A Role for A/T-Rich Sequences and Pit-1/GHF-1 in a Distal Enhancer Located in the Human Growth Hormone Locus Control Region with Preferential Pituitary Activity in Culture and Transgenic Mice

Yan Jin, Rama Mohan Surabhi, Agnes Fresnoza, Aristides Lytras and Peter A. Cattini

Department of Physiology University of Manitoba Winnipeg, Manitoba, Canada, R3E 3J7


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A region located remotely upstream of the human pituitary GH (GH-N) gene and required for efficient GH-N gene expression in the pituitary of transgenic mice was cloned as a 1.6-kb BglII (1.6G) fragment. The 1.6G fragment in the forward or reverse orientation increased -496GH-N promoter activity significantly in pituitary GC and GH3 cells after gene transfer. The 1.6G fragment was also able to stimulate activity from a minimal thymidine kinase (TK) promoter which, unlike -496GH-N, lacked any Pit-1/GHF-1 element. Enhancer activity was localized by deletion analysis to a 203-bp region in the 3'-end of the 1.6G fragment and was characterized by the presence of a diffuse 136-bp nuclease-protected site, observed with pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein. A major low-mobility complex was observed by electrophoretic mobility shift assay (EMSA) with GC cell nuclear protein, and the pattern was distinct from that seen with a HeLa cell extract. The nuclease-protected region contains three A/T-rich Pit-1/GHF-1-like elements, and their disruption, in the context of the 203-bp region fused to the TK promoter, reduced enhancer activity significantly in pituitary cells in culture. A mutation in this region was also shown to decrease enhancer activity in transgenic mice and correlated with a decrease in the 203-bp enhancer region complex observed by EMSA. The participation of Pit-1/GHF-1 in this complex is indicated by competition studies with Pit-1/GHF-1 elements and antibodies, and direct binding of Pit-1/GHF-1 to the A/T-rich sequences was shown by EMSA using recombinant protein. These studies link the A/T-rich sequences to the distal enhancer activity associated with the GH locus control region in vitro and in vivo.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human pituitary GH (GH-N) gene is expressed efficiently in pituitary somatotrophs and somatolactotrophs in vivo (1). Sequences located in the first 140 bp of upstream GH-N flanking DNA were implicated in pituitary-specific expression, based largely on the use of rat pituitary cell lines, hybrid reporter genes, and gene transfer (2, 3). Subsequently, a nuclear protein variously called Pit-1 or GHF-1 was shown to bind these sequences and direct efficient and pituitary-specific GH-N promoter activity (4, 5). However, these proximal promoter sequences were not sufficient to permit efficient or tissue-specific expression of GH-N (in transgenic mice) in vivo (6, 7), suggesting the absence and, thus, requirement for a locus control region (LCR). LCRs allow position-independent and efficient expression of their associated genes. The presence of deoxyribonuclease I (DNase I)-hypersensitive sites is a characteristic of LCRs (7, 8, 9). These sites often signal the presence of cis-acting sequences associated with a variety of mechanisms regulating gene expression. Two pituitary-specific hypersensitive sites (HS I and HS II) were detected in a 1.6-kb region, contained in a BglII (1.6G) fragment, and centred about 15 kb upstream of the GH-N transcription initiation site (7), in the 5'-flanking DNA of the adjacent B-lymphocyte-specific CD79b gene (10). GH-N transgenes including the 1.6G fragment were expressed efficiently and specifically in transgenic mice, suggesting that they constitute a necessary component of the GH LCR (7, 11). Both HS I and HS II were reconstructed in the transgenic mouse and were pituitary specific (7). LCRs are expected to possess at least two important activities to achieve position-independent high levels of expression. The first is the ability to establish an open chromosomal domain containing the gene or genes to be expressed, in this case, the GH-N gene in the pituitary, and the second is to promote high levels of expression (8) and, thus, possess enhancer activity.

In spite of the recent localization of enhancer activity to a 404-bp region of the larger 1.6G fragment (11), there is scant information about the sequences and factors involved in this component of the GH LCR, largely because of a paucity of in vitro studies. Specifically, the sequence of the 1.6-kb fragment containing HS I and HS II was not reported initially (7). Due to the pituitary-specific nature of HS I and HS II, it would be appropriate to question whether sites for the pituitary-specific factor Pit-1/GHF-1 are present in the 1.6G fragment. Also, the available data were obtained using the homologous human GH-N promoter to test the 1.6G fragment or subfragments. Thus, it was still unclear whether the enhancer activity, which is contained in the 5'-flanking DNA of the lymphocyte-specific CD79b gene, required the Pit-1/GHF-1 sites in the proximal promoter region for enhancer function in pituitary cells in vivo as well as in vitro.

We have cloned the GH-N gene and all upstream sequences to exon 9 of the SCN4A gene on chromosome 17 and characterized the 1605-bp 1.6G fragment of this clone, which is reported to contain HS I and HS II, as well as retain pituitary-specific activity in transgenic mice (7). We report the localization of enhancer activity to a 203-bp subfragment of the 1.6G fragment, which is characterized by the presence of a diffuse 136-bp nuclease-protected region using pituitary cell nuclear protein. Analysis of this region revealed three A/T-rich Pit-1/GHF-1-like elements. Mutation of these A/T-rich sites, in the context of the 203-bp subfragment, resulted in a decrease in enhancer activity in transfected GC cells; however, this decrease was most significant with the disruption of sequences at nucleotides 1426/1441. The effect of this mutation was also tested in vivo. Both the 1.6G fragment and the 203-bp subfragment were able to stimulate thymidine kinase (TK) promoter activity efficiently in the pituitary, and to a lesser extent in brain and testis, of founder transgenic mice. Mutation of these A/T-rich sequences resulted in an overall loss (>99%) of enhancer activity in the pituitary, and this correlated with a decrease in the levels of a specific major low-mobility complex observed between GC cell nuclear protein and the 203-bp subfragment, as seen by electrophoretic mobility shift assay (EMSA). Pit-1/GHF-1 binding was shown to contribute to complex formation. These data suggest the participation of A/T-rich sequences in the enhancer activity associated with a component of the GH LCR. Although our data implicate Pit-1/GHF-1 as a participant in the distal enhancer activity in the pituitary, it occurs independently of the homologous GH-N promoter and the presence of proximal Pit-1/GHF-1 sites.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning and Sequencing of 1.6G Fragment
A clone, P1–3 containing about 45 kb of sequences located upstream of the GH-N gene, was obtained by screening a P1 library of human genomic DNA with a 160-bp region of the GH-N 5'-flanking DNA (-3127/-2968) (12). The 1.6-kb BglII (1.6G) fragment, identified by Jones et al. (7), containing the pituitary-specific locus control activity and HS I and HS II, was isolated from the clone and sequenced (Fig. 1Go; EMBL/GenBank Data Library Accession AF010280).



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Figure 1. Nucleotide Sequence (Upper Strand) of the 1.6-kb BglII (1.6G) Fragment Located about 15 kb Upstream of the GH-N Transcription Initiation Site

No sequence corresponding to the consensus Pit-1/GHF-1 binding site (5'-WWTATNCAT-3') was identified. A 136-bp region protected by a pituitary GC cell nuclear extract from nuclease digestion is indicated by shading. Three A/T-rich regions containing Pit-1/GHF-1-like elements in the footprint region are indicated by double underlining. Details of the nucleotides mutated in M1, M2, M3, and M4 are indicated in the text; however, their positions are indicated by single overlining. A 72-bp CA-rich region containing a 6-bp stretch of reverse complementary TG residues is indicated by single underlining. The EMBL/GenBank Data Library accession number for these sequences is AF010280.

 
To establish the presence and location of the 1.6G fragment in the human genome, DNA from human choriocarcinoma JEG-3 cells was digested with BglII, EcoRI, HindIII, and HindIII/EcoRI and subjected to DNA blotting using the 1.6G fragment as a probe. Based on the restriction map, bands of predicted sizes (1.6 kb with BglII; 12 and 15 kb with EcoRI and HindIII; and 11.5 kb with HindIII/EcoRI) were detected in human genomic DNA (not shown).

