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Molecular Endocrinology, doi:10.1210/me.2005-0221
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Molecular Endocrinology 20 (3): 598-607
Copyright © 2006 by The Endocrine Society

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

Lisa D. Norquay1, Xiaoyang Yang1, Yan Jin, Karen A. Detillieux and Peter A. Cattini

Department of Physiology, University of Manitoba (X.Y., Y.J., K.A.D., P.A.C.), Winnipeg, Manitoba, Canada R3E 3J7; and Division of Endocrinology (L.D.N.), Children’s Hospital Boston, Harvard Medical School (L.D.N.), Boston, Massachusetts 02115

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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HUMAN GH/chorionic somatomammotropin (CS) locus is an excellent model system for examining the processes that regulate the development and maintenance of tissue-specific expression patterns. Located on chromosome 17, the GH/CS locus contains a family of five highly related genes. From 5' to 3', the genes within this locus are GH-N, CS-L, CS-A, GH-V, and CS-B. The GH-N gene is primarily expressed in somatotroph cells of the anterior pituitary, whereas the remaining genes of the locus (GH-V and the CS genes) are expressed in placental syncytiotrophoblasts (1). In the pituitary, the transcription factor Pit-1 is essential for GH-N expression (2), and functional Pit-1 binding sites are found in the promoter regions of all members of the GH/CS family (3, 4, 5, 6, 7). In addition, Pit-1 binding sites within a distal upstream nuclease-hypersensitive region (HS I/II) are required to achieve normal levels of human GH-N gene expression in transgenic mice (8). Current understanding of the GH/CS locus control region and the involvement of Pit-1 does not, however, explain the highly regulated expression of the human GH/CS locus. The HS I/II region is not a specific activator of the GH-N gene in the pituitary, because it is also able to enhance the activity of the minimal thymidine kinase promoter in both transgenic mice and cell culture (9). In addition, the immediate flanking regulatory regions of the GH/CS family members contain extensive sequence homology (1), and similar chromatin accessibility throughout the locus has been demonstrated (10, 11).

Within the locus, P sequence repeats are located approximately 2 kb upstream of each of the placental GH/CS genes, but are absent from the GH-N upstream region (1). We have shown that P sequence DNA has the capacity to repress GH/CS promoter activity in an in vitro pituitary cell model and localized the repressor activity to a 263-bp fragment (263P) of P sequence DNA (12). Recently, we have extended our analysis of P sequences to include identification of proteins that associate with this region in situ, using chromatin immunoprecipitation (ChIP) and human pituitary tissue (13). Based on in vitro assays that identified candidate P sequence binding proteins (13, 14), we demonstrated that the transcription factors nuclear factor-1 (NF-1) and regulatory factor X (RFX1) associate in situ with 263P in human pituitary tissue (13). This association corresponded with a lack of CS promoter hyperacetylation in the pituitary (13) and was not observed in placental tissue (Norquay, L. D., and P. A. Cattini, unpublished observations) where CS promoter chromatin is hyperacetylated (13). Based on these findings, a model was presented in which the presence of the 263P complex in the pituitary negated formation of a functional enhancer complex and/or hyperacetylated chromatin structure. This was proposed to contribute to the lack of placental gene expression in pituitary tissue.

Our previous characterizations of the pituitary repressor complex identified two regions of protein association with 263P, at P sequence element-A (PSE-A) and PSE-B. An analysis of protein binding events at PSE-A revealed multiple DNA-protein interactions, namely, the mutually exclusive binding of RFX1 and NF-1 (13), whereas an analysis of PSE-B provided evidence that NF-1 was the associating factor (14). These data raised the possibility that binding events at PSE-A had the capacity to be influenced by surrounding P sequences, specifically, the association of NF-1 with PSE-B (13, 14). We present evidence for the formation of at least two structurally and functionally independent pituitary complexes; a functional NF-1 repressor complex and a nonfunctional complex containing RFX1. A key component in regulating the formation of these complexes, and thus P sequence repressor function, is the presence of downstream 263P sequences. Through analysis of this region, we identify members of the hepatocyte nuclear factor/forkhead (HNF-3/fkh) family as 263P binding proteins that participate in protein-protein interactions with NF-1 family members. In addition, the repressor activity of P sequences is shown to be dependent on the presence of an intact Pit-1 binding site in the CS-A promoter. Taken together, these observations allow us to extend our understanding of the 263P pituitary repressor complex and develop the GH/CS locus regulatory model.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
P Sequences Have the Capacity to Form at Least Two Functionally and Structurally Independent Pituitary Complexes
Using competition of protein binding in EMSA and nuclease protection assay, DNA fragments containing both PSE-A and PSE-B were evaluated for participation of RFX1 and the NF-1 family. Initially, a 103-bp P sequence fragment (103P) containing the entire PSE-A and PSE-B nuclease protection regions was synthesized by PCR (Fig. 1Go). Four complexes (I–IV) were detected when 103P was used as a probe in an EMSA with rat pituitary GC cell nuclear extract (Fig. 2Go). PSE-A3, PSE-A4, and the RFX element were all efficient competitors of complexes I and II (Fig. 2AGo). In contrast, under these same conditions, neither PSE-B4 nor the NF-1 consensus element functioned as efficient competitors of 103P complexes (Fig. 2BGo).


