RFX1 and NF-1 Associate with P Sequences of the Human Growth Hormone Locus in Pituitary Chromatin
Lisa D. Norquay,
Xiaoyang Yang,
Patricia Sheppard,
Scott Gregoire,
Janice G. Dodd,
Walter Reith and
Peter A. Cattini
Department of Physiology (L.D.N., X.Y., P.S., S.G., J.G.D., P.A.C.), University of Manitoba, Winnipeg, MB, Canada R3E 3J7; and Départment de Génétique et Microbiologie (W.R.), Centre Médical Universitaire (CMU), Genéve CH-1211, Switzerland
Address all correspondence and requests for reprints to: P. A. Cattini, Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7. E-mail: Peter_Cattini{at}UManitoba.ca.
 |
ABSTRACT
|
---|
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 promoter activity in transfected pituitary (GC) cells. Regions of protein binding within 263P include P sequence elements A and B (PSE-A and PSE-B), and we reported nuclear factor-1 (NF-1) recognition of PSE-B. We now provide evidence for multiple interactions on PSE-A, including binding of the regulatory factor X (RFX) family. Disruption of the RFX site within 263P blunts repressor activity in transfected GC cells; however, repression is only abolished when both PSE-A/RFX and PSE-B/NF-1 sites are mutated. The capacity of RFX and NF-1 to participate in a novel common complex is further suggested by coimmunoprecipitation of RFX1 and epitope-tagged NF-1 family members. Finally, we confirm the association of NF-1 and RFX1 with P sequences in human pituitary tissue by chromatin immunoprecipitation. Taken together, our data suggest that an inverse relationship exists between 263P and CS promoter histone hyperacetylation and the association of these factors in vivo.
 |
INTRODUCTION
|
---|
THE HUMAN GH (hGH) gene family consists of five highly related genes contained within a single 47-kb locus on chromosome 17 (1). Despite a remarkable degree of similarity in gene structure and flanking DNA sequences, this family displays strictly regulated patterns of tissue-specific expression (1). GH-N is the only member of the family that is highly expressed in the pituitary (1), even though the entire locus is sensitive to endonuclease and thus appears to have an open chromatin structure (2). The remaining members of the family, GH variant (GH-V) and the chorionic somatomammotropins (CS-A, CS-B, and CS-L), are specifically expressed in placental syncytiotrophoblast (1). Conserved elements termed "P" sequences have been identified approximately 2 kb upstream of the transcription initiation site of each of the placental GH/CS genes, but not pituitary GH-N (1). The location of these repeats made them ideal candidates for involvement in local regulation of the GH locus. Further analysis of the P sequences identified a 263-bp core element (263P) based on its capacity to repress the in vitro pituitary activity of the CS-A promoter that is seen after gene transfer (3). To date, the ability of 263P to repress the activity of the CS-A promoter has not been tested in vivo; however, intriguing data regarding a potential role for 263P in placental activation of the locus have emerged. Analysis of histone acetylation levels within the GH locus of human term placenta chromatin has revealed a peak of histone hyperacetylation associated with 263P (4). The ability of 263P to repress CS-A promoter activity in pituitary cells in vitro, as well as its potential to enhance placental expression in vivo (implied by hyperacetylation), opens the possibility that this sequence may be an integral component in regulating tissue-specific expression of the human GH locus.
We have focused our attention on identifying the factors involved in 263P regulatory complexes. At the time 263P was originally identified, two regions of protein-DNA interactions were visualized by endonuclease protection assay, P sequence element-A (PSE-A) and PSE-B. It was not resolved whether these regions represented one or more protein-DNA interactions (3). Recently, we have shown that members of the nuclear factor-1 (NF-1) family of transcription factors, known to mediate both transcriptional activation and repression (5), can recognize PSE-B (6). The binding of NF-1 family members to PSE-A was not detected despite, paradoxically, the ability of a consensus NF-1 element to compete for the PSE-A footprint pattern in nuclease protection assays (6). We now provide evidence for multiple interactions at PSE-A, which offers an explanation for this paradox. More importantly, the ability of the NF-1 family and regulatory factor X1 (RFX1) to participate in the 263P complex in vitro, as well as in situ, and the capacity for interactions between RFX1 and NF-1 family members are described. These observations define the human GH locus as a novel regulatory target for the RFX family and allow us to propose a model for involvement of the 263P complex in repression of the placental GH/CS genes in the pituitary.