To determine the orientation of the 1.6G fragment within the genome, the clone P1–3 was digested with BglII, BglII/HindIII, and BglII/XhoI, transferred to nitrocellulose, and probed with a 27-bp oligonucleotide (5'-GCCTTCCAACCATGGCATGGGAGGTGG-3') corresponding to nucleotides 1629/1656 (relative to the 1–1605 sequence of the 1.6G fragment). This oligonucleotide sequence was determined by extending the sequence analysis outside of the 1.6G fragment in the P1–3 clone and corresponds to the sequence reported for the CD79b gene 5'-flanking DNA (10). Also, bands of 11.2 kb with BglII, 4.7 kb with BglII/HindIII, and 3.3 kb with BglII/XhoI were detected (not shown). Based on the reported restriction endonuclease map (7, 12, 13), these sizes correspond to those predicted for sequences downstream of the 1.6G fragment.

No Pit-1/GHF-1 element was identified in the 1.6G fragment after searching with a consensus sequence reported for Pit-1/GHF-1 (5'-WWTATNCAT-3')(14). However, A/T-rich domains, which might represent potential binding sites for homeodomain-containing proteins such as Pit-1/GHF-1, could be found throughout the fragment, including 21 bp (18 of 21, A or T), 29 bp (28 of 29, A or T), 12 bp (11 of 12, A or T), 18 bp (13 of 18, A or T), and 16 bp (14 of 16, A or T) observed at nucleotides 57/77, 894/922, 1368/1379, 1388/1405, and 1426/1441, respectively (Fig. 1Go). A 72-bp CA-rich region (58%) with 21 CA-dinucleotides, including a repeat of 14 bp, was identified at nucleotides 1086/1153 and contains the consensus CACC-binding protein site located at nucleotides 1141/1148. Interestingly, this CA-rich region also contains a 6 bp stretch of reverse complementary TG residues (nucleotides 1113/1118).

The 1.6G Fragment Confers Enhancer Activity on Minimal GH-N and TK Promoters
A hybrid luciferase gene directed by the (-496/+1) GH-N promoter (GHp.luc) was used to test the effect of the 1.6G fragment on expression in rat pituitary GC and GH3 cells after gene transfer. The 1.6G fragment was inserted upstream of GHp.luc in the forward (1/1605G.GHp.luc) and reverse (1605/1G.GHp.luc) orientation. A promoterless luciferase reporter gene (pXP1) was used as a control for random transcription initiation. All test plasmids were cotransfected with RSVp.cat, and chloramphenicol acetyltransferase (CAT) activity was used to control for variation in DNA uptake. Values were obtained as luciferase activity per µg lysate protein divided by CAT activity per µg lysate protein (luciferase/CAT activity). The results are expressed as fold effect of the 1.6G region on GH-N promoter activity (Fig. 2Go). The GH-N promoter was stimulated 5.1- and 2.6-fold (P < 0.005, n = 6) in the presence of the 1.6G fragment in the GC and GH3 cells, respectively. This enhancement of promoter activity was also seen when the 1.6G fragment was present in the reverse orientation (Fig. 2Go).



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Figure 2. The 1.6G Fragment Stimulates GH-N and TK Promoter Activity in Pituitary Cells

The effect of the 1.6G fragment, in the forward (1/1605G) and reverse (1605/1G) orientation, on -496/+1GH-N or -81/+52TK promoter activity was tested in pituitary and nonpituitary cells after gene transfer. The luciferase gene was used as the reporter, and cotransfection with RSVp.cat was used to control for DNA uptake. Promoter activity was determined as luciferase activity per µg lysate protein divided by CAT activity per µg lysate protein and expressed as fold effect of the 1.6G region on GH-N or TK promoter activity. The basal GH-N promoter activity in pituitary GC and GH3 cells was 1083.9 ± 46.5 (n = 6) and 1181.8 ± 280.4 (n = 6), respectively. The basal TK promoter activity in pituitary GC, cervical HeLa, and glial C6 cells was 146.6 ± 6.1 (n = 12), 30.7 ± 1.3 (n = 12), and 17.2 ± 0.5 (n = 3), respectively. Error bars represent SEM.

 
To assess the 1.6G fragment for activity on a heterologous promoter (lacking a Pit-1/GHF-1 element) as well as the possibility of a tissue-specific effect, the 1.6G fragment was inserted in the forward (1/1605G.TKp.luc) and reverse (1605/1G.TKp.luc) orientation upstream of a hybrid luciferase gene directed by a minimal (-81/+52) TK promoter and assessed in pituitary GC, cervical HeLa, or glial C6 cells after gene transfer. The data, expressed as the fold effect of the 1.6G region on TK promoter activity, are also shown in Fig. 2Go. The presence of the 1.6G fragment in GC cells resulted in about a 5- and 3-fold increase in heterologous promoter activity in the forward and reverse orientation, respectively (P < 0.0001, n = 12). In contrast, no significant effect was observed in HeLa or C6 cells with either hybrid gene (n = 3–12).

Enhancer Activity Can Be Localized to a 260-bp Subfragment (Nucleotides 1346/1605) at the 3'-End of the 1.6G Fragment
A series of deletions of the 1.6G fragment were made and inserted upstream of the hybrid TK/luciferase gene. These included 1) 5'-deletions of nucleotides 1/745, 1/917, and 1/1298 to generate 746/1605G.TKp.luc, 918/1605G.TKp.luc, and 1299/1605G.TKp.luc; respectively; 2) an internal deletion of nucleotides 601/1300 to generate {Delta}a601/1300G.TKp.luc; and 3) a 3'-deletion of nucleotides 1346/1605 to generate 1/1345G.TKp.luc (Fig. 3Go). These hybrid genes, together with the RSVp.cat gene, were used to transiently transfect rat pituitary GC cells to localize the enhancer activity. Values were generated as mean luciferase/CAT activity plus or minus SEM and are expressed as the fold effect of each of the truncated 1.6G fragments on TK promoter activity (Fig. 3Go). All 5'-deletions displayed significant (~5- to 6- fold) stimulatory activity (P < 0.001; n = 12–33), which was indistinguishable from that observed in the presence of the full-length 1.6G fragment (Fig. 3Go). Similarly, the internal deletion {Delta}a601/1300G did not affect enhancer activity in pituitary GC cells after gene transfer (4.1-fold, P < 0.01, n = 9). However, a deletion of nucleotides 1346/1605 at the 3'-end of the 1.6G fragment abolished all significant stimulatory activity (n = 9), localizing 93% of the enhancer activity to this 260-bp region (Fig. 3Go).



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Figure 3. Enhancer Activity Is Decreased Greater Than 90% with Deletion of Nucleotides 1346/1605 from the 3'-End of the 1.6G Fragment

Nucleotides 1/745, 1/917, 1/1297, 601/1300, and 1346/1605 were deleted from the 1.6G fragment and tested upstream of a hybrid TK promoter/luciferase gene in transiently transfected GC cells. Cells were cotransfected with the RSVp.cat gene. Data (luciferase/CAT activity) from at least nine experiments are expressed as the fold effect (of these truncated 1.6G fragments) on TK promoter activity ± SEM.

 
Detection of DNA-Protein Interactions in the Truncated Enhancer Region and Assessment of A/T-Rich Pit-1/GHF-1-Like Sequences Contained in This Region for Enhancer Function in Culture
DNase I protection assays were done in an attempt to provide a more focused basis for assessment of the 260-bp region. A fragment corresponding to nucleotides 1298/1605 was radiolabeled (separate reactions done to assess each strand), incubated without or with nuclear protein from rat anterior pituitary GC or human cervical carcinoma HeLa cells, treated with DNase I, and then subjected to denaturing gel electrophoresis and autoradiography (Fig. 4Go). A large region of 133–136 bp showed some protection with increasing amounts of GC but not HeLa nuclear protein. This protection was observed on both strands and affected nucleotides 1344/1476 and 1343/1478 on the upper and lower strands, respectively (shaded, Fig. 1Go).