Figure 1
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  		Fig. 1. Details of P Sequence Structure and Analysis

The 5'-3' sequence of the CS-A 263P fragment (–2245/–2283 in relation to the CS-A gene (1 ) is shown. Corresponding repeats for the remaining three placental genes are: CS-L, –2593/2327; GH-V, –2451/–2189; and CS-B, –2860/–2598. PSE-A and PSE-B nuclease protection regions are highlighted. The 103P fragment, generated by PCR, is underlined. The 41-bp PSE-C fragment is indicated by a dashed underline. Putative binding sites for HNF-3 and C/EBP are boxed.

 

Figure 2
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  		Fig. 2. Direct Binding of RFX1, But Not the NF-1 Family, to 103P

Radiolabeled 103P was used as an EMSA probe with pituitary GC nuclear extract. Complexes (I–IV) are indicated by arrowheads. A, Competitor oligonucleotides were preincubated with nuclear extract at 25-, 50-, and 100-fold (PSE-A3 and PSE-A4) or 25- and 50-fold (RFX) mass excesses of probe. B, Competitor oligonucleotides were preincubated with nuclear extract at 25- and 50-fold mass excesses of probe.

 
When radiolabeled 263P was used as an EMSA probe with pituitary GC nuclear extract, the RFX oligonucleotide was unable to compete, whereas the NF-1-binding element was an efficient competitor of 263P protein complexes (Fig. 3AGo). The RFX DNA element was also unable to compete the 263P nuclease protection pattern at a 5,000-fold or even as much as a 20,000-fold picomolar excess of probe (Fig. 3BGo).


Figure 3
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  		Fig. 3. Evidence for Direct Binding of the NF-1 Family, But Not RFX1, to 263P

A, The 263P fragment was radiolabeled at the 3' end and used as an EMSA probe with pituitary GC nuclear extract. Competitor oligonucleotides were included in the reactions at 20,000- and 50,000-fold picomolar excesses of probe. B, The 263P fragment was radiolabeled at the 3' end and incubated with or without pituitary GC nuclear extract before DNase I digestion. Competitor oligonucleotides were included in the reactions at 5,000-fold (NF-1 and PSE-A3), 5,000- and 10,000-fold (PSE-B4), or 5,000-, 10,000-, 15,000-, and 20,000-fold (RFX) picomolar excesses of probe. The nuclease-protected PSE-A and PSE-B regions are indicated.

 
The repressor function of P sequences has been well documented with the 263P fragment (12, 13, 14). To assess the effect of the 103P fragment on CS-A promoter function, a luciferase reporter gene construct (103PCSp.Luc) was made and tested for repressor activity in transiently transfected pituitary GC cells (Fig. 4AGo). As a positive control, the 263PCSp.Luc construct was included in the experiment and resulted in 36% of CSp.Luc activity (n = 6; P < 0.0005). In comparison, the presence of the 103P fragment (103PCSp.Luc) had no significant effect on CS-A promoter activity (n = 6).


