self-competed by the addition of a 10- and 50-fold molar excess of unlabeled F14.3 and F14.1 segments, indicating that the corresponding interactions were sequence-specific (Fig. 2 C, lanes 6–15). The F14.1 fragment was a weaker competitor than F14.3, consistent with the weaker intensity of these complexes on an F14.1 fragment probe (Fig. 2 B; compare lane 3 with lane 9). Thus, the F14 segment of the HS I,II LCR determinant bound to one or more pituitary-specific nuclear proteins, and these binding sites were localized to two subdomains, F14.1 and F14.3. The complexes that formed on the F14.1 and F14.3 probes were noted to be strikingly similar in gel migration to Pit-1 complexes (compare Fig. 2 C,lanes 3–5, with Fig. 2 A). In addition, the Pit-1, F14.1, and F14.3 probes all formed the intense doublet complex specifically with the nuclear extracts from the three pituitary cell types (double arrows in Fig. 2,A–C). This comparison suggested that the complexes forming on F14.1 and F14.3 contained Pit-1. This hypothesis was confirmed by supershift reactions using polyclonal and monoclonal Pit-1 antibodies. The intensity of the pituitary-specific complexes formed by all three pituitary cell nuclear extracts was diminished by the presence of the antibody, with the concomitant appearance of multiple supershifted complexes (Fig. 2 D and data not shown). The complexes formed by the NIH/3T3 nuclear extract were unaffected by the Pit-1 antibody (data not shown). As an additional test for the presence of Pit-1 in these complexes, the control oligonucleotide containing a known Pit-1 binding site was used as a cold competitor. The somatotrope-specific complexes were specifically competed by a 30-fold molar excess of the Pit-1 oligonucleotide (Fig. 2 D , lane 4). The high mobility Pit-1 complex specific for GH3 and mouse pituitary nuclear extracts was efficiently competed but inefficiently supershifted (Fig. 2 D, comparelanes 3 and 4), possibly indicating a minor Pit-1 isoform with a different topology than that of the major 30–33-kDa isoform immunogen. The same antibody supershift and competition experiments were performed with the labeled F14.1 subfragment probe, and the outcome was the same (Fig. 2 D,lanes 5–8). Therefore, the full set of EMSA studies indicated that the pituitary-specific complexes formed on the F14.3 and F14.1 probes were due to Pit-1 binding. The EMSA studies detailed above did not define whether Pit-1 was binding to a single site or to multiple sites within the target F14 probes. Because of the relatively loose consensus Pit-1 recognition motif (21Andersen B. Rosenfeld M.G. J. Biol. Chem. 1994; 269: 29335-29338Abstract Full Text PDF PubMed Google Scholar), it was difficult to predict the exact site(s) of Pit-1 binding. To directly sublocalize the Pit-1 binding site(s), F14.3 was divided into four partially overlapping subfragments. These subfragments were then characterized by EMSA, and Pit-1 content in complexes was confirmed by probe competition and antibody supershift studies. Strong somatotrope-specific complexes selectively formed on both the F14.3.1 and F14.3.4 fragments (Fig. 3 A). These complexes were identical with each of the two probes and were readily inhibited by a 30-fold molar excess of unlabeled Pit-1 probe (lanes 4 and 19) but not by the same probe carrying an inactivating Pit-1 mutation (lanes 5 and20). These complexes were also supershifted by the polyclonal Pit-1 antibody (lanes 6 and21) as well as by a monoclonal antibody raised against rat Pit-1 (lanes 7 and 22). The F14.3.2 fragment formed a very weak Pit-1-containing complex that could only be clearly documented by antibody supershift (lane 11). Using a similar scanning EMSA strategy, the Pit-1 binding seen with the F14.1 subfragment was localized to a single site (Fig. 3 B). Thus, the F14 segment of HS I,II contained a total of three strong Pit-1 binding sites: two within the central F14.3 subfragment and a third in the 5′-most F14.1 subfragment. The potential site within F14.3.2 was not pursued in this analysis due to the very weak Pit-1 binding as compared with the other three sites. The positions of the three Pit-1 sites within the F14 fragment were confirmed by DNase I footprinting. Regions of the F14 fragment protected from DNase I digestion by GH3 nuclear extract corresponded to the single Pit-1 site in F14.1 and the two Pit-1 sites in F14.3 (Fig. 4). Several additional footprints were also seen, as predicted by the presence of non-Pit-1 complexes on the EMSA (Fig. 2 C and data not shown). Thus, the EMSA and footprint analysis were internally consistent in demonstrating an array of three Pit-1 sites in the F14 segment of HS I,II (Fig. 4,bottom). Determinants sufficient to confer high level, position-independent, somatotrope-specific expression of a linked hGH-N gene in transgenic mice have previously been resolved to the F14 segment of HS I,II (11Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (55) Google Scholar). As described above, the pituitary-specific complexes formed on this fragment corresponded to three Pit-1 binding sites. The functional contribution of these Pit-1 sites to LCR function was next tested. We focused on the Pit-1 sites within the F14.3 sequence because they appeared by EMSA to demonstrate the highest binding affinity (Fig. 2 B, compare lanes 3 and 9; Fig. 2 D, compare lanes 2 and6). The entire F14.3 region was deleted from F14 to test the functional role of these two sites. The ability of the resultant F14Δ.3 to activate hGH-N transgene expression was compared with that of the intact F14 fragment using a previously validated transgenic founder assay (11Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (55) Google Scholar). The F14 and F14Δ.3 fragments were each ligated upstream of the genomic DNA fragment containing thehGH-N gene in native orientation. This hGH-N gene contained its own contiguous promoter within 0.5 kb of 5′-flanking sequence encompassing two functional Pit-1 sites (22Nelson C. Albert V.R. Elsholtz H.P. Lu L.I.-W. Rosenfeld M.G. Science. 1988; 239: 1400-1405Crossref PubMed Scopus (419) Google Scholar) (Fig. 5 A). Each construct was injected into fertilized oocytes, and reimplanted embryos were harvested at embryonic day 18.5. The transgenic status of each embryo was established by dot-blot analysis of placental DNA. Sagittally sectioned anterior segments of each embryo were fixed. Pituitaries were identified by light microscopy, and the presence of hGH and mGH staining was scored by immunostaining with antibodies specific for hGH or mGH. The immunohistochemical analysis was carried out without prior knowledge of the presence or identity of the corresponding transgene. The results of the immunostains, which could not be rigorously quantified due to the nature of the assay, were grouped into three categories based on the relative intensities of hGH and mGH signals: 1) hGH expression in somatotropes at levels str" @default.
• W1994501148 created "2016-06-24" @default.
• W1994501148 creator A5013949352 @default.
• W1994501148 creator A5042375028 @default.
• W1994501148 creator A5080563290 @default.
• W1994501148 creator A5090794174 @default.
• W1994501148 date "1999-12-01" @default.
• W1994501148 modified "2023-10-01" @default.
• W1994501148 title "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" @default.
• W1994501148 cites W1505648898 @default.
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