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Figure 4. A Large 133- to 136-bp Domain of the Enhancer Region Shows Some Protection with Increasing Amounts of GC but Not HeLa Cell Nuclear Extract

A subfragment (1 ng) containing nucleotides (A) 1298/1605 (SmaI/BglII), upper strand, or (B) 1605/1298 (BglII/SmaI), lower strand, was radiolabeled and incubated without or with nuclear extract and then digested partially with DNase I. Samples were run in a denaturing 5% or 8% acrylamide/8 M urea gel, dried, and assessed by autoradiography. Lane a: pBR322/HpaII; lane b: G+A sequencing reaction; lane c: G sequencing reaction; lane d: no nuclear protein; lanes e–g: 25, 12.5, and 6.3 µg of HeLa cell nuclear protein; and lanes h–j: 25, 12.5, and 6.3 µg of GC cell nuclear protein. The boxes indicate the footprinted (FP) region and the nucleotides protected.

 
Given the tissue-specific nature of the protected region as well as the activity of the 1.6G fragment in pituitary cells in vitro (Fig. 2Go) and in vivo (7), the possible participation of Pit-1/GHF-1 in the complex(es) formed in this region was investigated. Emphasis was given to the sequences 5'-ATGTTTATATTT-3' at nucleotides 1368/1379, 5'-TTTATTCCATGAACTGAA-3' at nucleotides 1388/1405, and 5'-AAATGTTTTTTCATTTg-3' at nucleotides 1426/1441 (double underlined, Fig. 1Go), because of the overrepresentation of A/T-rich sequences in binding sites for homeodomain proteins (14, 15, 16, 17). Indeed, a reexamination of these three sequences revealed that each contains a site, 5'-TATAaACAT-3', 5'-TTTatTCCAT-3', and 5'-TTTtTTCAT-3', respectively, that show some similarity with the consensus Pit-1/GHF-1 site, 5'-WWTATNCAT-3' (Fig. 5Go). To assess a possible role in enhancer function, site-directed mutagenesis was used to disrupt these three sequences, in the context of a 203-bp PCR product corresponding to nucleotides 1298/1500. This subfragment of the 1.6G fragment contains the entire nuclease-protected region at position 1343/1478. The element located at nucleotides 1368/1379 was converted to 5'-ATGgcggccgcT-3' (M1), the element at nucleotide 1388/1405 was converted to 5'-TTTATTCCgactctgtcA-3' (M2), and the element at 1426/1441 was converted to 5'-AAATGTTTTTTgtcgac-3' (M3). Sequences in an adjacent region corresponding to nucleotides 1444/1450 (5'-AACATCT-3') at the 3'-end of the footprint were also mutated to 5'-AACgcgT-3' (M4) for comparison. The wild-type and mutated fragments were then inserted upstream of TKp.luc to generate 1298/1500G.TKp.luc, M1–1298/1500G.TKp.luc, M2–1298/1500G.TKp.luc, M3–1298/1500G.TKp.luc, and M4–1298/1500G.TKp.luc, respectively, and tested for activity in transfected GC cells. The expression for each of these constructs was corrected using RSVp.cat activity, and the results are presented relative to 1298/1605TKp.luc which, based on the results presented in Fig. 3Go, was set to 100% activity (Fig. 6Go). As expected, the truncated region 1298/1500 containing the intact nuclease protected domain retained (96%) enhancer activity. Although 58% and 56% reductions in enhancer activity were observed with the disruption of sequences in M1 and M2 (P < 0.05, n = 18), the most significant decrease (75%) was seen with the modification resulting in M3 sequences (P < 0.001, n = 30). In contrast, only a 15% decrease in enhancer activity was seen with the disruption of sequences in M4 DNA.



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Figure 5. Sequence of Pit-1/GHF-1-Like Sites Found in the Region Protected by Pituitary GC Cell Nuclear Protein

A, Nucleotides identical to those in the consensus Pit-1/GHF-1 element (14 ) are indicated by uppercase letters. The sequences of previously characterized Pit-1/GHF-1 elements from the proximal promoter region of the human GH-N (hGH-N) gene (18 ) and the proximal promoter region from the rat PRL (rPRL) gene (19 ) are indicated. The rPRL sequence was shown to represent a high-affinity binding site due to the presence of the upstream A/T-rich sequences (underlined) and is capable of supporting binding of Pit-1/GHF-1 as a dimer (19 ). B, Sequence of the region protected by pituitary GC cell nuclear protein corresponding to nucleotides 1343/1478. The three boxed domains indicate the boundaries of the A/T-rich regions, A/T-1, A/T-2, and A/T-3, and the Pit-1/GHF-1-like sequences are underlined. The region described as A/T-1+2 corresponds to a fragment spanning nucleotides 1344/1425.

 


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Figure 6. Enhancer Activity Is Decreased Greater Than 50% with Disruption of A/T-Rich Regions Containing Pit-1/GHF-1-Like Elements

A fragment corresponding to nucleotides 1298/1500 and containing the intact nuclease-protected region 1343/1478 was generated by PCR. Site-directed mutagenesis was used to disrupt the A/T-rich Pit-1/GHF-1-like elements located at nucleotides 1368/1379 (M1), 1388/1405 (M2), and 1426/1441 (M3), as well as an adjacent region 1464/1470 (M4) for comparison. The wild-type and mutated fragments were then inserted upstream of TKp.luc to generate 1298/1500G.TKp.luc, M1–1298/1500G.TKp.luc, M2–1298/1500G.TKp.luc, M3–1298/1500G.TKp.luc, and M4–1298/1500G.TKp.luc, respectively, and tested for activity in transfected GC cells. The results are expressed as a percentage of the enhancer activity observed with 1298/1605TKp.luc, which was set to 100%. Error bars represent SEM from at least six experiments.

 
Disruption of 5'-AAATGTTTTTTCATTT-3' in M3 Interferes with Enhancer Function in Transgenic Mice
Founder transgenics were used to assess the result of disrupted sequences, referred to as M3, in vivo. Since previous studies of enhancer function in vivo were restricted to using the homologous human GH-N promoter, which contains Pit-1/GHF-1 DNA elements, the opportunity was also taken to examine the effect of the 1.6G fragment on heterologous promoter activity in pituitary and nonpituitary tissue. The hybrid genes 1/1605G.TKp.luc, 1298/1500G.TKp.luc, M3–1298/1500G.TKp.luc, and TKp.luc were introduced into zygotes of CD1 mice by pronuclear injection. Tissues were taken at embryonic day 19 or term, and luciferase activity was determined. The results expressed as luciferase activity per µg protein (per 30-sec period of measurement) are presented in Table 1Go. The identification (or confirmation) of a transgenic mouse as well as an estimate of copy number was done by DNA blotting using genomic tail DNA. Even in the absence of consensus Pit-1/GHF-1 sites within the context of the TK promoter, appreciable activity was detected in the pituitary of transgenic mice in the presence of the 1.6G fragment. However, significant activity was also seen in multiple tissues, particularly the brain and testis. Although there was more variability, a similar pattern of transgene expression was observed using the 203-bp subfragment of the 1.6G fragment. However, enhancer activity was disrupted by modification of sequences, M3, in the context of the 203-bp subfragment. Three of the four founders identified using M3–1298/1500G.TKp.luc as the transgene had control levels of activity in the pituitary (similar to that seen with the TKp.luc gene). In contrast, one of the four transgenics appeared to retain enhancer activity in the pituitary; however, levels were almost 4 times lower than seen in the brain of this animal. The mean results from 20 mice processed in an identical manner during the course of this study but determined not to be transgenic are included for comparison.


