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Molecular Endocrinology, doi:10.1210/me.2003-0405
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Molecular Endocrinology 18 (3): 574-587
Copyright © 2004 by The Endocrine Society

Binding of AP-2 and ETS-Domain Family Members Is Associated with Enhancer Activity in the Hypersensitive Site III Region of the Human Growth Hormone/Chorionic Somatomammotropin Locus

Yan Jin, Lisa D. Norquay, Xiaoyang Yang, Scott Gregoire and Peter A. Cattini

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

Address all correspondence and requests for reprints to: Peter A. Cattini, Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7. E-mail: Peter_Cattini{at}UManitoba.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human GH gene family is specifically expressed in somatotrophs of the anterior pituitary and placental syncytiotrophoblast. Two nuclease-hypersensitive sites, HS III and HS V, are associated with a region of chromatin located 28 and 30 kb upstream of the pituitary GH gene transcription initiation site (+1) in both pituitary and placenta nuclei. A role for this region in pituitary GH gene expression has been reported, but the potential relevance to placental gene expression has not been determined. Deletion analysis of a 5.2-kb region (nucleotides - 27,568/-32,746) containing HS III to V-related sequences localized significant enhancer activity to a 574-bp HS III fragment (nucleotides -27,676/-28,249) in multiple transfected cell lines. Four nuclease-protected regions [footprints (FP) 1–4] were identified in the 574-bp fragment. FP2 and FP3 were detected with placenta cell nuclear protein, whereas FP1 and FP4 were observed with placental and nonplacental cell nuclear extract. Disruption of FP1 had no effect on heterologous promoter activity in transfected pituitary and placental cells, whereas loss of FP2 and FP3 resulted in modest increases in placental cells, reflecting the presence of repressor activity associated with these regions in vitro. In contrast, disruption of the FP4 region by mutation or deletion significantly reduced enhancer activity. As a result, 30-fold enhancer activity was localized to a 41-bp region in transfected placental tumor cells. Binding of candidate proteins, activator protein (AP)-2 (FP3) and Elk-1 (FP4), was confirmed using competition assays with specific oligonucleotides and antibodies. Moreover, these factors were associated with the hyperacetylated HS III region in human pituitary [activator protein 2 (AP-2) and Elk-1] and term placenta (AP-2) chromatin. These data implicate AP-2 and ETS-domain family members in the regulation of the GH/CS locus and raise the possibility that different complexes form in the HS III region in placenta and pituitary cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE FIVE HUMAN GH and chorionic somatomammotropin (CS) genes are contained within 47 kb at a single locus on chromosome 17q22-q24 (1). The most upstream gene, GH-N, is found in vertebrates but the remaining four genes are peculiar to primates (2). Duplication of the GH gene is believed to have occurred based on the relatively recent arrival of the remaining GH/CS genes and a greater than 95% sequence similarity within the family (1, 2). In spite of their similarities, GH-N is expressed in pituitary somatotrophs, whereas CS and GH-V are expressed specifically in the villus syncytiotrophoblast of the placenta (1). Regulatory control of this locus appears to involve a distal upstream locus control region (LCR) where five nuclease hypersensitive (HS) sites, termed HS I-V, have been identified (3). Hypersensitive sites in chromatin often signal the presence of cis-acting sequences that may be associated with transcriptional regulation (4, 5). As seen with the GH/CS expression pattern, some HS sites are tissue specific. HS I and HS II are located approximately 15 kb upstream of the GH-N transcription initiation site and are pituitary specific, whereas HS IV is reported to be 30 kb upstream and seen only in placenta (3). The remaining two hypersensitive sites, HS III and HS V, flank the HS IV region and are found in both pituitary and placenta chromatin (3).

Assessments of HS I/II function associated this region with preferential pituitary enhancer activity in transfected pituitary cells in culture as well as transgenic mice (6, 7). We were able to link this activity to binding of the pituitary-specific POU-homeodomain protein Pit-1 (7), which was confirmed subsequently by others (8, 9). The HS I/II region alone, however, does not recapitulate appropriate expression of GH-N in the pituitary of transgenic mice. Despite pituitary-specific GH-N expression from a construct containing HS I/II in 22 kb of GH-N 5'-flanking DNA, levels of expression were variable and produced a poor correlation between serum GH and GH-N RNA (3). Appropriate pituitary expression of GH-N was only reported when sequences including HS III and HS V were included (3, 10, 11).

A role for the HS III to HS V region in appropriate expression of the GH/CS family in the placenta has also been implied. Unlike pituitary expression of GH-N, the mechanism for placental gene expression is not as well defined. Multiple elements and corresponding factors have been implicated in placental CS gene regulation, including the upstream P sequences (12) and the downstream transcription enhancer factor-related placental enhancer region (13, 14, 15, 16, 17, 18, 19, 20, 21, 22). However, efficient and consistent CS-A promoter activity was not seen in transgenic mouse placenta when a transgene was used that contained sufficient 5' (5.4 kb) and 3' (7.2 kb) flanking DNA to include both P sequences and the CS-A enhancer (3). Appropriate expression of CS-A in the mouse placenta labyrinth, the functional equivalent of the human placental villus syncytium (23, 24), has only been reported with an 87-kb GH/CS transgene, which included the entire GH/CS LCR (11), suggesting a requirement for remote upstream sequences.

Taken together, the data to date strongly support an ability of the distal HS III to HS V region to function as part of a human GH/CS LCR in both pituitary and placenta. A role for these elements in regulating GH/CS gene expression in both tissues was further supported by the observation that the chromatin within HS III and HS V is hyperacetylated in both human pituitary adenoma and placenta tissue samples (12, 25).