Figure 4
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  		Fig. 4. Functional Analysis of P Sequence Elements in Transiently Transfected Pituitary Cells

Hybrid Luc genes were used to assess the effects of P sequence elements on CS-A promoter (–496/+6; CSp.Luc) activity in transiently transfected pituitary GC cells. To control for DNA uptake, cells were cotransfected with pRLTKp.Luc. Corrected values are expressed as a percentage of CSp.Luc activity, which was arbitrarily set at 100%. In all experiments, 263PCSp.Luc was included for comparison. Bars represent the SEM. A, Evaluation of 103P. The mean Luc value for CSp.Luc was 1.33 ± 0.10 (n = 6). ***, P < 0.001, comparing 263PCSp.Luc with CSp.Luc. B, PCR-based, site-directed mutagenesis was used to introduce mutations into the PSE-C region of 263PCSp.Luc and generate 263CmCSp.Luc. The mean luciferase value for CSp.Luc was 1.79 ± 0.25 (n =15). *, P < 0.05, comparing 263CmCSp.Luc to 263PCSp.Luc, indicating a significant loss of repressor activity. C, A deletion was introduced into the Pit-1 binding site of the CS-A promoter (CSp{Delta}Pit-1.Luc). The effect of 263P on non-Pit-1-mediated activity of the CS-A promoter in pituitary cells was assessed with 263PCSp{Delta}Pit-1.Luc. The corrected basal activity for CSp.Luc was 3.22 ± 0.15 (n = 6). ***, P < 0.001, comparing 263PCSp.Luc to CSp.Luc.

 
Sequences in PSE-C and an Intact Pit-1 Binding Site in the CS Promoter Contribute to 263P Pituitary Repressor Activity in Transiently Transfected Pituitary Cells
Previous evidence suggested the presence of 263P-binding proteins at sites in addition to PSE-A and PSE-B (see Discussion). Based on this, the 41-bp region downstream of PSE-A, corresponding to nucleotides 229–263 of 263P, was named PSE-C (Fig. 1Go). Sequence analysis using the MatInspector 2.2 binding site detection program based on the TRANSFAC 4.0 database (15, 16) was performed using PSE-C as a query. This analysis revealed putative binding sites for members of the CCAAT enhancer binding protein (C/EBP) and HNF-3/fkh families of transcription factors in PSE-C (Fig. 1Go).

To assess the possible contribution of PSE-C to 263P repressor activity, specific mutations in this region were introduced into 263P by site-directed mutagenesis (PSE-Cm). The mutations effectively eliminated both the C/EBP and HNF-3/fkh binding sites, without the creation of additional binding sites (as assessed by MatInspector 2.2 and the TRANSFAC 4.0 database) (15, 16). The mutated 263P fragment was placed upstream of the CS-A promoter to generate 263CmCSp.Luc. Introduction of this mutation resulted in 61% of CSp.Luc activity in transiently transfected pituitary GC cells (Fig. 4BGo). Although the repressor activity of 263CmCSp.Luc was significant (P < 0.05 compared with CSp.Luc; n =15), it also represented a significant reduction in repressor capacity compared with that of 263PCSp.Luc (P < 0.05 comparing 263 and 263Cm; n =15).

Efficient CS-A promoter activity in transfected pituitary GC cells is driven by the transcription factor Pit-1 (6). Disruption of the proximal Pit-1 DNA element has been shown to eliminate both Pit-1 binding and CS-A promoter activities (6, 17). Thus, a mechanism through which P sequences might repress placental CS-A promoter activity in pituitary cells is interference with Pit-1 action. To assess this possibility, a deletion was introduced into the proximal Pit-1 binding site of the CS-A promoter upstream of the Luc reporter gene (CSp{Delta}Pit-1p.Luc). The effects on Luc activity with and without 263P were assessed (Fig. 4CGo). The absence of an intact Pit-1 binding site resulted in expression levels that were 25% of CSp.Luc activity. For comparison, the Luc activity of Tkp.Luc, a Luc reporter driven by the minimal thymidine kinase promoter was 3.5% of CSp.Luc (data not shown), confirming that CSp{Delta}Pit-1p.Luc is a viable promoter in pituitary GC cells. Removal of the CS-A promoter Pit-1 site effectively eliminated the ability of 263P to function as a transcriptional repressor, because the activities of 263PCSp{Delta}Pit-1p.Luc and CSp{Delta}Pit-1p.Luc were not significantly different (n =12).

A Member of the HNF-3/fkh, But Not the C/EBP, Family Associates with PSE-C
Protein binding to PSE-C was assessed using EMSA with pituitary GC nuclear extract and specific oligonucleotide competitors (Fig. 5Go). Three specific PSE-C complexes (I–III) were identified (Fig. 5Go, arrowheads) by virtue of competition with excess unlabeled PSE-C and lack of competition with PSE-Cm. When a consensus C/EBP DNA-binding element was used, no evidence of competition was observed. In contrast, an HNF-3-binding site was an efficient competitor of all three PSE-C protein complexes.