 |
RESULTS
|
---|
Specific Complex Formation on PSE-A Is Seen by EMSA
When 263P was incubated with pituitary GC cell nuclear extract in a nuclease protection assay, two characteristic footprint regions, PSE-A and PSE-B, were observed (Fig. 1A
). As previously reported, both PSE-A and PSE-B oligonucleotides compete for both protected regions (3, 6). Analysis of PSE-A by EMSA revealed several DNA-protein complexes with pituitary cell nuclear protein (Fig. 1B
). Complexes IIV were readily competed by a 5-fold excess of unlabeled PSE-A competitor, but not by a 50-fold excess of unlabeled PSE-B competitor, suggesting specific, high-affinity protein binding events.

View larger version (86K):
[in this window]
[in a new window]
|
Figure 1. Specific Complex Formation on PSE-A
Panel A, Characteristic footprint regions spanning PSE-A and PSE-B seen when 263P is incubated with pituitary GC nuclear extract in a nuclease protection assay. Competition of both PSE-A and PSE-B protected regions with a 5000-fold pmol excess of PSE-A oligonucleotide is indicated. Panel B, EMSA done with pituitary GC nuclear extract and radiolabeled PSE-A as a probe. Competition was performed with double-stranded PSE-A (5-, 25-, and 50-fold mass excess of probe) and PSE-B (50-fold mass excess of probe) oligonucleotides. Arrowheads indicate specific, high-affinity complexes (IIV).
|
|
Determination of the Core Sequences Within PSE-A for Specific Pituitary Complex Formation
Examination of PSE-A sequences for known protein binding DNA elements (7) reveals several candidates, including a putative RFX site and a half-site for NF-1 binding (Fig. 2A
). To determine essential or core sequences involved in the formation of both PSE-A and 263P specific complexes, a series of scanning mutations were created within PSE-A (Fig. 2A
). These mutations did not create additional known recognition sequences based on database analysis. Using EMSA and pituitary GC cell protein, the mutated oligonucleotides were then tested for their ability to compete complexes IIV from the PSE-A probe (Fig. 2B
). Whereas none of the PSE-A mutations were able to efficiently compete complexes III and IV, Am1, Am4, Am5, and Am6 were competitors of complex I with incomplete competition of complex II. The Am2 and Am3 mutations, however, were unable to effectively compete for either complex I or II and thus defined a core region within PSE-A for their formation. This core region localizes to a putative X-box motif [5'-GTNRCC(03N)RGYAAC-3'] (8) recognized by the RFX family of transcription factors (Fig. 2A
).

View larger version (115K):
[in this window]
[in a new window]
|
Figure 2. Determination of the Essential Sequences within PSE-A for Specific Pituitary Complex Formation Reveals Two Core Regions
Panel A, The sequence of PSE-A is shown with a putative binding site for the RFX family (solid line) and a half-site for the NF-1 family (broken line) underlined. Scanning mutations were created throughout PSE-A, and the mutated sequences are in upper case with the name of each mutant indicated to the right of the sequence. Panel B, EMSA was done using pituitary GC cell nuclear extract and radiolabeled PSE-A as a probe. Arrowheads indicate specific, high-affinity complexes (IIV). Double-stranded mutant PSE-A oligonucleotides were used as competitors at 25- and 50-fold mass excess of probe. Panel C, Competition of 263P protected regions in a nuclease protection assay with pituitary GC nuclear extract was performed with double-stranded PSE-A and PSE-A mutant oligonucleotides as competitors at 1000-fold pmol excess of 263P probe.
|
|
To determine those PSE-A sequences that were important for competition of 263P footprint patterns, the PSE-A scanning mutations, along with a 3' truncated form of PSE-A (PSE-Adel), were used as competitors in a nuclease protection assay (Fig. 2C
). Mutations PSE-Am2 and PSE-Am3, which encompass the putative RFX binding site, as well as wild-type PSE-A, PSE-Am1, PSE-Am4, and PSE-Am5, were able to compete the 263P footprint pattern (Fig. 2C
). In contrast, the 3'-mutation (PSE-Am6) and truncation (PSE-Adel) of PSE-A, which disrupt the NF-1 half-site (Fig. 2A
), were able to compete the nuclease protection pattern at 1000-fold pmol excess of the 263P probe, although not as efficiently as wild-type PSE-A (Fig. 2C
). At 5000-fold pmol excess, all of the PSE-A mutations competed the nuclease protection pattern (data not shown).