Here, we have used a combination of gene transfer and binding studies to assess the HS III–V region and, specifically, the function of HS III-related sequences in placental as well as pituitary cells. The HS III region was originally identified approximately 28 kb upstream of the GH-N transcription initiation site (+1) (3). Hyperacetylation of the HS III region in human pituitary adenoma and term placenta tissue samples was reported using a probe spanning -28 kb (25), and thus, we have characterized HS III-related sequences spanning this region. We have identified both positive and negative regulatory elements in the HS III region in vitro and demonstrated association of the transcription factors activator protein 2 (AP-2) and Elk-1. Furthermore, the observation of HS III region hyperacetylation was extended to normal human pituitary tissue samples taken post mortem, and association of Elk-1 and/or AP-2 with these sequences was confirmed in situ using specific antibodies and chromatin immunoprecipitation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A 574-bp HS III Fragment Functions as an Enhancer in Placental and Nonplacental Cell Lines
A 5.2-kb region of the human chromosome 17 spanning nucleotides -27,568/-32,746 of the human GH-N gene (Fig. 1AGo) and containing HS III to HS V-related sequences was isolated and inserted upstream of a minimal thymidine kinase (tk) promoter fused to the firefly luciferase (luc) gene (HSIII-Vtkpluc). This region was used to generate deletions, and hybrid tk genes containing a 2.9-kb HS III/HS IV (-27,568/-30,450) fragment as well as long (L, -27,568/-29,344) and short (-27,676/-28,249) 1.8-kb and 574-bp fragments of HS III-related sequences; these hybrid genes are referred to as HSIII/IVtkpluc, HSIII(L)tkpluc, and HSIIItkpluc, respectively. Hybrid tk genes, without (tkp.luc) or with HS III–V regions, were used to investigate functional activity in transiently transfected placental JAR and pituitary GC cell lines (Fig. 1BGo). Cells were cotransfected with a Renilla luciferase (pRL-TK) gene as a control for DNA uptake. Luciferase activity was measured as a ratio of firefly/Renilla luciferase activity, and the results are expressed as the fold effect of the HS III–V region on tk promoter activity in each cell line; in each case, tk promoter activity has been arbitrarily set to 1.0. Significant, approximately 4-fold enhancer activity was observed with inclusion of the HS III–V region in JAR but not GC cells. Deletion of sequences corresponding to HS V (HSIII/IVtkpluc) and HS IV-related sequences (HSIII(L)tkpluc) resulted in modest changes to the patterns of expression in both cell types (Fig. 1BGo). However, assessment of a 574-bp HS III-truncated fragment (nucleotides -27,676/-28,249), representing further deletion of intervening sequences between HS IV and HS III (HSIIItkpluc), resulted in significant 15.1- and 6.4-fold increases in enhancer activity in both placental JAR and pituitary GC cells, respectively (Fig. 1BGo).



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  		Fig. 1. Assessment of the 574-bp HS III Region on Heterologous tk Promoter Activity in Transfected Cell Lines

A, Schematic showing the HS III region spanning -28 kb relative to the human GH-N transcription initiation site (+1). The location is based on the human genomic clone hRPK.214_C_8 (GenBank accession no. AC005803), where bp 162,473 = - 487 bp upstream of GH-N. B, Firefly luciferase (luc) reporter gene expression is corrected with Renilla luciferase values as a control for DNA uptake. Promoter activity is expressed as the fold effect of the HS III–V, HS III/IV, HS III(L), and HS III regions, as indicated, on tk promoter activity, which was arbitrarily set to 1.0 (-). The basal tk promoter activity (corrected) in human choriocarcinoma placental JAR and rat pituitary GC cells was 0.83 ± 0.10 (n = 8) and 0.14 ± 0.01 (n = 12), respectively. C, Promoter activity is expressed as the fold effect of the 574-bp HS III region on tk promoter activity. The basal tk promoter activity (corrected) in rat pituitary GC cells was 0.14 ± 0.01 (n = 12), in human choriocarcinoma placental cells JAR, JEG, and BeWo were 0.83 ± 0.10 (n = 8), 0.55 ± 0.01 (n = 4), and 0.58 ± 0.02 (n = 5), respectively. In the unrelated human cells, uterine HEC-1A, cervical HeLa, breast MCF-7, embryonic kidney 293, and liver SK-HEP-1 cells, the basal tk promoter activities (corrected) were 2.43 ± 0.17 (n = 4), 0.16 ± 0.01 (n = 8), 0.16 ± 0.02 (n = 5), 0.87 ± 0.04 (n = 5), and 1.02 ± 0.08 (n = 5), respectively. Error bars represent SEM.

 
To further characterize the 574-bp HS III fragment the HSIIItkpluc gene was tested for enhancer activity in additional placental as well as several nonplacental cell lines. Cells were again cotransfected with a Renilla luciferase (pRL-TK) gene as a control for DNA uptake. Luciferase activity was measured as a ratio of firefly/Renilla luciferase activity, and the results are expressed as the fold effect of the 574-bp HS III fragment on tk promoter activity in each cell line; in each case, tk promoter activity has been arbitrarily set to 1.0 (Fig. 1CGo). In human placental cell lines, significant enhancer activity was observed in JEG-3 (13-fold) and BeWo (12.8-fold) as well as JAR cells in the presence of the 574-bp HS III region. Several nonplacental cell lines of human origin were also assessed, and significant enhancer activity (4.2- to 10.3-fold) was observed in four of the five cell lines tested; enhancer activity was not observed in liver Hep SK-HEP-1 cells.

Detection of Multiple DNA-Protein Interactions in the 574-bp HS III Region
DNA-protein interactions on the 574-bp HS III fragment were assessed using nuclease protection assays (Fig. 2Go). Each strand of the fragment was radiolabeled and incubated with or without nuclear protein (25–50 µg) from pituitary GC cells or placental JAR cells before nuclease treatment, denaturing gel electrophoresis, and autoradiography. With pituitary GC nuclear extract, two protected regions were detected, footprint (FP)1 (Fig. 2AGo) and FP4 (Fig. 2CGo). These two regions did not appear to be specific for pituitary nuclear proteins as they were also detected with placental JAR (Fig. 2Go, B and C) and cervical HeLa (data not shown) nuclear extracts. The placental JAR extract allowed for the identification of two additional regions of protection, FP2 and FP3 (Fig. 2BGo). These two regions of protection may have some degree of placental specificity as they were also detected with JEG-3 and human placental tissue nuclear extracts (data not shown) but not pituitary GC or cervical HeLa cell nuclear proteins (Fig. 2AGo and data not shown). The four protected regions correspond to nucleotides located at -28,151/-28,114 for FP1, -28,113/-28,085 for FP2, -28,072/-28,040 for FP3, and -27,704/-27,686 for FP4 relative to the transcription initiation site (+1) of the GH-N gene (Fig. 2DGo).