Figure 5
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  		Fig. 5. HNF-3, But Not C/EBP, Associates with PSE-C in Pituitary GC Nuclear Extracts

The PSE-C fragment was radiolabeled and incubated with nuclear extract from pituitary GC cells. Complexes I–III are indicated by arrowheads. Competitor oligonucleotides were used at 50- and 100-fold mass excesses of probe.

 
Identification of HNF-3{alpha} in Rat and Human Pituitary Cells
The HNF-3/fkh family is comprised of multiple members. To identify candidate family members that may associate with PSE-C, an RT-PCR-based screening approach was devised for rat pituitary GC cells. Primers were designed for the conserved DNA-binding domain of rat HNF-3{alpha}, -ß, and -{gamma}. The expected 250-bp amplicon was isolated (Fig. 6AGo) and sequenced. Sequence analysis indicated a predominance of HNF-3{alpha} in pituitary GC cell RNA, because HNF-3ß or {gamma} sequences were not detected in our amplification (data not shown). In addition to HNF-3{alpha}, the family member hepatocyte nuclear factor-3 homolog (HFH-B3), previously reported to be expressed in rat brain (18), was also detected.


Figure 6
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  		Fig. 6. Identification of HNF-3 Family Members in Rat and Human Pituitary

A, RT-PCR for HNF-3 family members. Total RNA from rat pituitary GC cells or two separate human pituitary tissue samples (hPit) were used. Primers that amplify the conserved DNA-binding domain of rat HNF-3{alpha}, -ß, and -{gamma} (rat HNF3 DNA BD), HNF-3{alpha} (rat/human HNF3{alpha}), and HFH-B3 (rat HFHB3) are listed in Table 1Go. M, 100-bp DNA marker. B, Protein blotting for HNF-3{alpha}. Nuclear extracts (20 µg) from human cervical cancer cells (HeLa), rat pituitary cells (GC), and human pituitary tissue (hPit), were probed with HNF-3{alpha} antibody (C-20, Santa Cruz Biotechnology, Inc.).

 
To confirm the presence of these HNF-3/fkh family members, both RT-PCR and protein immunoblotting were used. Primers to divergent regions of HNF-3{alpha} and HFH-B3 were designed and used for RT-PCR. Diagnostic amplicons of 648 bp for HNF-3{alpha} and 291 bp for HFH-B3 were both observed in pituitary GC cell RNA (Fig. 6AGo). Additionally, the presence of HNF-3{alpha} in human pituitary RNA was confirmed by RT-PCR (Fig. 6AGo). Using protein blotting with HNF-3{alpha} antibody, a band of the expected 52K size was detected in both rat pituitary GC cell and human pituitary nuclear proteins (Fig. 6BGo).

HNF-3{alpha} Associates with the P Sequence Region in Human Pituitary Chromatin
ChIP was used to assess the association of HNF-3{alpha} with P sequences in human pituitary chromatin (Fig. 7Go). Immunoprecipitation with two separate HNF-3{alpha} antibodies resulted in a bound to input (B/I) ratio for the 263P region that was 6- to 7-fold higher than that for exon 3 of the unrelated fibroblast growth factor-16 (FGF-16) gene, which was used as a negative control. This result was reproduced in repeat ChIP assays for each antibody. In contrast, B/I ratios for both the CS-A and GH-N promoter regions, which would not be expected to associate with HNF-3{alpha}, were in a range similar to or less than that of FGF-16 exon 3.


Figure 7
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  		Fig. 7. HNF-3{alpha} Associates with P Sequences in Human Pituitary Chromatin

Nuclei were isolated from three pooled postmortem human pituitary samples and used for ChIP. A, Representative PCR results from ChIP assays using two separate HNF-3{alpha} antibodies (C-20 and H-120). DNA from both the immunoprecipitation input (I) and the bound (B) fractions were amplified by PCR with primer pairs for 263P, the CS-A promoter, the GH-N promoter, and exon 3 of FGF-16. B, The B/I ratio for FGF-16 exon 3 was arbitrarily set at 1.0, and the relative ratios for GH-N, CS-A, and 263P PCRs are shown.