RFX1 Is Capable of Binding to PSE-A in Pituitary Extracts in Vitro
To assess the ability of the RFX family to directly bind to PSE-A, the EF-C/MDBP RFX1 element and RFX1 antibodies (9) were used as specific competitors in EMSA (Fig. 3
). The EF-C/MDBP element was previously shown to bind a chemically synthesized RFX1 DNA binding domain (10) and was used as a representative RFX1 binding site in the absence of a defined RFX consensus sequence. The wild type, but not a mutation of the EF-C/MDBP site, was able to efficiently compete for both complexes I and II when PSE-A was used as a probe with pituitary GC cell nuclear protein (Fig. 3A
, closed arrowheads). These same complexes were also competed by RFX1 antibodies, with evidence of supershifted aggregates in the well, as previously observed with this antibody (11). Neither competition of complexes I and II nor supershifts were observed when RFX2 and RFX3 antibodies were used with pituitary GC nuclear extract and the PSE-A probe in EMSA (Fig. 3B
).

View larger version (86K):
[in this window]
[in a new window]
|
Figure 3. Direct in Vitro Binding of RFX1 from Pituitary GC Cell Nuclear Extract to PSE-A
EMSA was performed using pituitary GC cell nuclear extract and radiolabeled PSE-A as a probe. Competition with specific double-stranded oligonucleotides (PSE-A, RFX, and RFXm) or RFX (1, 2, and 3) antibodies (Ab) and normal rabbit serum (NRS) is indicated. For panel A, PSE-A competitor at 100-fold mass excess of probe was used as a positive control. RFX and RFXm oligonucleotides were used at 10-, 25-, and 100-fold mass excess of probe. Specific high-affinity complexes [(I and II) and (III and IV)] are indicated by closed and open arrowheads, respectively.
|
|
NF-1 Can Interact with PSE-A in Pituitary Extracts in the Absence of RFX1 Binding
Our previous investigation was unable to detect direct binding of the NF-1 family to PSE-A (6). To assess the possibility that NF-1 can interact with PSE-A in the absence of RFX1 binding, PSE-Am2 was used as a probe for EMSA. The mutation contained in PSE-Am2 is located within the putative RFX site (Fig. 2A
) and was unable to compete for complexes I or II from a PSE-A probe (Fig. 2B
). When the PSE-Am2 oligonucleotide was used as a probe for EMSA with pituitary GC nuclear extract, complexes I and II were not detected (Fig. 4A
); however, an additional complex was observed compared with the pattern seen with the wild-type PSE-A probe (compare Fig. 4A
with Fig. 3
). A consensus, but not a mutant, NF-1 DNA element was able to efficiently compete for this additional complex (Fig. 4A
, open arrowhead). This same complex was also competed by NF-1 antibodies with evidence of a supershifted band (Fig. 4B
, closed arrowhead). In contrast to the PSE-Am2 probe, no evidence of NF-1 binding was detected when PSE-Am6 was used as an EMSA probe (Fig. 4B
). The PSE-Am6 mutation disrupts the NF-1 1/2 site (Fig. 2A
), and the pattern of complexes on this probe resembles that of the wild-type PSE-A (compare Fig. 4B
with Fig. 3
).