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Table 1. Luciferase activity (per µg protein per 30 sec) in Tissues Taken at the Time of Birth of Founder Transgenic Mice

 
Disruption of Sequences at Nucleotides 1426/1441 in M3 Interferes with Complex Formation on the 203-bp Enhancer Fragment, Correlating with a Loss of Function
The effect of the M3 mutation on protein/DNA complex formation with the 203-bp fragment was assessed by EMSA (Fig. 7Go). The patterns generated between the wild-type 203-bp fragment, containing the intact nuclease protected region 1343/1478, and GC vs. HeLa cell nuclear protein were distinct, although some bands/complexes with similar mobilities were observed. Most notably, a major high-mobility (smaller) and low-mobility (larger) complex was detected with the pituitary GC cell extract (Fig. 7Go, lane b). Mutation of the A/T-rich region at the core of the footprint region at nucleotides 1426/1441, referred to as M3, resulted in a decrease in the levels of the low-mobility complex (see arrow; Fig. 7Go, compare lanes b and e). The result of the M4 modification of sequences (nucleotides 1447/1449) was also assessed for comparison. In contrast to M3, this mutation appeared to have little effect on the pattern of complexes observed (Fig. 7Go, compare lanes b and h).



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Figure 7. Complex Formation on the 203-bp Subfragment with Pituitary Nuclear Protein Is Decreased after Mutation of Sequences Associated with the A/T-Rich Pit-1/GHF-1-like Element (1426/1441) but Not Adjacent Sequences (1464/1470)

EMSAs were done by incubating 32P-labeled wild-type 203-bp fragment (wt) corresponding to nucleotides 1298/1500 (a–c), as well as the mutant form of this fragment M3 (e–g) or M4 (h–j), without (a, e, and h) or with pituitary GC (b, f, and i) or cervical HeLa (c, g, and j) cell nuclear extract (6 µg) in the presence of 2 µg of poly (dI-dC). Complexes detected after gel electrophoresis and autoradiography are shown. The major specific LMC formed between GC nuclear protein and wild-type 203-bp fragment is indicated (arrow).

 
The 203-bp Enhancer Region Contains Pit-1/GHF-1 Binding Sites
EMSA of the 203-bp enhancer region with pituitary GC nuclear protein was done in combination with DNA competitors for low- and high-affinity Pit-1/GHF-1 binding. Pit-1/GHF-1 elements were used from the human GH-N gene (18) and rat PRL (19) proximal promoter regions (Fig. 5Go). The PRL sequence was shown to be a high-affinity site capable of binding Pit-1/GHF-1 as a dimer (19). An unrelated RF-1 element (20) was used as a negative control. The low-mobility complex was competed efficiently with the PRL (high affinity) but not GH-N Pit-1/GHF-1 or RF-1 sites (Fig. 8Go). Three overlapping fragments containing one or more of the A/T-rich regions (Fig. 5BGo) corresponding to nucleotides 1344/1425, which contain two putative elements at 1368/1379 and 1388/1400 (A/T-1+2), 1378/1412 (A/T-2, not shown) and 1416/1455 (A/T-3), containing the Pit-1/GHF-1-like elements in the 203-bp enhancer region, were generated and also used as competitors. Efficient competition was seen only with the A/T-1+2 region (Fig. 8Go).



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Figure 8. A Major Complex Formed on the 203-bp Subfragment with Pituitary Nuclear Protein Is Competed Using a Pit-1/GHF-1 DNA Element

EMSA of the 203-bp enhancer region (a) with pituitary GC nuclear protein (b,r) was done in combination with a 50, 100, and 200 pM excess of the following DNA competitors: rat PRL Pit-1/GHF-1 element (c–e); human GH-N Pit-1/GHF-1 element (f–h); nucleotide region 1416/1455 (i–k); nucleotide region 1344/1425 (l–n); and RF-1 element (o–q). The major specific LMC formed between GC nuclear protein and wild-type 203-bp fragment, as seen in Fig. 7Go, is indicated (arrow).

 
The overlapping fragments corresponding to A/T-1+2 (nucleotides 1344/1425), A/T-1 (nucleotides 1365/1379), A/T-2 (nucleotides 1378/1412), and A/T-3 (nucleotides 1416/1455) were also used as probes and competed with the GH-N and PRL Pit-1/GHF-1 elements (Fig. 9Go). For the fragment A/T-1+2 multiple specific complexes were observed based on their competition by excess unlabeled fragment (Fig. 9AGo). Both low- and high-mobility bands, reflecting larger, low-mobility (LMC) and smaller (SMC) complexes, respectively, were competed with the PRL Pit-1/GHF-1 element (Fig. 9AGo). In contrast, the GH-N Pit-1/GHF-1 element was only able to compete the smaller high-mobility complexes. A specific intermediate complex (IMC) with a mobility between those indicated (LMC and SMC) was not competed with either Pit-1/GHF-1 elements (Fig. 9AGo). For the A/T-1 region, a specific intermediate mobility complex was observed (Fig. 9BGo). This complex was competed by the GH-N Pit-1/GHF-1 site to the same extent as observed with the wild-type unlabeled probe fragment, but more efficiently by the PRL Pit-1/GHF-1 element. A minor low- (LMC) and two major high-mobility complexes (SMC) were observed with A/T-2 (Fig. 9CGo), which contains the second putative A/T-rich Pit-1/GHF-1-like element at nucleotides 1388/1400 in the A/T-1+2 fragment (Fig. 5Go). These complexes all showed some degree of competition, and thus specificity, with excess unlabeled fragment. Evidence for competition of the high-mobility complexes (SMC) was observed with the GH-N Pit-1/GHF-1 element, and to a greater extent with the PRL Pit-1/GHF-1 element (Fig. 9CGo). For the A/T-3 region, which contains a putative Pit-1/GHF-1-like element at nucleotides 1426/1441 (Fig. 5Go), multiple specific complexes ranging in mobility/size were detected (LMC, IMC1, IMC2, and SMC; Fig. 9DGo). Complexes represented by IMC2 and SMC were competed by the PRL Pit-1/GHF-1 element. Competition of these complexes by the GH-N Pit-1/GHF-1 site was also observed, although competition of SMC was to a lesser extent (Fig. 9DGo).



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Figure 9. Multiple Complexes Are Associated with A/T-Rich Regions in the 136-bp Nuclease-Protected Domain

Pituitary GC cell nuclear extract (5 µg) was incubated with 2 µg of poly (dI-dC) and 32P-labeled DNA probes (1 ng) corresponding to nucleotides 1344/1425 (A/T-1+2), 1365/1379 (A/T-1), 1378/1412 (A/T-2), and 1416/1455 (A/T-3). For competition assays, EMSA of each fragment with pituitary GC nuclear protein was done in combination with DNA competitors for Pit-1/GHF-1 binding. Pit-1/GHF-1 elements were used from the human GH-N (18 ) and rat PRL (19 ) gene proximal promoter regions (Fig. 5Go). Large, intermediate, and small complexes were detected after gel electrophoresis and autoradiography and are indicated by LMC, IMC, and SMC, respectively. A, 1344/1425 probe (a); probe/pituitary protein extract (b) in the presence of 25 (c), 50 (d), and 100 (e) pM excess 1344/1425 fragment, or 25 (f), 50 (g), and 100 (h) pM excess PRL Pit-1/GHF-1 oligonucleotide, or 25 (i), 50 (j), and 100 (k) pM excess GH-N Pit-1/GHF-1 oligonucleotide. B, 1365/1379 probe (a); probe/pituitary protein extract (b) in the presence of 50 (c), 100 (d), 250 (e), and 500 (f) pM excess 1365/1379 fragment, or 100 (g), 250 (h), and 500 (i) pM excess PRL Pit-1/GHF-1 oligonucleotide, or 100 (j), 250 (k), and 500 (l) pM excess GH-N Pit-1/GHF-1 oligonucleotide. C, 1378/1412 probe (a); probe/pituitary protein extract (b) in the presence of 25 (c), 50 (d), and 100 (e) pM excess 1378/1412 fragment, or 25 (f), 50 (g), and 100 (h) pM excess PRL Pit-1/GHF-1 oligonucleotide, or 25 (i), 50 (j), and 100 (k) pM excess GH-N Pit-1/GHF-1 oligonucleotide. D, 1416/1455 probe (a); probe/pituitary protein extract (b) in the presence of 25 (c), 50 (d), and 100 (e) pM excess 1416/1455 fragment, or 25 (f), 50 (g), and 100 (h) pM excess PRL Pit-1/GHF-1 oligonucleotide, or 25 (i), 50 (j), and 100 (k) pM excess GH-N Pit-1/GHF-1 oligonucleotide.