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  		Fig. 2. Nuclease Protection Assays Reveal Four Major Protected Regions in the 574-bp HS III Fragment

A–C, The 574-bp HS III fragment (0.5 ng) was radiolabeled on both strands and incubated with or without (-) nuclear extract in nuclease protection assays. Nuclear extracts (rat pituitary GC and human choriocarcinoma JAR) were used at 50 and 25 µg. C, The probe labeled at the 3'-end includes -81/+38 of the tk promoter. D, The location of the nuclease protection regions within the 574-bp HS III fragment. Regions that were protected with both GC and JAR nuclear proteins are underlined. Highlighted regions of protection were observed only with placental nuclear proteins. Candidate binding sites for NF-1, AP-2, and Elk-1 in FP2–4 that were identified by database analysis (26 27 ) are indicated by lowercase lettering.

 
Mutation of FP2, FP3, and FP4 Affect Function of the 574-bp HS III Fragment
To test for a possible contribution to HS III enhancer activity (Fig. 1Go), the FP1–4 regions within the 574-bp HS III fragment were disrupted by mutation and inserted upstream of the tk promoter to generate HSIII/mFP1tkp.luc, HSIII/mFP2tkp.luc, HSIII/mFP3tkp.luc, and HSIII/mFP4tkp.luc, respectively. The modified 574-bp HS III fragments were assessed by nuclease protection assays, and loss of DNA-protein interactions in the mutated regions was confirmed (data not shown). The activities of these modified fragments were compared with wild-type (wt) HSIIItkp.luc gene activity after transient transfection (Fig. 3Go). Results are expressed as a percentage of the activity detected with the HSIIItkp.luc gene, which has been arbitrarily set to 100%. The four modified constructs were first assessed in transiently transfected placental JAR cells (Fig. 3AGo). Despite the loss of nuclease protection at FP1, the activity of HSIII/mFP1tkp.luc was not significantly different from that of HSIIItkp.luc. In contrast, disruption of the placental-restricted FP2 and FP3 protected regions resulted in an approximately 2-fold increase in enhancer activity (Fig. 3AGo). This increase in activity suggests that, at least in vitro, these regions modify 574-bp element enhancer function through a repressor activity. As in placental cells, disruption of the FP1 region did not affect the function of the 574-bp HS III element in transiently transfected pituitary GC cells (Fig. 3BGo). Instead, the enhancer activity in the 574-bp region localized to the ubiquitous FP4 region in both pituitary and placental cells. Mutation of FP4 in placental JAR cells resulted in a greater than 87% loss of enhancer activity (Fig. 3AGo) and in pituitary GC cells a more than 50% loss of enhancer activity (Fig. 3BGo).



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  		Fig. 3. Contributions of the Nuclease-Protected Regions to Functional Activity

FP1, FP2, FP3, and FP4 regions within the 574-bp fragment were disrupted by site-directed mutagenesis and inserted upstream of tkp.luc to generate HSIII/mFP1tkp.luc, HSIII/mFP2tkp.luc, HSIII/mFP3tkp.luc, and HSIII/{Delta}FP4tkp.luc, respectively. Luciferase (luc) reporter gene activity was assessed in transiently transfected placental JAR (panel A) and pituitary GC (panel B) cells. Firefly luciferase values are corrected with Renilla luciferase values as a control for DNA uptake. Promoter activity was assessed after gene transfer, and corrected values are expressed as a percentage of the enhancer activity observed with HSIIItkp.luc, which was arbitrarily set to 100%. The basal HSIIItkp.luc activity was 17.0 ± 1.38 (n = 8) for JAR, and 1.74 ± 0.08 (n = 8) for GC cells. Error bars represent SEM.

 
The Transcription Factors AP-2 and Elk-1 Associate with HS III-Related Sequences in Vitro
Data base analysis (26, 27) of the 574-bp HS III region identified multiple putative protein binding sites including those for nuclear factor 1 (NF-1), AP-2, and Elk-1 at FP2, FP3, and FP4, respectively (Fig. 2DGo). To assess the presence and possible binding of these transcription factors, EMSAs with competitor oligonucleotides were used. When a 29-bp oligonucleotide, corresponding to the FP2 region, was used as a radiolabeled probe with JAR nuclear extract, no evidence of NF-1 association with this fragment was evident, despite the use of a reported high-affinity NF-1 binding site (28) as a competitor (data not shown).

In contrast, the binding of AP-2 to FP3 was confirmed. A 33-bp oligonucleotide, corresponding to the FP3 region was used as an EMSA probe with placental JAR nuclear extract (Fig. 4AGo). Competition of two specific complexes was evident when a wt AP-2 oligonucleotide, but not wt Elk-1 oligonucleotide, was used as a specific competitor (Fig. 4AGo, closed arrowheads). AP-2 association with the FP3 probe was confirmed by the observation that AP-2-specific antibodies (C-18, AP-2{alpha},ß,{gamma} or H79, AP-2{alpha}) resulted in a supershifted band of lower mobility (Fig. 4AGo, open arrowhead). The ability of AP-2 to participate in the 574-bp region complex was further demonstrated through competition of the FP2 as well as FP3 nuclease protection regions with AP-2-specific oligonucleotides (Fig. 4BGo). Consistent with its inability to compete for FP2 complexes in EMSAs, competition of the FP2 region with a consensus NF-1 oligonucleotide was not detected (Fig. 4BGo).