View larger version (115K):
[in this window]
[in a new window]
|
Figure 4. Direct Binding of NF-1 to PSE-A in the Absence of RFX1
Pituitary GC cell nuclear extract and radiolabeled (A) PSE-Am2 and (B) PSE-Am6 probes were used in EMSA. Competition with specific double-stranded oligonucleotides (NF-1 and NF-1m) at 50-fold mass excess of probe, or NF-1 antibodies (Ab) and normal rabbit serum (NRS) is indicated. NF-1 complexes and supershifted bands are indicated by open and closed arrowheads, respectively.
|
|
Both the PSE-B/NF-1 Binding Site and the RFX Site within PSE-A Contribute to the Pituitary Repressor Function of 263P
To determine whether the RFX site within PSE-A contributes to 263P repressor activity, mutations within 263P were created and tested in transfected rat pituitary GC cells. The PSE-Am2 mutation, shown to be unable to form complexes I or II and thus bind RFX1 (Fig. 4A
), was introduced into 263P by site-directed mutagenesis. The resulting fragment was placed upstream of the CS-A promoter to create Am2.CSp.Luc. A PSE-B mutation, shown previously to be unrecognized by members of the NF-1 family (6), was likewise introduced into 263P upstream of the CS-A promoter to generate Bm.CSp.Luc. A significant loss of repressor function was seen with Am2.CSp.Luc (P < 0.001, n = 9) and Bm.CSp.Luc (P < 0.001, n = 9) compared with wild-type 263P (263PCSp.Luc) (Fig. 5
).

View larger version (27K):
[in this window]
[in a new window]
|
Figure 5. RFX/PSE-A and NF-1/PSE-B Sites within 263P Are Both Necessary to Achieve Full CS-A Promoter Repression in Pituitary GC Cells
Hybrid luciferase (Luc) genes were used to assess the effect of introducing nonbinding RFX1 and NF-1 mutations within 263P on -492/+6 CS-A promoter (CS-Ap) activity in GC cells. DNA uptake was controlled through cotransfection with pRL-TKp. Results, corrected, are expressed as a percentage of the activity of CSp.Luc, which was arbitrarily set to 100%. The basal firefly Luc/Renilla Luc and firefly Luc values for CSp.Luc were 2.56 ± 0.10 and 70,000 ± 3,600 (n = 9), respectively. Bars represent SEM.
|
|
The relative luciferase activity of Am2.CSp.Luc and Bm.CSp.Luc was also compared with the luciferase activity of CSp.Luc (arbitrarily set to 100%) to assess whether either of the single mutations had completely abolished 263P repressor function. This was not the case, as both single mutants displayed a significant decrease in activity compared with the CSp.Luc control (Fig. 5
). Specifically, Am2.CSp.Luc and Bm.CSp.Luc repressed CS-A promoter activity by 21% (P < 0.01, n = 9) and 25% (P < 0.001, n = 9), respectively. In contrast, introduction of mutations at both the RFX1/PSE-A and NF-1/PSE-B sites within 263P (Am2/Bm.CSp.Luc) abolished 263P repressor activity, as Am2/Bm.CSp.Luc and CSp.Luc showed no significant difference in activity (n = 9).
RFX1 Has the Capacity to Interact with Members of the NF-1 Family
The proximity of the PSE-A/RFX and PSE-B/NF-1 sites in 263P, as well as the ability of each element to compete for the characteristic footprint pattern on 263P, raises the possibility of an interaction between RFX1 and one or more members of the NF-1 family (NF-1A, NF-1B, NF-1C, and NF-1X). This was investigated by immunoprecipitation and protein blotting. In the absence of specific antibodies to each of the different NF-1 family members, c-myc epitope-tagged NF-1s as well as RFX1 were coexpressed in JAR cells. Overexpression of each of the NF-1 family members was detected in the lysates by protein blotting and immunodetection with a c-myc antibody (Fig. 6A
). RFX1 overexpression above endogenous levels was also detected in lysates from transfected JAR cells (Fig. 6B
). The ability of RFX1 antibodies to immunoprecipitate RFX1 was confirmed (Fig. 6C
). The myc epitope tag of each NF-1 species was assessed by immunoblotting following immunoprecipitation with polyclonal RFX1 or, as an unrelated control, dorsolateral protein (DLP)-specific antibodies. In the case of immunoprecipitation with RFX1 antibodies, bands corresponding to tagged NF-1A, NF-1B, NF-1C, and NF-1X were detected with antibodies to c-myc (Fig. 6D
). In contrast, none of the bands corresponding to tagged NF-1s were seen as a result of immunoprecipitation with DLP antibodies (Fig. 6E
).