 
HNF-3{alpha} Associates with the NF-1 Family in Rat and Human Pituitary
Coimmunoprecipitation was used to assess the potential of HNF-3{alpha} to associate with NF-1 family members. Human pituitary nuclear extracts were immunoprecipitated with either HNF-3{alpha} antibody (experimental) or nonimmune goat serum (control). The presence of NF-1 family members in HNF-3{alpha} immunoprecipitate was observed by immunoblotting with an NF-1-specific antibody (Fig. 8AGo).


Figure 8
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  		Fig. 8. Protein Interactions between HNF-3{alpha} and the NF-1 Family

A, Coimmunoprecipitation of NF-1 with HNF-3{alpha}. Human pituitary nuclear extract was immunoprecipitated (IP) with nonimmune goat serum or HNF-3{alpha} antibody. Purified proteins from immunoprecipitations as well as 20 µg human pituitary nuclear extract were resolved by SDS-PAGE and immunoblotted (IB) with NF-1 antibody. The arrowhead indicates the NF-1 band of approximately 50 kDa. B, Affinity purification of P sequence-binding proteins using PSE-C and RF-1 (control) DNA elements. Double-stranded oligonucleotides were labeled with biotin and coupled to streptavidin magnetic beads. After incubation with rat pituitary GC nuclear proteins (50 µg), binding complexes were eluted, resolved by SDS-PAGE, and immunoblotted for HNF-3{alpha} and the NF-1 family.

 
To confirm the presence of this interaction in the context of P sequence binding events, protein complexes were purified using magnetic DNA affinity columns. Double-stranded PSE-C oligonucleotide was labeled with biotin and coupled to streptavidin magnetic beads. After incubation with rat pituitary GC nuclear extract, P sequence complexes were purified, resolved by SDS-PAGE, and immunoblotted. As a control, an unrelated oligonucleotide (named RF-1) from the CS-B enhancer region (19) was also used. HNF-3{alpha} and NF-1 were detected in eluent from the PSE-C, but not the RF-1, affinity column (Fig. 8BGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have used both in vitro and in situ techniques to examine the structure of the 263P pituitary complex and investigate repressor function. Previously, we demonstrated that 263P contains two regions of protein binding, designated PSE-A and PSE-B (12, 14). Although NF-1 was shown to bind to PSE-B (14), evidence indicated the mutually exclusive association of NF-1 and RFX1 at PSE-A (13, 14). In this study we first assessed protein binding to 103P, a subfragment of 263P, containing both PSE-A and PSE-B. Direct association of RFX1 with 103P, but not binding of NF-1, was detected with pituitary GC cell nuclear extracts. This subfragment, however, was unable to repress CS-A promoter activity. In contrast, NF-1 binding to 263P was observed through competition of EMSA and nuclease protection patterns. These results linked NF-1 with the repressor function of 263P, suggesting that at least two complexes have the capacity to associate with P sequences: a repressor complex that contains NF-1 and a nonfunctional RFX1-containing complex.

The lack of repressor activity seen with 103P vs. 263P raised the possibility that sequence information outside of the PSE-A and PSE-B regions is required for pituitary repression. We hypothesized that an additional binding factor(s) could favor NF-1 vs. RFX1 binding and thus determine pituitary repressor function. It was presumed that should this be the case, the participation of additional factors would be reflected in the 263P nuclease protection pattern. In consideration of the region upstream of PSE-B (nucleotides 1P-127P), the presence of additional nuclease protection regions was not observed in either the original characterization of 263P, which included assessment of the opposite strand (12), or the current nuclease protection experiments. In contrast, an extended footprint has been observed for the sequences downstream of the PSE-A nuclease protection region. In a previous attempt to purify P sequence-binding factors, a DNA affinity column was made with PSE-B sequences. Enrichment for what was then an unknown binding factor resulted in an extension of the PSE-A nuclease protected region (20). This implied that the factor(s) responsible for the extended footprint and the factor associating with PSE-B were interacting and would be consistent with the hypothesis that additional 263P-binding factors exist that favor or stabilize the formation of a functional NF-1 repressor complex. Based on this evaluation, we titled the region downstream of PSE-A as PSE-C and through mutation in this region demonstrated its importance for the repressor activity of 263P.