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  		Fig. 4. Evidence for AP-2 Association with FP3 Based on Competition of EMSA and Nuclease Protection Patterns

A, The FP3 oligonucleotide was used as an EMSA probe with placental JAR nuclear extract. Competitor oligonucleotides were included at 2.5- and 5-fold mass excess of the probe. Specific FP3 complexes, defined as those competed by cold FP3 competitor are indicated by closed arrowheads. A supershifted complex of lower mobility was evident with the inclusion of an AP-2-specific antibody and is indicated by an open arrowhead. B, The 574-bp HS III fragment (0.5 ng) was radiolabeled at the 5'-end and incubated with (+) or without (-) nuclear extract in nuclease protection assays. The FP3, FP2, and portion of the FP1 region are indicated. Competitor oligonucleotides were included at 10,000- and 50,000-fold excess of probe. The NF-1 competitor was the commercially available NF-1 consensus binding site.

 
Several specific complexes were observed when a 41-bp oligonucleotide containing the FP4 was used as a probe for EMSA with rat pituitary GC nuclear extract. A 7-bp mutation was created in the FP4 probe sequence that disrupted the core of the ETS-domain binding site recognized by Elk-1 (mFP4; 5'-CtcgctgaG-3' vs. wt 5'-CGGAAGTGG-3'). When the mFP4 oligonucleotide was included as a competitor, competition of FP4 complexes, including two major complexes indicated by closed arrowheads, was no longer observed. One of these specific complexes, with the lowest mobility, was competed by the addition of a consensus Elk-1 DNA element (Fig. 5AGo, closed arrowhead); the predominant complex with the higher mobility was not competed, suggesting binding of an ETS-domain factor(s) in addition to Elk-1. The FP4 oligonucleotide probe was also used with placental JAR nuclear extract in an EMSA (Fig. 5BGo). Placental JAR complexes were competed with an Elk-1 consensus DNA element, but not the mFP4 oligonucleotide (Fig. 5BGo, closed arrowhead). Association of Elk-1 with FP4 was confirmed by the ability of specific antibodies to Elk-1, but not Egr-1, to either compete for a specific complex (Fig. 5AGo, closed arrowhead) or to generate a supershifted EMSA complex (Fig. 5BGo, open arrowhead). The ability of Elk-1 to participate in the 574-bp HS III region complex was further suggested through competition of FP4 with the Elk-1 DNA element (Fig. 5CGo).



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  		Fig. 5. Evidence for the Association of Elk-1 with FP4

The FP4 oligonucleotide was used as an EMSA probe with pituitary GC (panel A) and placental JAR (panel B) nuclear extracts. Competitors were included at 50- and 100-fold mass excess of probe. Complexes that were competed by the Elk-1 consensus binding site are indicated by closed arrowheads, and supershifted bands are indicated by open arrowheads. C, The 574-bp fragment (0.5 ng) was radiolabeled at the 3'-end and incubated with (+) or without (-) pituitary GC nuclear extract in a nuclease protection assay. This probe includes -81/+38 of the tk promoter. The FP4 region is indicated. Competitor oligonucleotides were included at 10,000- and 50,000-fold excess of probe. The NF-1 competitor was the commercially available NF-1 consensus binding site. Ab, Antibody.

 
Binding to the ETS-Domain Binding Site in FP4 Contributes to Enhancer Activity of the 574-bp HS III Region
In contrast to FP1–3, mutation of FP4 decreased the enhancer activity of the HS III region in both transfected placental JAR and pituitary GC cells (Fig. 3Go). Given binding of Elk-1, a member of the ETS-domain family, to the FP4 region (Fig. 5Go), the contribution of the DNA element for the ETS-domain (GGAA/T) to enhancer function was also assessed. A single-base pair substitution (GGAA to aGAA) was made within the 574-bp HS III fragment in the core of the ETS-domain binding site (Fig. 6Go) and inserted upstream of the tk promoter to generate HSIIIm2FP4tkp.luc. The activity of this modified construct was compared with HSIIItkp.luc in transiently transfected placental JAR cells (Fig. 6AGo). Results are expressed as a percentage of the activity detected with HSIIItkp.luc, which has been arbitrarily set to 100%. As observed with HSIIImFP4tkp.luc, disruption of the Elk-1 binding site in HSIIIm2FP4tkp.luc resulted in significantly reduced levels of enhancer activity; for HSIIImFP4tkp.luc, reporter gene was reduced by more than 86%, and for HSIIIm2FP4tkp.luc, reporter gene was reduced by more than 93% (n = 4).



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  		Fig. 6. The FP4/Elk-1 Region Enhances Heterologous Promoter Activity

The contribution of Elk-1 to enhancer activity was assessed in transiently transfected placental JAR cells. Firefly luciferase (luc) reporter gene expression is corrected with Renilla luciferase values as a control for DNA uptake. Corrected values are expressed as the percentage of HSIIItkp.luc activity, which was arbitrarily set to 100%. Error bars represent SEM. A, The FP4/Elk-1 binding site within the 574-bp fragment was disrupted by a 7-bp mutation (HSIIImFP4HSIIItkp.luc) or a single-base pair substitution (HSIIIm2FP4HSIIItkp.luc). The basal HSIIItkp.luc activity was 15.07 ± 0.62 (n = 4). B, The 41-bp FP4 and FP4m2 elements were inserted upstream of tkp.luc to generate wtFP4tkp.luc and m2FP4tkp.luc, respectively. FP4m2 contains a single-base pair substitution in the core of the Elk-1 binding site (GGAA to aGAA). The basal HSIIItkp.luc activity was 10.06 ± 0.54 (n = 8).

 
The 41-bp FP4 and m2FP4 elements were inserted upstream of the tkp.luc reporter gene to generate wtFP4tkp.luc and m2FP4tkp.luc, respectively. The activity of these constructs was assessed in placental JAR cells and corrected for DNA uptake through cotransfection with Renilla luciferase (Fig. 6BGo). Results are expressed relative to the activity of HSIIItkp.luc, which has been arbitrarily set to 100%. The presence of the ETS-domain binding site in wtFP4tkp.luc resulted in a 1.8-fold increase relative to HSIIItkp.luc activity and a 30-fold increase in enhancer activity relative to tkp.luc (n = 6). When the Elk-1/ETS-domain site was disrupted in m2FP4tkp.luc, reporter gene activity was reduced by more than 98% relative to HSIIItkp.luc activity (n = 6).