View larger version (52K):
[in this window]
[in a new window]
|
Figure 6. Interactions Between RFX1 and the NF-1 Family Can Be Detected by Coimmunoprecipitation
Panel A, Lysates (50 µg) from JAR cells transfected with myc-NF-1A, myc-NF-1B, myc-NF-1C, or myc-NF-1X were assessed by immunoblotting using a c-myc antibody. The approximate sizes of the NF-1 proteins were: NF-1A, 60 kDa; NF-1B, 57 kDa; NF-1C, 54 kDa; and NF-1X, 50 kDa. Panel B, Lysates (50 µg) from JAR cells transfected with HA-RFX1 (JAR-RFX1) or nontransfected (JAR) were probed with RFX1 antibody to visualize overexpressed and endogenous RFX1 protein. The approximate size of RFX1 was 96 kDa. Panel C, Lysates from JAR cells transfected with HA-RFX1 were immunoprecipitated with RFX1 antibody and probed with RFX1 antibody to confirm detection of RFX1 after immunoprecipitation. Panel D, JAR cells transfected with HA-RFX1 and myc-NF-1A, myc-NF-1B, myc-NF-1C, or myc-NF-1X were immunoprecipitated with RFX1-specific antibody. NF-1A, NF-1B, NF-1C, and NF-1X were detected by immunoblotting with c-myc antibody. Panel E, JAR cells transfected with HA-RFX1 and myc-NF-1A, myc-NF-1B, myc-NF-1C, or myc-NF-1X were immunoprecipitated with DLP-specific antibody as a control. NF-1 family members were not detected when the immunoprecipitates were probed with c-myc antibody.
|
|
NF-1 and RFX1 Associate with 263P Sequences in Human Pituitary Chromatin
Chromatin immunoprecipitation (ChIP) assays were used to investigate the ability of NF-1 and RFX1 to associate with 263P in human pituitary tissue samples. The ability of the NF-1 antibody to immunoprecipitate all four NF-1 family members as well as a commercial RFX1 antibody to immunoprecipitate RFX1 was confirmed (Fig. 7
). These same conditions were then used to immunoprecipitate cross-linked chromatin from human pituitary nuclei. Following cross-link reversal and DNA isolation, 263P and fibroblast growth factor (FGF)-16 exon 3 sequences were amplified from both input and immunoprecipitated (bound) DNA samples by PCR. FGF-16 is a member of the FGF family expressed specifically in embryonic brown adipose and adult cardiac tissue (12); PCR with the FGF-16 exon 3 primers was thus used as a measure of nonspecific sequences present in our bound sample. PCR products were electrophoresed and densitometry used to assess levels from digital images. To correct for differences between primer pair efficiency, bound values are expressed as a ratio of the input value. When the NF-1 antibody was used for ChIP, the mean bound/input (B/I) ratio for 263P was 0.4, which was significantly higher than the mean FGF-16 exon 3 B/I ratio of 0.18 (P < 0.01, n = 4) (Fig. 8
). A 263P B/I ratio of 0.63 was also significantly higher than the FGF-16 exon 3 B/I ratio of 0.31 (P < 0.05, n = 4) when the RFX1 antibody was used for ChIP (Fig. 8
).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 7. Specificity of NF-1 and RFX1 Antibodies
Panel A, Immunoprecipitations with NF-1-specific antibody were run alongside lysates (100 µg) from JAR cells transfected with myc-NF-1A (A), myc-NF-1B (B), myc-NF-1C (C), or myc-NF-1X (X). A longer exposure of NF-1 immunoprecipitation from myc-NF-1A JAR lysate is also shown to the left. Panel B, Lysate (100 µg) from JAR cells transfected with myc-RFX1 was run alongside RFX1 immunoprecipitation. Both blots were probed with c-myc antibody to assess the specificity of the NF-1 and RFX1 antibodies for use in ChIP assays.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Figure 8. NF-1 and RFX1 Associate with 263P in Human Pituitary Chromatin
Panel A, Representative PCR results from pituitary ChIP assays. Immunoprecipitation (IP) of cross-linked chromatin from human pituitary nuclei was done with NF-1 and RFX1 antibodies. DNA from both the IP input (I) and the IP bound (B) fractions was amplified by PCR with primer pairs for 263P and FGF-16 exon 3. Panel B, Mean B/I ratios from panel A and three additional pituitary ChIP assays. Bars represent SEM.