 
Pit-1/GHF-1 Participates in Complexes Formed on Subfragments of the 203-bp Enhancer Region
Antibodies to GHF-1 were used in an attempt to obtain a more direct assessment of the participation of GHF-1/Pit-1 in complexes formed on the A/T-1+2 (nucleotides 1344/1425) and A/T-3 (nucleotides 1416/1455) subfragments, which contain the A/T-rich Pit-1/GHF-1-like elements identified in the 1.6G fragment (Figs. 1Go and 5Go). More specifically, antibodies to GHF-1 were used to compete complexes formed with these regions and GC cell nuclear extract in a gel mobility shift assay. Evidence for competition and the appearance of low- mobility complexes was detected with these fragments in the presence of GHF-1 antibodies but not normal rabbit serum (Fig. 10Go). Most notably, complexes identified as IMC2 in the A/T-1+2 region and SMC in the A/T-3 region that were competed with a Pit-1/GHF-1 DNA element (Fig. 9Go) were also competed by the GHF-1 antibodies (Fig. 10Go).



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Figure 10. EMSA and Competition with Specific Antibodies Indicates that Pit-1/GHF-1 Participates in Complexes Formed on Nucleotide Regions 1344/1425 and 1416/1455

Radiolabeled DNA probes (1 ng) corresponding to nucleotide regions (A) 1344/1425 (A/T-1+2) and (B) 1416/1455 (A/T-3) were incubated with 2 µg poly (dI-dC) and pituitary GC cell nuclear extract in the absence (a) or presence (b) of GHF-1 antiserum or (c) normal rabbit serum (NRS). Some competition of bands and the appearance of LMCs are detected in the presence of GHF-1 antibodies but not NRS with both fragments.

 
The A/T-1+2, A/T-1, A/T-2, and A/T-3 fragments were used with purified Pit-1/GHF-1 in EMSAs to assess any interaction in the absence of additional nuclear proteins. Specific binding of a commercially available rat Pit-1 preparation to well characterized Pit-1/GHF-1 elements was established initially. Pit-1/GHF-1-specific bands were observed when PRL and GH-N Pit-1/GHF-1 DNA elements were used as probes, but not with the unrelated RF-1 site (Fig. 11Go). Assessment of the A/T-rich regions revealed evidence of Pit-1 binding to A/T-1+2 and A/T-3 sequences (Fig. 11Go). A band corresponding to the higher mobility Pit-1/GHF-1-specific complex was also seen with the A/T-2 fragment on prolonged autoradiographic exposure.



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Figure 11. Pit-1 Binds to A/T-Rich Regions of the 136-bp Nuclease-Protected Domain in the Absence of Other Nuclear Proteins

Radiolabeled DNA probes (1 ng) corresponding to Pit-1/GHF-1 elements from the rat PRL and human GH-N gene proximal promoter regions, the RF-1 element (RF-1E, negative control), as well as the A/T-1+2, A/T-1, A/T-2, and A/T-3 regions of the distal enhancer were incubated in the absence (-) or presence (+) of 714 ng Pit-1. In each panel, the open arrowheads indicate Pit-1/GHF-1-specific complexes observed with the rPRL Pit-1/GHF-1 element. The solid arrowhead indicates a, presumably, DNA-dependent complex seen with the A/T-3 region with a mobility that would, unfortunately, obscure one of the Pit-1/GHF-1-specific complexes. The lower-mobility Pit-1/GHF-1-specific complex is, however, observed with the A/T-3 region.

 
Pit-1/GHF-1 Alone Is Not Sufficient to Stimulate 1.6G Enhancer Activity
To further assess a possible contribution of Pit-1/GHF-1 to 1.6G enhancer activity, possibly through protein-protein interactions, expression of GHp.luc and 1/1605G.GHp.luc was compared in HeLa cells cotransfected with increasing amounts of a Pit-1/GHF-1 cDNA expression vector (RSVp.GHF-1). Results for promoter activity, corrected using RSVp.cat activity, were expressed as mean luciferase/CAT activity ± SEM (Fig. 12Go). Comparison of GHp.luc and 1/1605G.GHp.luc expression in the absence of Pit-1/GHF-1 reveals only a 1.2-fold increase (not significant) in (albeit low levels of) basal expression in the presence of the 1.6G fragment. Overexpression of Pit-1/GHF-1 using 5 and 10 µg of expression vector resulted in 3.2- and 15.5-fold increases in GHp.luc activity, respectively (Fig. 12Go). Similar increases (3.6- and 13.6-fold) in 1/1605G.GHp.luc expression were also observed with Pit-1/GHF-1 overexpression (Fig. 12Go). The increase in GH-N promoter activity observed as a consequence of Pit-1/GHF-1 overexpression is Pit-1/GHF-1 DNA element dependent, since the corresponding effect of Pit-1/GHF-1 overexpression using 5 and 10 µg of expression vector on Rous sarcoma virus (RSV) promoter activity (expressed as cpm/mg protein) was a 2-fold decrease and only a 1.5-fold increase (not significant), respectively (n = 6).



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Figure 12. Pit-1/GHF-1 Coexpression in HeLa Cells Results in a Similar Level of TK Promoter Activity in the Presence or Absence of the 1.6G Fragment

Expression of GHp.luc and 1.6G/GHp.luc were compared in HeLa cells cotransfected with 0, 5, or 10 µg of RSVp.GHF-1 to increase Pit-1/GHF-1 levels. Cells were also cotransfected with RSVp.cat. Promoter activity from at least six experiments is expressed as mean luciferase/CAT activity ± SEM.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we have done sequencing, gene transfer, and protein/DNA interaction experiments to investigate the enhancer activity associated with a component (1.6G) of the GH LCR, located remotely (15 kb) upstream of the GH-N transcription initiation site (7, 10). The 1.6G region was reported to contain two pituitary-specific hypersensitive sites (HS I and HS II) (7), and enhancer activity was localized to a 404-bp region (11). However, a more detailed investigation of the sequences and protein/DNA interactions involved in enhancer activity was lacking. In this study we have characterized functional and structural properties of a 203-bp distal GH-N enhancer region. We report that the 1.6G fragment, in both the forward and reverse orientation, was able to significantly enhance the GH-N promoter in two rat pituitary cell lines (GC and GH3) after gene transfer (Fig. 2Go). Thus, 1.6G enhancer activity can be observed in both transient transfection as well as transgenic mouse experiments. Similarly, enhancer sequences within the context of the globin genes LCRs, function efficiently both in vitro and in vivo (7, 21, 22, 23). The ability of the 1.6G fragment to act as an enhancer in a promoter-independent manner and without a requirement for a Pit-1/GHF-1 element in the proximal promoter region, was confirmed when the 1.6G fragment was shown to stimulate a heterologous (minimal TK) promoter in pituitary cells in culture (Fig. 2Go) and transgenic mice (Table 1Go). In the case of the latter, enhancer activity was detected in tissues other than pituitary, most notably the brain and testis. However, on a per µg protein basis, levels were significantly less than measured in the pituitary of all three founder transgenic mice (Table 1Go).