AP-2 and Elk-1 Associate with the Hyperacetylated HS III Region in Human Pituitary Chromatin
Chromatin immunoprecipitation (ChIP) was used to assess GH/CS LCR hyperacetylation in human pituitary chromatin obtained from whole human pituitaries taken post mortem (Fig. 7AGo). Previously, histone hyperacetylation of the GH/CS LCR was reported for tissue samples where GH expression is abnormally high (10, 25). A specific antibody against hyperacetylated histone H4 was used to immunoprecipitate cross-linked and mechanically sheared human pituitary chromatin. PCR was performed on both input and immunoprecipitated (bound) chromatin fractions with primers specific for HS V, HS IV, HS III, HS I/II, and fibroblast growth factor-16 (FGF-16) exon 3. FGF-16 is a member of the FGF family, which is specifically expressed in embryonic brown adipose and adult cardiac tissue (29); PCR with the FGF-16 exon 3 primers was used as a measure of non specific sequences present in the bound samples. Values were obtained by electrophoresis and densitometry from digital images. The results are expressed as bound/input (B/I) ratios to correct for possible PCR primer pair variation. Immunoprecipitation with hyperacetylated H4 antibody revealed a consistent and significant increase in the B/I ratio of HS I/II (1.25 ± 0.2) HS III (2.23 ± 0.17), and HS IV (1.27 ± 0.23) compared with background FGF-16 exon 3 levels (0.35 ± 0.12) (n = 3). Although an increase in the B/I ratio was also seen overall for HS V (1.44 ± 0.56), this reflected an increase in two of the three samples only. Consistent with our previous report (30), hyperacetylation of P sequences (263P), which are located upstream of each of the placentally expresssed members of the GH/CS family, was not observed in pituitary samples.



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  		Fig. 7. Association of AP-2 and Elk-1, But Not NF-1, with Hyperacetylated HS III in Human Pituitary Chromatin

A, Histone H4 hyperacetylation in human pituitary chromatin. The mean B/I ratios for each primer set are shown. Significant increases over the FGF-16 exon 3 background indicates hyperacetylation of the region and is denoted by gray bars. Bars represent SEM. B, Representative PCR results from pituitary ChIP assays with antihyperactylated H4 antibody. C, Association of AP-2 and Elk-1 with the HS III region in human pituitary chromatin. The mean B/I ratios for FGF-16 exon 3 and HS III region PCR are shown from immunoprecipitations (IP) with NF-1, AP-2, and Elk-1 antibodies. Bars represent SEM. D, Representative PCR results from pituitary ChIP assays.

 
Specific antibodies for NF-1, AP-2, and Elk-1 were also used to determine whether association of these factors with hyperacetylated HS III regions could be detected in human pituitary chromatin (Fig. 7CGo). Consistent with the in vitro binding data, association of NF-1 with the HS III region was not detected. As a positive control for the NF-1 ChIP, NF-1 association was detected at 263P (30). In the NF-1 immunoprecipitation the B/I ratio for the HS III region was 0.25 ± 0.03, and the B/I ratio for FGF-16 exon 3 was 0.18 ± 0.02 (n = 4). In contrast, significant increases in the bound/input (B/I) ratios were observed for the HS III region relative to FGF-16 exon 3 for both the AP-2 and Elk-1 immunoprecipitations. In the AP-2 immunoprecipitations the mean B/I ratio for FGF-16 exon 3 was 0.23 ± 0.02 and for HS III was 0.48 ± 0.09 (P < 0.05, n = 5). In the Elk-1 immunoprecipitations the mean B/I ratio for FGF-16 exon 3 was 0.21 ± 0.03 and for HS III was 0.39 ± 0.03 (P < 0.005, n = 5).

AP-2 Associates with the Hyperacetylated HS III Region in Human Term Placenta Chromatin
ChIP was used to assess whether association of NF-1, AP-2, or Elk-1 with the HS III region could be detected in human placental chromatin (Fig. 8Go). The specific antibody to hyperacetylated histone H4 was included to assess hyperacetylation of the HS III region, which was previously reported (12). Immunoprecipitation with the hyperacetylated histone H4 antibody resulted in a B/I ratio of 1.30 ± 0.14 for the HS III region, which was significantly higher than that of the FGF-16 exon 3 backgound levels (0.29 ± 0.05, P < 0.0005, n = 5). In contrast, immunoprecipitation with either NF-1- or Elk-1-specific antibodies did not result in B/I ratios for the HS III region that were significantly different from background levels. In the NF-1 immunoprecipitation the B/I ratio for the HS III region was 0.19 ± 0.02, and the B/I ratio for FGF-16 exon 3 was 0.17 ± 0.03; in the Elk-1 immunoprecipitation the B/I ratio was 0.32 ± 0.07 for the HS III region and 0.26 ± 0.07 for FGF-16 exon 3 (n = 6). Immunoprecipitation of placental chromatin with an AP-2-specific antibody (C-18 will detect AP-2{alpha}, -ß, and -{gamma}) resulted in a B/I ratio of 1.16 ± 0.09 for the HS III region. This was significantly higher than the B/I ratio of 0.24 ± 0.02 for FGF-16 exon 3 from the same immunoprecipitations (P < 0.0005, n = 6). The association of AP-2{alpha} with the hyperacetylated HS III region in placental chromatin was confirmed by a similar result with a second AP-2-specific antibody (H-79, which will detect AP-2{alpha}), where the B/I ratio for the HS III region was 0.76 ± 0.15 and for FGF-16 exon 3 was 0.18 ± 0.01 (P < 0.05, n = 3).