|
|
The Detection of the NF-1/RFX1 263P Complexes in Pituitary Tissue Correlates with a Lack of Both 263P and CS Promoter Hyperacetylation
ChIP assays with an antibody to the hyperacetylated form of histone H4 were used to assess the hyperacetylation status of various regions of the GH/CS locus in human pituitary tissue as well as human term placenta. When cross-linked chromatin from pituitary tissue was immunoprecipitated with the hyperacetylated H4 antibody, a significant increase in the mean B/I of the GH promoter (1.31) over the mean (control) FGF-16 exon 3 B/I (0.35) was detected (P < 0.001, n = 3), whereas no significant difference was seen for the mean B/I of either the CS promoter (0.32) or 263P (0.62) above the FGF-16 background in these same assays (Fig. 9
).

View larger version (25K):
[in this window]
[in a new window]
|
Figure 9. Hyperacetylation of 263P and CS Promoter Sequences in Human Term Placenta But Not Pituitary Chromatin
Panel A, Representative PCR results from pituitary and placenta ChIP assays. Immunoprecipitation (IP) of cross-linked chromatin from human pituitary and term placenta nuclei was done with an antibody to the hyperacetylated form of histone H4 (acetyl-H4). DNA from both the IP input (I) and the IP bound (B) fractions was amplified by PCR with primer pairs for 263P, the CS promoter (CSp), the GH promoter (GHp), and FGF-16 exon 3. Panel B, Mean B/I ratios from pituitary and placenta ChIP assays. Bars represent SEM.
|
|
In term placenta chromatin, evidence of histone H4 hyperacetylation at both the CS and GH promoters, as well as 263P, was evident (Fig. 9
). ChIP with hyperacetylated H4 antibody yielded a mean B/I of 1.77 for the GH promoter, 2.57 for the CS promoters, and 1.8 for 263P, all of which were significantly higher than the FGF-16 exon 3 B/I of 1.03 (P < 0.05, P < 0.05, and P < 0.01, respectively; n = 3).
 |
DISCUSSION
|
---|
P sequences, and specifically the 263P region, have been implicated in the expression and regulation of the human GH family in vitro and in vivo (3, 4, 6). The 263P region contains two sites of direct protein-DNA interactions that are visualized by nuclease protection, namely PSE-A and PSE-B. We have shown previously that PSE-B is a binding site in vitro for NF-1, a family of transcription factors known to be involved in both activation and repression of genes (5). In the present study we used a combination of structural and functional assays to assess PSE-A, with a view to further characterize the 263P complex. Our data define the human GH locus as a novel regulatory target for the RFX winged-helix family and provide evidence for multiple interactions at PSE-A. In addition, we have described, for the first time, the potential for NF-1 and RFX1 to participate in a common regulatory complex and have identified association of these factors with 263P in pituitary chromatin. We also observed an inverse relationship between association of NF-1 and RFX1 and the presence of 263P and CS promoter hyperacetylation. Taken together, our data support the idea that the presence of the NF-1/RFX1 263P complex in vivo negates the formation of a potential enhancer complex and/or hyperacetylated chromatin structure, leading to the lack of placental gene expression in pituitary tissue.