Deletion analysis with subsequent testing of truncated fragments upstream of the minimal TK promoter in GC cells localized the enhancer activity, at least in part, to a 203-bp region corresponding to nucleotides 1298/1500 in the 3'-end of the 1.6G fragment (Figs. 3Go and 6Go). These sequences were also capable of stimulating TK promoter activity in founder transgenic mice (Table 1Go). A similar pattern to that seen with the 1.6G fragment was observed, as three of the four founders displayed more efficient enhancer activity in the pituitary vs. brain, testis, and other tissues, including kidney, liver, placenta, and spleen. The 1.6G fragment was shown previously to contain insufficient information to permit copy number-dependent transgene expression (7). Consistent with this result, we did not observe copy number-dependent luciferase gene expression and, thus, integration independence with the 203-bp subfragment. Considerable variation in expression was seen with three founders determined to have a single copy of the transgene inserted, and a fourth with five copies was determined to display the lowest level of stimulation (Table 1Go). Given this observation, the fifth founder (), which showed efficient enhancer activity in the pituitary but also a comparable level in the brain, likely reflects a consequence of site of integration.

Nuclease protection assay of the 203-bp region using pituitary GC cell nuclear protein resulted in the detection of a large 136-bp footprint, which appears diffuse because of difficulty in detecting any clear internal boundaries. However, this level of protection was evident on both strands, corresponding to nucleotides 1344/1476 and 1343/1478 on the upper and lower strands, respectively (Fig. 4Go). This footprint was not seen with nonpituitary HeLa cell nuclear extract (Fig. 4Go). Although some of these results were obtained using human DNA sequences with proteins isolated from rat pituitary cells, both human pituitary-specific hypersensitive sites (HS I and HS II) were reconstructed in the transgenic mouse pituitary when the 1.6G-containing fragment, including the GH-N promoter, was used as a transgene (7). Thus, protein(s) responsible for generating the chromosomal organization at HS I and HS II that is recognized by nuclease in the human pituitary must be conserved in the mouse pituitary. By extension, it is likely then that equivalent proteins are present in the rat anterior pituitary GC cells used for our studies.

The size and nature of the nuclease-protected region suggested that more than one protein, a complex, likely involving DNA-protein as well as protein-protein interactions, was responsible for this event and, presumably efficient enhancer function. However, this also excluded any simple rational approach for mutagenesis to investigate protein/DNA interactions. The idea of a multicomponent complex was supported, however, by the detection of a major low-mobility (larger) band with pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein by EMSA (Fig. 7Go). The failure to see a similar level of nuclease protection or pattern of shifted bands with cervical HeLa cell nuclear protein correlates with the lack of any stimulatory activity in these cells after gene transfer (Fig. 2Go). Given the broad range of enhancer activity seen in various tissues (Table 1Go), it is unclear why no stimulatory activity was seen in HeLa or C6 cells. However, although cervical tissue and glial cells specifically (as opposed to brain) were not assessed for luciferase activity in transgenic mice, the lack of activity observed is likely due to the enormous difference in sensitivity between the in culture vs. in vivo assay. This difference is reflected in the levels of enhancer activity seen in pituitary GC cells vs. pituitary tissue (Fig. 2Go and Table 1Go). A difference between transient transfection in culture and stable integration in the transgenic mice could also contribute to this effect.

The apparent tissue-specific nature of the 136-bp footprint and the efficient enhancer activity observed in pituitary cells suggested the possible involvement of the POU-homeodomain protein Pit-1/GHF-1, which plays an essential role in the normal development and function of the pituitary. No consensus Pit-1/GHF-1 DNA element of the form 5'-WWTATNCAT-3' is present in the 136-bp nuclease-protected region (Fig. 1Go). However, examination of the sequences in this region did reveal three relatively A/T-rich sequences that contained Pit-1/GHF-1-like DNA elements (Fig. 5Go). Mutation of each of these three elements resulted in some, but not total, loss of enhancer function in the context of the 203-bp subfragment (Fig. 6Go). The greatest and most significant loss was observed with disruption of the Pit-1/GHF-1-like element at nucleotides 1426/1441 within A/T-3. This element can be distinguished from the other two by virtue of a sequence (5'-AAATGTTTTTTCATTT-3') with the capacity to form a stem-loop structure, which was also disrupted by the mutation, M3. In the context of the ß-globin locus, palindromes with the potential to form cruciform structures under torsional stress were found preferentially near clusters of possible homeodomain protein-binding sites and or matrix attachment sites (MARs)(16).

The involvement of A/T-3 in enhancer activity was confirmed in transgenic mice. As a result of the A/T-3 mutation (M3), three of the four founders displayed only background levels of activity, comparable to that seen using the minimal TK promoter alone, in the pituitary as well as other tissues (Table 1Go). A fourth founder () retained stimulatory activity in the pituitary, but the level was relatively lower than seen in all but one of the nine founders expressing the wild type 1.6G or 203-bp enhancer transgenes. Also, the pattern of enhancer activity was different from the majority of founders, as more than 3 times the activity was seen in the brain and, thus, it is assumed that this reflects a consequence of site of integration. Although not shown, three founders () were also generated with the double mutation corresponding to M1 and M3, and none displayed pituitary-enhancer activity (14.0–8.4 luciferase units/µg protein).

The loss of enhancer activity in culture, as well as transgenic mice with the M3 mutation, correlates with a decrease in the levels of a major low-mobility specific pituitary complex seen with a 203-bp subfragment bearing the M3 mutation (Fig. 7Go). This decrease was not observed with mutation (M4) of adjacent sequences (nucleotides 1444/1450). The Pit-1/GHF-1-like site in A/T-3 lies at the core of the footprint domain detected with pituitary nuclear protein at position 1343/1478 (Figs. 4Go and 5Go). The efficient enhancer activity observed in pituitary cells and knowledge that A/T-rich sequences are a common feature of homeodomain protein-binding sites raised the possibility that Pit-1/GHF-1 or a Pit-1/GHF-1-like protein interacts with this DNA. Data from EMSAs done using the 203-bp fragment and pituitary nuclear extract, in combination with competitors of Pit-1/GHF-1 binding, indicated that Pit-1/GHF-1 binds with high affinity to the major LMC (Fig. 8Go). Given the competition using the rat PRL, but not the human GH-N Pit-1/GHF-1 element, the binding likely reflects formation of a dimer (19). When the three A/T-rich regions were used as competitors, only the A/T-1+2 region (nucleotides 1344/1425) competed the same complex effectively (Fig. 8Go). This suggested that these sequences might contain a high-affinity Pit-1/GHF-1 binding site. This was confirmed by EMSA and competition with the rat PRL Pit-1/GHF-1 element (Figs. 8Go and 9Go). Pit-1/GHF-1 binding to A/T-3 (nucleotides 1416/1455), the region containing the putative stem-loop structure, as well as A/T-1 (nucleotides 1365/1379) and A/T-2 (nucleotides 1378/1412), were also indicated by competition with Pit-1/GHF-1 elements (Fig. 9Go). Indeed, these appeared to contain low-affinity Pit-1/GHF-1 binding sites when assessed individually, as homologous competition was relatively weak. Furthermore, competition was not only possible by the high-affinity PRL Pit-1/GHF-1 site but also by the GH-N Pit-1/GHF-1 element. The participation of Pit-1/GHF-1 in complexes formed on fragments containing these sequences was confirmed using antibodies to GHF-1 (Fig. 10Go) and through their ability to directly interact with recombinant rat Pit-1 in the absence of additional nuclear protein (Fig. 11Go). Interestingly, when the A/T-1+2 region, which contains two of the Pit-1/GHF-1-like elements, was used as a probe or competitor, evidence for high-affinity Pit-1/GHF-1 binding was obtained. The presence of Pit-1/GHF-1 binding sites spanning a significant portion of nuclease-protected region raises the possibility that there is interaction between these sites and is consistent with the formation of a large complex on the 203-bp enhancer fragment. Of course this does not rule out the participation of proteins other than Pit-1/GHF-1 in this complex. Indeed, the detection of bands formed on the A/T-1+2 (IMC, Fig. 9AGo), A/T-2 (LMC, Fig. 9CGo), and A/T-3 (LMC and IMC1, Fig. 9DGo) regions, which are not competed by a Pit-1/GHF-1 element (under the EMSA conditions employed), indicates the participation of other protein(s) (Fig. 9Go). The importance of a factor(s) in addition to Pit-1/GHF-1 for distal enhancer activity was suggested by transfection studies using nonpituitary HeLa cells overexpressing Pit-1/GHF-1. Although there was sufficient overexpression of Pit-1/GHF-1 to see a stimulation of basal GH-N promoter activity, there was no further increase resulting from the inclusion of the 1.6G fragment (Fig. 12Go). This additional participant might be tissue- or cell-specific, but the possibility that it is present at low levels or modified in the HeLa cells cannot be ruled out. A review of the properties of POU domain transcription factors like Pit-1/GHF-1 supported the possibility that Pit-1/GHF-1 might act as a coactivator but that the specificity of its function would be determined in part by the unique configuration it assumes on binding its own DNA element (24). With regard to the 203-bp enhancer fragment, the need for multiple binding events to produce a stable and functional complex allowing pituitary enhancer activity is indicated not only by the 136-bp nuclease-protected region (nucleotides 1343/1478; Fig. 5Go) but also by the functional assessment of mutant enhancer regions (Fig. 6Go and Table 1Go). Also, although not shown, two transgenic founders () were generated using the A/T-1+2 region (nucleotides 1344/1425) alone to stimulate TK/luciferase gene expression. No pituitary enhancer activity was detected with either 385–3 (3.8 luciferase units/µg protein) or 398–3 (0.03 luciferase units/µg protein). The case for multiple binding events with the GH-N distal enhancer region may be analogous to the situation observed with both human and rat PRL genes. Their distal enhancers are both Pit-1/GHF-1-dependent, and multiple Pit-1/GHF-1 elements are required for efficient enhancer activity (25, 26). However, both contain additional protein-binding sites within these regions and, in the case of the rat gene, these are necessary to confer regulatory potential (25, 26).