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  		Fig. 8. AP-2 Association with Hyperacetylated HS III Is Detected by ChIP Assay in Human Term Placenta Chromatin

A, Association of AP-2 with the HS III region in human term placenta chromatin. The mean B/I ratios for FGF-16 exon 3 and HS III region PCR are shown from immunoprecipitations (IP) with NF-1, AP-2, and Elk-1 antibodies. Bars represent SEM. B, Representative PCR results from placenta ChIP assays.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Evidence predominantly from transgenic mouse studies suggests that the mechanisms that regulate expression of the GH/CS locus in the pituitary and placenta are distinct. In the pituitary, the importance of the distal HS I/II region for GH-N expression, and the role of the transcription factor Pit-1 in this process, is now well established (3, 7, 8, 9, 10). In contrast, evidence suggests that in the placenta, relatively more proximal sequences, such as the P sequences (12) and downstream CS enhancer regions (13, 19, 22, 31), may play a role in activated expression of this gene family. These regions are not sufficient, however, to confer appropriately regulated expression in transgenic mice, which requires the inclusion of additional remote sequences (3, 11, 12). In both pituitary adenoma and term placenta, these remote sequences contain the distal HS III–V region (3). Although factors that associate with HS I/II, P sequences (30, 32), and downstream CS enhancer regions (13, 14, 17, 18, 31, 33) have been identified, there are no reports of potential binding factors for the HS III–V region. In the present study, we have used a combination of gene transfer as well as binding studies in vitro and in situ to characterize HS III-related sequences contained in the region spanning -28 kb of the human GH/CS locus. Our results indicate that the 574-bp HS III fragment contains significant enhancer activity in transfected placental and pituitary cells, and that levels are regulated by members of the ETS-domain and AP-2 families of transcription factors. Although not pursued, our data also suggest significant repressor activity in the HS III–V region outside of the 574-bp HS III fragment.

Enhancer activity in the HS III–V region of the human GH/CS locus was attributed to a 574-bp HS III fragment containing a binding site for the ETS-domain transcription factor family and capable of binding a member, Elk-1 (34). Interestingly, whereas competition of Elk-1 binding from pituitary GC and placental JAR cells to the FP4 domain was observed with both an Elk-1 DNA element and antibody, a supershift was observed only with the placental nuclear extract. This, presumably, reflects the lower mobility (larger size) of the specific Elk-1 complex observed with GC cell protein and, thus, any supershifted complex may have been too large to enter the gel. Alternatively, the nature of the two complexes may be such that, with placental JAR protein, the antibody may bind without interfering with DNA binding but in the case of pituitary GC cell protein, antibody binding may preclude interaction with the DNA. Although both raise the possibility of distinct pituitary and placental complexes at the HS III region, a direct link between the nuclease-protected region FP4 and the in vitro enhancer activity was indicated by mutation and functional analyses (Figs. 3Go, 5Go, and 6Go).

In contrast to the FP4 region, disruption of FP2 and FP3 in the 574-bp HS III fragment resulted in an approximately 2-fold increase in promoter activity (Fig. 3Go), suggesting they may contribute to regulating the level of enhancer activity through repressor function. This is also consistent with the approximately 2-fold increase in enhancer activity seen with the truncated 41-bp FP4 fragment lacking FP2 and FP3 (Fig. 6BGo). Screening of a transcription factor database (26, 27) revealed multiple putative DNA binding sites in FP2 and FP3 including partial sites for NF-1 and AP-2 (Fig. 2Go). When these factors were assessed, only the binding of AP-2 to FP3 was confirmed (Fig. 4Go and data not shown). The competition of both FP2 and FP3 regions with an AP-2 element suggests that either 1) FP2 and FP3 both result from AP-2 association, or 2) that the factor associating with FP3 interacts strongly with AP-2 and may involve cooperative binding. However, mutation of the AP-2 binding site in FP3 in HSIII/mFP3tkp.luc did not disrupt nuclease protection at FP2 (data not shown), which would imply that if the factors are separate, then cooperative binding is not required. Thus, it is possible that AP-2 association accounts for both FP2 and FP3 protection in the context of the 574-bp fragment.

The placenta-specific nature of FP2 and FP3 is intriguing given the link between AP-2{alpha} and AP-2{gamma} in the regulation of placental genes including human chorionic gonadotropin, placental leucine aminopeptidase, and CS (35, 36, 37, 38). AP-2{alpha} and AP-2{gamma} are expressed in human placenta (36) but there is evidence that AP-2{gamma} expression decreases whereas AP-2{alpha} synthesis increases with human trophoblast differentiation and syncytiotrophoblast formation (35, 36, 37). Consistent with this pattern of expression, synthesis of the human chorionic gonadotropin subunits in cytotrophoblasts is associated with AP-2{gamma} binding (36), whereas placental leucine aminopeptidase, which is expressed preferentially in syncytiotrophoblasts, has been linked to increased AP-2{alpha} binding (35). The latter, as well as the predominant expression of AP-2{alpha} at term (39), would be consistent with our data and the association of AP-2{alpha} with the HS III region in human placenta chromatin (Fig. 8Go), as human CS is also expressed preferentially by syncytiotrophoblasts. Thus, whereas AP-2{gamma} (as well as AP-2{alpha}) has been implicated in the expression of human placental genes including CS (37, 38) and can bind to the HS III region in vitro (supershift of JAR nuclear protein with AP-2{gamma} antibodies, H-77; data not shown), appropriate levels of expression in vivo may require the presence and increase in AP2-{alpha} levels. This appears to contrast with the situation in mice where a lack of AP-2{alpha} has no effect on placentation or implantation, whereas AP-2{gamma} is expressed in all trophoblast lineages during placental development (40). As a result, a lack of AP-2{gamma} in mice leads to a failure to form a labyrinth layer resulting in malnutrition and death of the embryo (40).