Analysis of PSE-C sequences (nucleotides 229P-263P) revealed the presence of both C/EBP and HNF-3/fkh binding sites. Both HNF-3/fkh (21, 22, 23, 24, 25, 26) and C/EBP (27, 28, 29, 30) families have previously been demonstrated to be involved in regulatory complexes that include NF-1. Our EMSA data, however, support binding of the HNF-3/fkh family, but not C/EBP proteins, to PSE-C. Through the use of RT-PCR and immunoblotting, we observed a predominance of HNF-3{alpha} in rat pituitary GC cells and confirmed the presence of this factor in human pituitary RNA. This analysis enabled us to focus on the potential participation of HNF-3{alpha} in the 263P pituitary repressor complex, and as a result, we demonstrated association of HNF-3{alpha} with P sequences in human pituitary chromatin using ChIP assays. Although potential involvement of other HNF-3/fkh family members at PSE-C cannot be ruled out in the human pituitary, the relative abundance of HNF-3{alpha} as well as its association with P sequences in human pituitary chromatin collectively link HNF-3{alpha} with P sequence repressor function in the human system, as seen in our rat GC in vitro model.

The proteins containing the distinct forkhead DNA-binding domain comprise a large family of regulatory factors involved in the control of cell type-specific expression (18). Like the NF-1 family (31), the capacity exists to both repress and enhance transcription (32). This flexibility is intriguing in relation to P sequences, which have the potential for a dual role in regulating expression of the human GH/CS gene family. Although it is a repressor of promoter activity in pituitary cells, the 263P fragment has been shown to enhance expression in the placentas of transgenic mice (33). In addition, the chromatin structure of P sequences contains hyperacetylated histones in human placental samples (13), a modification often correlative with the enhancement of gene expression. The identification of HNF-3{alpha} as a component of the P sequence complex in the pituitary expands the list of candidate factors in placental enhancement. In the placenta, the repertoire of HNF-3/fkh, NF-1, and even RFX family members is likely to be distinct from that in the pituitary. The differential expression or relative abundance of these factors may therefore underlie the tissue-specific function of 263P. Certainly, then, an evaluation of the HNF-3/fkh, NF-1, and RFX families in pituitary and placental tissues is an important future direction.

Detection of HNF-3{alpha} at P sequences in human pituitary cell nuclei in situ suggests that pituitary repression and the mechanism described from work with GC cells may apply in vivo. In somatotrophs of the anterior pituitary, GHRH signaling results in both the release of GH from intracellular secretory granules and an increase in Pit-1 gene transcription (34, 35). The need for repression of the placental GH/CS promoters in the pituitary should coincide with conditions that lead to GH-N transactivation. The HNF-3/fkh family has been identified in mediating genetic responses to peptide hormone-induced signals (36, 37). In the GHRH signaling pathway, increased Pit-1 gene transcription follows elevated cAMP levels that increase phosphorylation and activation of cAMP response element-binding protein-binding protein (35, 38). The activity of HNF-3/fkh family members has also been linked to increases in cAMP response element-binding protein-binding protein activity (39). From a physiological standpoint, involvement of the HNF-3/fkh family in stabilization of the P sequence repressor complex may provide a mechanism to ensure that under conditions where GH transactivation can be increased (for example, in the presence of increased Pit-1 levels in response to GHRH signaling), a consequent activation of placental GH/CS gene activity does not occur. Competition studies have suggested the participation of Pit-1 and the P sequence factors in a common complex (12), and our current functional data indicate that repression of CS promoter activity by 263P is dependent on an intact Pit-1 binding site. Establishment of a direct link, however, between the P sequence repressor complex and Pit-1 binding to the CS promoter(s) along with the examination of chromatin effects resulting from increased Pit-1 activity will be instrumental in obtaining support for this hypothesis.

In summary, we have identified HNF-3{alpha} as a novel member of the pituitary P sequence repressor complex. Based on the data we have presented, our working model for pituitary repression is that HNF-3{alpha}, through binding to PSE-C, favors the association of NF-1 over RFX1 with PSE-A, and that this interaction is a key component in regulating the ability of 263P to function as a transcriptional repressor. We support this model by demonstrating that in the absence of HNF-3{alpha} binding, either through deletion (103PCSp.Luc) or mutation (263CmCSp.Luc), full repressor function does not occur. We also observed protein-protein interactions between NF-1 and HNF-3{alpha} through coimmunoprecipitation from pituitary nuclear extracts, and the presence of this interaction in the context of P sequence binding events was confirmed through the purification of both HNF-3{alpha} and NF-1 from a PSE-C DNA affinity column.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Gene Transfer
Rat anterior pituitary tumor GC cells were maintained as monolayers in DMEM supplemented with 8% (vol/vol) fetal bovine serum and antibiotics in a humidified atmosphere at 37 C with 5% CO2. Gene transfer, with 10 µg test (firefly Luc) plasmid and 25 ng/plate control pRL-TKpLuc (Renilla Luc) plasmid (Promega Corp., Madison, WI), was performed using the calcium phosphate/DNA precipitation method, as previously described (14). The Luc values were determined using the dual-Luc assay system (Promega Corp.) according to the manufacturer’s instructions, with the exception of cell lysis, which was performed in a Tris-Triton lysis buffer [100 mM Tris-HCl (pH 7.8) and 0.1% Triton X-100] as previously described (40). Values are expressed as a percentage of the CSp.Luc activity and are the mean of at least three separate precipitations.