The 1.6G fragment is located in the 5'-flanking DNA of the B-lymphocyte-specific CD79b gene (10). The ability of these sequences to stimulate not only the GH-N promoter, but also the minimal TK promoter (Fig. 2Go), raises the possibility that they might also be able to influence the expression of the CD79b gene in lymphocytes, where the B lymphocyte-specific CD79b gene locus is open. Furthermore, we and others have detected GH-N in lymphocytes (27, 28), which raises the question of whether the 1.6G fragment participates in the activation of the GH-N gene in these cells. Interestingly, the presence of Pit-1/GHF-1 transcripts in lymphocytes has been reported (29). Thus, it is possible that sequences in the 1.6G region might serve two functions. The first relates to a role in the GH LCR where it contributes to the establishment of an open chromatin conformation in pituitary. This is betrayed by the generation of pituitary-specific hypersensitive sites, which presumably involves chromatin reorganization, perhaps through association with the nuclear matrix. The second could involve stimulation of promoter activity outside as well as inside the pituitary. These functions would be consistent with multiple regulatory roles associated with A/T-rich sequences, which include binding sites for homeobox-containing transcription factors, as well as MARs. A striking similarity has been suggested between the A/T-rich sequence motifs present in MARs and homeobox-containing transcription factors (15). Indeed, the POU-specific domain in Pit-1/GHF-1 has been shown recently to contain a necessary and sufficient signal for targeting to the nuclear matrix (30). Among several functions ascribed to MARs is their ability to mediate cell-specific expression and define the borders of chromatin domains (Ref. 15 and references therein and Ref. 16). We have not examined whether the A/T-rich Pit-1/GHF-1-like sequences that we identified within the distal GH enhancer located in the GH LCR participate in changing chromatin configuration and the establishment of an open locus. However, the A/T-rich Pit-1/GHF-1-like sequences identified in the 136-bp protected region are excellent candidates as mediators of the enhancer activity associated with the GH LCR, since we have demonstrated that their disruption diminishes enhancer function in pituitary in vivo.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning the 1.6-kb BglII Fragment of the GH Locus and Sequencing
A P1 library containing human genomic DNA was screened by PCR using an amplicon corresponding to 160 bp located in a region 3 kb upstream of the GH-N transcription initiation site (Genome Systems Inc., St. Louis, MO). The amplicon was generated using the primers: 5'-CAGCCTCTGATCTCAAGGAAG-3' and 5'-GTGGGGTTGAGGACGATCAC-3', and a clone was obtained (P1–3; clone address: DMPC-HFF#1–1434-A). The boundaries of P1–3 were confirmed by sequence analysis, since both the 5'- and 3'-ends reside in reported sequences (exon 9 of SCN4A and downstream of the GH-N gene, respectively)(12, 31). P1–3 was digested with BglII and electrophoresed, and the desired 1.6-kb BglII (1.6G) fragment size was eluted and subcloned into the BglII site of pSP73. Nucleotide sequence was determined by the dideoxy method using the f-mol sequencing kit (Promega Corp., Madison, WI). Analysis of the sequence for consensus elements was done using GeneWorks v2.4 (Intelligenetics, Inc., Mountain View, CA) including a consensus sequences for Pit-1/GHF-1 (14).

DNA (Southern) Blotting
Human genomic DNA was isolated from choriocarcinoma JEG-3 cells (32), digested with various restriction endonucleases, electrophoresed in 0.7% (wt/vol) agarose gels, and blotted to nitrocellulose. Fragments or oligonucleotides used as probes were radiolabeled routinely to a specific activity of approximately 1 x 109 cpm/µg or 1.6 x 107 cpm/µg using [32P]dATP or [32P]ATP, respectively. DNA blots were hybridized to radiolabeled probes at 42 C (DNA fragments) or 48 C (oligonucleotides) in the presence of 50% formamide for 20–24 h. For DNA fragment probes, blots were washed three times for 15 min each time at 65 C in 0.1x SSC (20x SSC: 3 M sodium chloride, 0.3 M sodium citrate) with 0.1% SDS, and for oligonucleotides, washed for 10 min each with 6x SSC/0.1% SDS and 2x SSC/0.1% SDS. Blots were visualized by autoradiography.