With regard to the HS I/II region, in vitro studies have been instrumental for identifying the essential role of the transcription factor Pit-1 in pituitary locus regulation (3, 7, 8, 10). Consistent with this approach, our current analysis of sequences from the HS III region has provided candidates for regulation of the GH/CS locus in vivo. The ability of the 574-bp HS III fragment to stimulate heterologous promoter activity was observed in transiently transfected cell lines derived from several tissues including kidney (293) as well as placenta and pituitary (Fig. 1Go). The presence of enhancer activity but lack of pituitary or placenta specificity shows some similarity to results obtained when an isolated HS III to HS V region was used to influence human GH-N gene expression in transgenic mice in vivo. Although the presence of these sequences was sufficient to induce GH-N transgene expression in the pituitary, the highest levels were detected in the kidney (3). Further similarities were provided through extension of the in vitro binding assays to the in situ analysis, where Elk-1 and/or AP-2 association was demonstrated (Figs. 7Go and 8Go).

Like, the GH/CS locus, regulation of the ß-globin gene cluster involves distal HS sites. In the case of ß-globin, these regions were believed to modify chromatin structure for opening of the locus in erythroid cells, and hence the term "locus control region" (LCR). More recent studies, however, indicate that the LCR in its native context is not required to open the locus but, rather, to act as an enhancer of tissue- and developmental-specific gene expression (41, 42, 43, 44, 45, 46). The function of the pituitary-specific HS I/II region, as an enhancer of transgene activity in vitro and in transgenic mice, is consistent with this current notion of LCR function. In the present study, normal human pituitaries taken post mortem were used for the ChIP assays. In contrast to previous reports, a peak of hyperacetylation at HS I/II was not detected (10, 25). Instead, each of the HS sites demonstrated roughly equivalent levels of hyperacetylation (Fig. 7AGo) with the possible exception of HS V, where two of the three experiments showed hyperacetylation. It should be noted that, in the studies in which significantly greater hyperacetylation was observed at the HS I/II region, the chromatin used was from pituitary tissue with abnormally high GH expression (10, 25). Hyperacetylation throughout the LCR is consistent with transgenic data, where the full set of HS sites appears to be required for accurate and efficient expression (3, 11).

In contrast to the ß-globin-regulatory system, evidence for the GH/CS locus continues to support the notion that distal elements, such as the HS III–V region, play a dominant role in production of an independent chromatin domain in vivo. Although HS III-related enhancer activity in vitro required an intact ETS-domain binding site, it was AP-2 association with the HS III region that correlated with histone H4 hyperacetylation in both human pituitary and placenta chromatin samples. This correlation is supported by previous reports, which have documented the ability of AP-2 to recruit the histone acetyltransferase coactivator p300/cAMP response element-binding protein-binding protein (47, 48). Therefore, although the contribution of AP-2 to the activity of the 574-bp HS III fragment activity in vitro was to derepress enhancer activity, raising the possibility of a repressor function, the role of this region and factor in the context of chromatin may differ. The association of Elk-1 in situ was observed in pituitary but not placental chromatin samples. It has been demonstrated previously that Elk-1 is activated by GHRH in pituitary cells (49), and activated Elk-1 has the capacity to recruit p300/cAMP response element-binding protein-binding protein (50, 51, 52). Although it is possible that Elk-1 does not bind at the HS III region in placenta chromatin, we cannot rule out the possibility that Elk-1 is associated but was not detected, perhaps due to epitope masking in the bound complex, or alternatively that another member of the ETS-domain family may occupy the site. This family of transcription regulators now numbers 45, each capable of binding to a consensus DNA element centered on the core sequence 5'-GGA(A/T)-3' through the highly conserved ETS DNA-binding domain (53). Certainly, data from the competition of the ETS-domain binding site in FP4 with pituitary protein support the potential involvement of alternative ETS-domain family member(s). Specifically, the predominant ETS-domain-related complex identified by competition with FP4, but not mFP4 (in which the ETS-domain binding site was disrupted), was not competed by the Elk-1 DNA element (Fig. 5AGo). Regardless, our gene transfer data strongly support a role for the core 5'-GGAA-3' sequence and, thus, binding of an ETS-domain family member(s) for enhancer activity. Taken together, our observations support the possibility that mechanisms that regulate GH/CS expression in the pituitary and placenta are distinct.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Oligonucleotide Sequences
Double-stranded DNA elements were generated by synthesizing and annealing sense and antisense oligonucleotides (Invitrogen, Burlington, Ontario, Canada). Commercially available NF-1 and AP-2 DNA elements were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The sense strand for each element is provided in Table 1Go. The sequences of primers used for PCR are also given in Table 1Go.


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  		Table 1. Oligonucleotide and Primer Sequences

 
Plasmid Construction and Site-Directed Mutagenesis
A P1 library containing human genomic DNA was screened as previously described, and a clone was obtained (P1–3; clone address: DMPC-HFF#1–1434-A) (7). P1–3 was digested with XbaI, and a 10-kb fragment (10X) was subcloned into pBluescript II+ to generate 10XpBSII+. A 5.2-kb fragment containing HS III–V sequences (nucleotides -27,568/-32,746) and a 2.9-kb fragment containing HS III/IV sequences (nucleotides -27,568/-30,450) was generated by XhoI and BamH1 digestion of 10XpBSII+, respectively. Both fragments were inserted into XhoI or BamH1 sites of pT81Luc, upstream of a minimal thymidine kinase (tk) promoter and the firefly luciferase (luc) reporter gene (tkp.luc), to generate HSIII-Vtkp.luc and HSIII/IVtkp.luc, respectively. To delete HS IV-related sequences, HSIII/IVtkp.luc was digested with SmaI and religated to retain a 1.8-kb region (nucleotides -27,568/-29,344) and generate HSIII(L)tkp.luc. Based on primers used previously to detect HS III sequences and/or reported fragment sizes (3, 25), a 574-bp HS III fragment (5' to 3', -28,249/-27,676) was generated by PCR. Taq DNA polymerase (QIAGEN, Mississauga, Ontario, Canada) was used for all PCR reactions. The primers used for amplifying the wt 574-bp fragment, 574(R) and 574(F), are shown in Table 1Go. The 574-bp HS III fragment was inserted into HindIII/SacI sites of pT81Luc to generate HSIIItkp.luc.