ChIP assay
Nuclei from postmortem human pituitary tissue samples (obtained from the Human Pituitary Repository, Protein and Polypeptide Laboratory, University of Manitoba) were isolated, and ChIP experiments were performed as previously described (13). Specific antibodies for HNF-3{alpha} (50 µl; C-20 and H-120; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used for immunoprecipitation. PCR was carried out with 10 ng input DNA or 5 µl eluted (bound) DNA/PCR (Qiagen, Mississauga, Canada) at an annealing temperature of 55 C for 27 cycles. Results are expressed as B/I ratios to correct for normal variation in primer pair efficiencies.

DNA-Protein Binding Assays
Deoxyribonuclease (DNase) I protection and EMSAs were performed as previously described (14). For DNase I assays, 25 µg pituitary GC cell nuclear protein was used with 0.5 ng radiolabeled 263P fragment as a probe.

Immunoprecipitation and Protein Immunoblotting
For detection of HNF-3{alpha}, nuclear extracts (20 µg) from human pituitary tissue and GC and HeLa cells were boiled for 5 min in Laemmli sample buffer [2% sodium dodecyl sulfate, 10% glycerol, 100 mM dithiothreitol, 60 mM Tris (pH 6.8), and 0.001% bromophenol blue] and resolved by SDS-PAGE in a 12% gel. Gels were transferred and immobilized onto polyvinylidene difluoride membranes (Roche, Laval, Canada) and blocked with 5% milk-Tris-buffered saline-Tween 20 (TBST). Immunoblotting with C-20 HNF-3{alpha} antibody (Santa Cruz Biotechnology, Inc.) occurred for 1 h at room temperature, and detection of antibody-antigen complexes with Supersignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Nepean, Canada) was performed according to the manufacturer’s instructions. Complexes were visualized on Kodak Biomax film (Amersham Biosciences, Baie d’Urfé, Canada).

For immunoprecipitation experiments, all steps were conducted at 4 C. Initially, 200 µg human pituitary nuclear extract was precleared for 1 h with 20 µl protein A/G plus agarose, prepared according to the manufacturer’s instructions (Santa Cruz Biotechnology, Inc.). Precleared extracts were equally divided into control and experimental groups for immunoprecipitation with non-immune goat serum (2 µL) or C-20 HNF-3{alpha} antibody (2 µg, Santa Cruz Biotechnology Inc.), respectively. Immunoprecipitation was carried out overnight and the following day, extracts were incubated with prepared protein A/G plus agarose for 3 h. Washes were done for 2 min as follows: supplemented NET Buffer (50 mM Tris, pH 7.5; 500 mM NaCl; 0.1% NP-40; 1 mM EDTA), followed by NET Buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% NP-40; 1 mM EDTA), and a final wash buffer (10 mM Tris; 0.1% NP-40). Agarose pellets were resuspended in 40 µl of Laemmli sample buffer, boiled for 5 min, before resolution of 20 µL aliquots on 10% SDS-PAGE gels. Gels were transferred and immobilized on Immobilon-P transfer membrane (Millipore Corporation, Nepean, ON Canada) and blocked with 5% milk-TBST. Immunoblotting with 1 µg NF-1 antibody (H-300; Santa Cruz Biotechnology Inc.) occurred for 1 h at room temperature and detection of antibody-antigen complexes was performed as described above.