Plasmid Construction and Site-Directed Mutagenesis
The 1.6G fragment was blunted into the HindIII site of pBR322 in the forward and reverse orientation, with the 2.6 kb GH-N gene located downstream in the EcoRI site (33). The 1.6G fragments (forward and reverse) with the GH-N promoter region (-496/+1) were released by BamHI digestion and introduced in the BamHI site of pXP1 (34) to generate 1/1605G.GHp.luc and 1605/1G.GHp.luc, respectively. To generate GHp.luc, the 1.6G fragment was removed from 1/1605G.GHp.luc by EcoRV/ClaI digestion. The 1.6G fragment was blunted and inserted into the SmaI site of pT81luc (34) in the forward (1/1605G.TKp.luc) and reverse (1605/1G.TKp.luc) orientation. To generate 5'-deletions, the 1.6G fragment was first blunted into the SmaI site of pUC19. A PstI digestion was done to remove an internal 745-bp sequence containing a SacI site and then reclosed resulting in a subfragment of the 1.6G fragment corresponding to nucleotides 741/1605. This vector was then digested with PstI (blunted)/SacI, SspI/SacI or SmaI/SacI. The resulting subfragments of the 1.6G fragment were introduced into the SmaI/SacI sites of pT81luc to generate 746/1605G.TKp.luc, 918/1605G.TKp.luc, and 1298/1605G.TKp.luc, respectively. The plasmid containing 1/1605G.TKp.luc was digested with SmaI or NcoI/XhoI and religated to generate the internal deletion {Delta}a601/1300GTKp.luc, or the 3'-deletion 1/1345G.TKp.luc, respectively. A 203-bp subfragment of the 1.6G fragment corresponding to nucleotides 1298/1500 was synthesized by PCR with HindIII and SacI ends and inserted into pT81luc cut with HindIII/SacI to generate 1298/1500G.TKp.luc. Site-directed mutagenesis (35, 36) was done to disrupt sequences in the 203-bp region (1298/1500) of the 1.6G fragment by PCR, using 5 ng of template and the primers: M1, 5'-CGGGCCCATGGGCCTCAAGCTGACCTCAGGTGATGgcg-gccgcTCTGAGCTGTTTATTCC-3'; M2, 5'-CGGGCCCATGGGCCTCAAGCTGACCTCAGGTGATGTTTATATTTCTGA-GCT GTTTATTCCgactctgtcACATCTGACAGCTTTTC-3'; M3, 5'-TAAGGTGAGCTCCGAGGAAC AGCCCGTTCCGGGCAGCCCCAGATGTTCTTTCTTGTTTCCAGATGTTCgtcgac AA-AAAACATTTCTCT-3'; and M4 5'-TAAGGTGAGCTCCGAGGAACAGCCCGTTCCGGGC AGCCCCAGATGTTCTTTCTTGTTTCCAcgcGTTCCAAATGAAAAAAC-3'. The M1 and M2 primers were paired with the reverse primer 5'-TAAGGTGAGCTCCGAGGAACAGCCCGTTCC G-3', and M3 and M4 were paired with the forward primer 5'-GATATCAAGCTTCC CGGGTCAGTCTCTCTCCAG-3'. After an initial step at 94 C for 4 min, amplification was done (in 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 50 mM KCl, 200 µg/ml gelatin, 50 µM of each deoxynucleotide triphosphate, 1 µM of each primer, and 2 U Taq polymerase in a final volume of 50 µl) with 29–39 cycles of denaturation at 95 C for 1 min, annealing at 55–58 C for 45 sec, and extension at 72 C for 90 sec. In the final cycle the extension time was increased to 10 min. The products were digested with either NcoI/SacI (M1 and M2) or HindIII/SacI (M3 and M4), resolved by 4% agarose gel electrophoresis, isolated, and inserted upstream of 1298/1605G.TKp.luc cut with NcoI/SacI (M1 and M2) or pT81luc cut with HindIII/SacI (M3 and M4). The chloramphenicol acetyltransferase (CAT) gene directed by the RSV promoter (RSVp.cat) was described elsewhere (37). The expression vector containing the rat GHF-1 cDNA directed by the RSV promoter (RSVp.GHF-1) was a generous gift from Dr. M. Karin (University of California, San Diego, CA).

Cell Culture and Gene Transfer
Monolayer cultures of rat anterior pituitary GC and GH3 cells, as well as glioma C6 cells, and human cervical carcinoma HeLa cells were grown on 100-mm dishes and maintained at 37 C in 8% FBS-DMEM medium at a density of 1 x 106 cells per plate. Cells, in triplicate, were transfected with 10 µg of test (luciferase) plasmid DNA and 2 µg of RSVp.cat 18–24 h after plating, by the calcium phosphate/DNA precipitation method as previously described (38). Cells were harvested 48 h after transfection. Luciferase activity per µg of lysate protein was determined using the Luciferase Assay System (Promega Corp.) and a luminometer (ILA911 Luminometer, Tropix Inc., Bedford, MA) according to manufacturer’s instructions. CAT activity was measured using a modification of the two-phase fluor diffusion assay (39). Quantitative values for CAT activity were determined by regression analysis to give counts per min/µg of cell lysate protein. Values for promoter activity are expressed as the mean (luciferase/CAT) ± SEM.

Nuclease Protection Assay
Nuclear extracts were made from GC and HeLa cell lines according to published protocols (40). A subfragment of the 1.6G fragment containing nucleotides 1298/1605 (SmaI/BglII) was radiolabeled at convenient restriction enzyme sites in adjacent vector sequences using Klenow and [32P]dATP. For the protection assay, 0.5–1 ng of radiolabeled DNA was incubated without or with nuclear extract (6.25, 12.5, and 25 µg) on ice for 30 min, and then at room temperature for 5 min. Each sample was treated with 0.05 U of deoxyribonuclease I (DNase I) (Promega Corp.) for 90 sec. The DNase I digestion was stopped with 1% (wt/vol) SDS, 0.1 M NaCl, 0.02 mM EDTA, 10 µg proteinase K, and 4 µg tRNA and incubated at 37 C for 30 min. Samples were extracted once with phenol-chloroform-isoamyl alcohol and precipitated with 2 volumes of ethanol. Pellets were resuspended in 80% formamide, 1 mM EDTA, 0.1% (wt/vol) xylene cyanol and 0.1% (wt/vol) bromophenol blue, and run in a denaturing 5% acrylamide/8 M urea gel, and assessed by autoradiography.

Gel Mobility Shift Assay
For the gel mobility shift assay (41), pituitary GC or cervical HeLa cell nuclear protein (2 or 6 µg) was incubated with 2 µg of poly (dI-dC) and 32P-labeled DNA fragments (250 pg; 1 x 104 cpm). Reactions were done in binding buffer (10 mM HEPES-NaOH, pH 7.9, 50 mM KCl, 2.5 mM EDTA, 10% glycerol, and 1 mM dithiothreitol) for 30 min at room temperature. For competition assays, competitor double-stranded oligonucleotides were added with nuclear extract for 10 min at room temperature and then radiolabeled fragment for a further 20 min. The specific Pit-1/GHF-1 competitors, corresponding to the proximal promoter site in the human GH-N gene, as described previously (18), and the proximal promoter site in the rat PRL gene, a high-affinity DNA element capable of supporting Pit-1/GHF-1 dimerization (19), were synthesized (Fig. 5Go). A nonspecific RF-1 competitor corresponding to a region in the 3'-flanking region of the CS-B gene, described previously (20), was also synthesized as a control. For supershift assays, rabbit GHF-1 antiserum or normal rabbit serum (1 µl) was added to the binding reaction (20 µl final volume) after 20 min preincubation of the other components at room temperature and incubated for a further 10 min. Antibodies to the carboxy-terminal region (amino acids 274–285) of GHF-1 (lots 5032–2 and 5033–3) were kindly provided by Drs. M. Karin and C. Caelles (University of California, San Diego, La Jolla, CA). Full-length rat Pit-1 produced in Escherichia coli as a 40-kDa polyhistidine-tagged fusion protein was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Pit-1 binding assay was done essentially as described (19) except that reactions were done with 714 ng of Pit-1 protein at room temperature for 20 min in 20 µl of 20 mM HEPES, pH 7.9, 1 mM EDTA, 0.1% NP-40, 15% glycerol, and 2 µg of poly-(dIdC). The DNA-protein complexes were resolved in nondenaturing 5% polyacrylamide gels.

Transgenic Mice
All animal experiments were done in accordance with the standards of the Canadian Council for Animal Care. The plasmids 1/1605G.TKp.luc, 1298/1500G.TKp.luc, M3–1298/1500G.TKp.luc, and pT81luc were linearized with BamHI and PvuI and introduced into the pronuclei of single-cell zygotes from CD1 mice. Injected embryos were subsequently transferred to the oviduct of surrogate mothers and brought to embryonic day 19 or term. Genomic DNA was extracted from tail tissue using Proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation. The DNA was blotted to nitrocellulose membrane as well as an amount of each of the transgenes estimated to reflect one or five copies in the genome. The presence of the transgene was determined by probing with a 834-bp PvuI/PstI fragment of pTK81. The fragment was labeled with 32P using the random priming method (Promega Corp.) and Prime-A-Gene Kit Fisher Scientific, Pittsburgh, PA).

Statistics
Statistical analysis of the data was done using the Mann-Whitney (nonparametric) test. Alternatively, the Kruskal-Wallis (nonparametric) ANOVA with Dunn’s multiple comparisons post hoc test was employed. In all cases, a value was considered statistically significant if P was determined to be <0.01.


    FOOTNOTES
 
Address requests for reprints to: Peter A. Cattini, Gene Technology and Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E.

This work was supported by a Medical Research Council of Canada grant (MT-10853). P.A.C. is the recipient of a Medical Research Council of Canada Scientist award.

Received for publication March 15, 1999. Revision received May 3, 1999. Accepted for publication May 14, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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