Putative DNA elements/footprints (FP1–4) within the 574-bp HS III fragment were disrupted by PCR site-directed mutagenesis using HSIIItkp.luc as a template. The primer pairs are described in Table 1Go. PCR products were purified, annealed, and extended, and the final products were amplified using 574(R) and 574(F) primers. To subclone the modified 574-bp HS III fragments, PCR products were digested with HindIII/SacI, isolated, and inserted upstream of pT81.luc to generate HSIII/mFP1tkp.luc, HSIII/mFP2tkp.luc, HSIII/mFP3tkp.luc, and HSIII/mFP4tkp.luc. To generate wtFP4tkp.luc and mFP4tkp.luc, 41-bp fragments corresponding to nucleotides -27,716/-27,676 and containing the FP4 region were synthesized with and without mutations in an Elk-1 binding site, and inserted into the SmaI site of pT81.luc.

Cell Culture and Gene Transfer
Monolayer cultures of rat pituitary GC, human uterine HEC-1A, cervical HeLa, breast MCF-7, embryonic kidney 293, liver SK-HEP-1, and choriocarcinoma JAR, JEG, and BeWo tumor cells were grown in a humidified atmosphere on 100-mm dishes and maintained at 37 C in 10% fetal bovine serum-DMEM at a density of 0.8–1 x 106 cells per plate, with the exception of BeWo cells, which were grown in 10% fetal bovine serum-RPMI. Cells, in triplicate or quadruplicate, were transfected with 10 µg test (luciferase) plasmid DNA and 20 ng Renilla luciferase (pRL-TK, Promega Corp., Madison, WI) for 18–24 h after plating, by the calcium phosphate/DNA precipitation method as previously described (7). Cells were harvested 72 h after DNA addition. The ratio of firefly/Renilla luciferase activity was determined using the Dual-Luciferase Assay System (Promega Corp.) and a luminometer (ILA911 Luminometer, Tropix, Inc., Bedford, MA) according to manufacturer’s instructions, with the exception of cell lysis, which was performed as described previously (54). Values for promoter activity are expressed as the mean (luciferase/Renilla luciferase) ± SEM.

Deoxyribonuclease 1 (Nuclease) Protection Assay
Nuclear extracts were made from human placental tissue, JAR, GC, and HeLa cell lines according to published protocols (55) and dialyzed as previously described (20). Radiolabeled probe (0.5 ng) was incubated without or with nuclear extract (25 and 50 µg), and nuclease protection was carried out as previously described (7).

EMSA
EMSA was performed as previously described (7). Briefly, nuclear protein (4–5 µg) was incubated with 2 µg poly (dI-dC) and 1 ng labeled oligonucleotide for 20 min at room temperature. For competition assays, competitor double-stranded oligonucleotides or antiserum, 2 µg of Elk-1 antiserum (Santa Cruz), NF-1 antiserum (Santa Cruz), AP-2 antiserum (C-18, H-77, H-79; Santa Cruz) or, as a negative control, Egr-1 antiserum (Santa Cruz), were preincubated with nuclear exacts for 5 min at room temperature before the addition of probe. The DNA-protein complexes were resolved in nondenaturing 5% polyacrylamide gels and visualized by autoradiography.

ChIP Assay
Nuclei from human placenta and pituitary tissue samples were isolated and ChIP experiments performed as previously described (30, 56). Briefly, nuclei were resuspended in HEPES buffer (10 mM HEPES, 10 mM NaCl, 3 mM MgCl2, 1 mM phenylmethylsulfonylfluoride; pH 7.5) to approximately 20 A260 U/ml and cross-linked in 1% formaldehyde (final concentration) for 5 min at room temperature. For immunoprecipitation, 5-ml samples of lysed and sonicated nuclei were prepared in dilution buffer (Complete Mini protease inhibitor cocktail with 16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% sodium dodecyl sulfate, 1 mM phenylmethylsulfonylfluoride) at an A260 of 2 U/ml. Samples were precleared for 3 h at 4 C using 300 µl of pretreated protein A sepharose (Amersham Pharmacia Biotech, Québec, Canada) and 25 µg sheared salmon sperm DNA. Specific antibodies were added for overnight incubation as follows: 25 µl antihyperacetylated (penta) histone H4 (Upstate Biotechnology) 50 µl Elk-1 (I-20) (Santa Cruz), 50 µl NF-1 (H-300) (Santa Cruz), 50 µl AP-2 (C-18) (Santa Cruz), or for confirmation 50 µl AP-2 (H-79) (Santa Cruz). The following day, 300 µl protein A sepharose and 50 µg sheared salmon sperm DNA were added for 1 h. Samples were washed and eluted as described previously (30) before cross-links were reversed for 6 h at 68 C. DNA was isolated using QIAquick columns (QIAGEN) according to the manufacturer’s instructions. PCR was carried out with 10 ng of input DNA or 5 µl eluted (bound) DNA per PCR (Taq DNA polymerase; QIAGEN) at 55 C annealing temperature for 28 cycles. PCR primers are listed in Table 1Go.

Statistics
Statistical analysis of the data was done using a two-tailed, unpaired Student’s t test and ANOVA with a post hoc Bonferroni test. A value of P < 0.05 is considered statistically significant. In figures * represents P < 0.05; ** is P < 0.01; and *** indicates P < 0.005.


   FOOTNOTES
 
This work was supported by Grant MT-10853 from the Canadian Institutes of Health Research (CIHR). L.D.N. is the recipient of a CIHR Doctoral Studentship.

Abbreviations: AP-2, Activator protein 2; B/I ratio, bound/input ratio; ChIP, chromatin immunoprecipitation; CS, chorionic somatomammotropin; FGF, fibroblast growth factor; FP, footprint; HS, hypersensitive; LCR, locus control region; luc, luciferase; NF-1, nuclear factor-1; tk, thymidine kinase; wt, wild type.

Received for publication October 20, 2003. Accepted for publication December 4, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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