Magnetic DNA Affinity Purification
Forty picomoles of double-stranded biotinylated oligonucleotides (PSE-C and RF-1; Table 1Go) were coupled to 0.2 mg streptavidin magnetic beads (Promega Corp.) and washed three times with binding buffer [10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride]. A 5-min preincubation of rat pituitary GC nuclear extract (50 µg) with 5 µg polydeoxyinosinic-polydeoxycytidylic acid was carried out at room temperature. After preincubation, nuclear proteins were incubated with PSE-C-coupled beads for 25 min at room temperature, with rotation every 3–5 min. Beads were then washed three times for 5 min each time using 500 µl binding buffer for each wash. Bound protein complexes were removed from the beads by boiling for 5 min in Laemmli sample buffer, then were resolved by SDS-PAGE and immunoblotted as described above.


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  		Table 1. Oligonucleotide and Primer Sequences

 
Oligonucleotide sequences
Double-stranded DNA elements were generated by synthesizing and annealing sense and antisense oligonucleotides (Invitrogen Life Technologies, Inc., Burlington, Canada). The sense strand for each element is provided in Table 1Go. The sequence of the HNF-3 binding element is from a high affinity site in the transthyretin gene (41). The sequences of primers used for PCR of rat and human HNF3/fkh family members as well as primers used for ChIP assay are also given in Table 1Go.

Plasmid Constructions
The immediate 5'-flanking region of the CS-A gene (–492/+6) upstream of the firefly Luc gene (CSp.Luc) as well as 263PCSp.Luc were previously described (14). A two-step PCR approach with 263P primers [263P(F) and 263P(R); Table 1Go] and the PSE-Cm oligonucleotide was used to introduce the PSE-C mutation into 263P. The final PCR product was inserted as a BamHI fragment upstream of the CS-A promoter in CSp.Luc to generate 263CmCSp.Luc. To generate CSp{Delta}Pit-1.Luc and 263PCSp{Delta}Pit-1.Luc, the CSp.Luc and 263PCSp.Luc plasmids were digested with NsiI, staggered 3' ends were removed with Klenow fragment, and plasmids were religated. All constructs were sequenced for verification.

Preparation and Fractionation of Nuclear Extracts
Nuclear protein extracts from rat pituitary GC cells and human cervical cancer (HeLa) cells were made according to published protocols (42, 43) and were dialyzed as previously described (44). A modification of this procedure was performed for human pituitary tissue using the same buffers. In brief, tissue was scissor-minced in buffer A, mechanically homogenized, and strained through three layers of cheesecloth. Loose cells were pelleted at 2000 rpm for 10 min at 4 C, resuspended in buffer A, and treated as described for cell lines. The protein concentration of nuclear extracts was assessed using a protein assay (Bio-Rad Laboratories, Inc., Mississauga, Canada) with BSA as a standard. Extracts were stored as aliquots at –70 C.

RT-PCR
Total RNA was isolated from rat pituitary GC cells with guanidinium thiocyanate. Total RNA from postmortem human pituitary tissue samples was isolated using the RNeasy Fibrous Tissue Midi Kit (Qiagen). For RT, cDNA was synthesized from 5 µg total RNA with oligo(deoxythymidine), pdN6 primer, and Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Inc.) according to the manufacturer’s recommendations. Subsequently, PCR was carried out with 10 ng input DNA/PCR at a 50–60 C annealing temperature for 30 cycles. PCR primer pairs are listed in Table 1Go.

Statistical Analysis
Statistical analysis was performed using a multivariate ANOVA, followed by a Bonferroni post hoc test as required. P < 0.05 was considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank Drs. R. P. C. Shiu and Michel Chrétien for human pituitary tissue samples.


   FOOTNOTES
 
First Published Online October 20, 2005

1 L.D.N. and X.Y. contributed equally to this manuscript. Back

Abbreviations: B/I, Bound to input; C/EBP, CAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation, CS, chorionic somatomammotropin; DNase, deoxyribonuclease; FGF-16, fibroblast growth factor-16; HFH-B3, hepatocyte nuclear factor-3 homolog; HNF-3/fkh, hepatocyte nuclear factor-3/forkhead protein family; HS, hypersensitive site; IB, immunoblot; IP, immunoprecipitation; Luc, luciferase; NF-1, nuclear factor-1; PSE, P sequence element; RFX1, regulatory factor X1.

This work was supported by a grant from the Canadian Institutes of Health Research (10853). X.Y. is the recipient of a Manitoba Health Research Council Studentship, and L.D.N. was the recipient of a Canadian Institutes of Health Research Doctoral Research Award.

The authors have nothing to declare.

Received for publication June 6, 2005. Accepted for publication October 11, 2005.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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