Analysis of PSE-A in vitro has revealed a core sequence for pituitary protein-DNA interactions that localizes to a region containing an X-box motif (5'-GTNRCC(03N)RGYAAC-3') recognized by the regulatory factor X (RFX) family of transcription factors (Fig. 2
, A and B) (8). Through EMSA, we have confirmed that PSE-A is an RFX1 DNA element (Fig. 3
) and that this element can compete the 263P footprint pattern (Fig. 1A
). In addition, our functional data indicate a role for RFX in the repressor action of 263P in pituitary cells after gene transfer (Fig. 5
), an activity that helps define this element (3). The human GH family, and specifically PSE-A, represents a novel regulatory target for the RFX family. In mammals, the RFX family consists of five members (13). RFX1, RFX2, and RFX3 are expressed ubiquitously (14, 15), whereas RFX4 is expressed specifically in testis (16), and RFX5 has been identified as being essential for expression of the tissue-specific MHC class II genes (17). Candidate target genes for RFX1 include ribosomal protein L30, interleukin-5 receptor
, and proliferating cell nuclear antigen (11, 18, 19); targets have not been identified for RFX-2 or RFX-3. RFX1, but not RFX2 and RFX3, binding to PSE-A was detected (Fig. 3
); however, a negative result does not rule out participation of either RFX2 or RFX3 in a 263P complex. These family members may be absent, present at low levels, or modified and unable to bind. Nonetheless, the competition of pituitary RFX-related complexes by the RFX1 antibody appears complete, suggesting that RFX1 is the predominant species, at least in pituitary cells.
In addition to binding RFX1, PSE-A is also a binding site for NF-1 (Fig. 4
). However, binding of RFX1 and NF-1 to PSE-A appears to be mutually exclusive, as NF-1 was detected only after disruption of the RFX site (Fig. 4A
). This suggests that RFX1 binding is the favored event under the in vitro conditions used and is consistent with our previous inability to see direct binding of NF-1 to PSE-A (6). By extension, the capacity of PSE-A to bind either RFX1 or NF-1 implies that the nuclease protection pattern at PSE-A likely represents a composite of both interactions. There are numerous examples of composite footprint patterns, including one derived from Pit-1 and Sp1 binding in the GH-N proximal promoter region (20, 21). This suggests that when RFX1 is competed, the binding of NF-1 to PSE-A may still be visualized, explaining why individual RFX elements, such as the PSE-Am6 mutation, appear to be relatively weak competitors of the nuclease protection pattern (Fig. 2C
).
Previous observations of the RFX family have provided evidence that RFX binding can be modified by formation of a complex (reviewed in Ref. 22). The proximity of PSE-A and PSE-B raises the possibility that RFX1 and NF-1 can interact, and as a result modify their affinity/specificity for 263P. Our ability to coprecipitate NF-1 family members with RFX1 suggests that an interaction between these factors can occur and may contribute to a mechanism regulating gene expression (Fig. 6
). In addition, the relatively large amounts of oligonucleotide required to see efficient competition of complexes formed on 263P in nuclease protection assays, as compared with individual elements in EMSA, suggest differences in the affinity/specificity of NF-1 and/or RFX1 complexes under these two assay conditions (Fig. 1
). Regardless of whether RFX1 and NF-1 interact and modify their binding, our assessment of human pituitary chromatin indicates both RFX1 and NF-1 can associate with the 263P region (Fig. 8
).
The presence of P sequences upstream of each member of the human GH gene family that is expressed in the placenta (CS-L, CS-A, GH-V, and CS-B) makes them ideal candidates for involvement in local tissue-specific gene regulation of the GH locus. The detection of 263P hyperacetylation in human placenta, but not pituitary tissue (Fig. 9
and Ref. 4) supports a hypothesis whereby P sequences are not only repressors of placental gene expression in the pituitary, but play a role as an activator in the placenta. The capacity to form distinct pituitary and placental complexes is observed by EMSA using pituitary GC and placental JAR nuclear protein (Norquay, L. D., unpublished results) and supports the potential for separate repressor and activator complexes. It may, however, be inappropriate at this time to limit the analysis of P sequences in the placenta to only 263 bp, as this element was defined based on pituitary repressor function (3, 6). Although 263P was shown previously to enhance gene expression in the placenta of transgenic mice, the capacity to function in this way was not seen in all lines and was exerted on the pituitary expressed GH-N gene (4). No consistent activation was seen when full-length P sequences were tested in their native context with the placental CS-A promoter (23). It therefore remains to be determined what functional role P sequences play in the placenta. Nonetheless, the observation of placental hyperacetylation (Fig. 9
) is intriguing, and clearly further structural and functional characterizations of a potential placenta P sequence complex are required.
Previous investigation of the human GH/CS locus revealed two upstream pituitary-specific hypersensitive sites, HS I/II (23), subsequently shown to be essential for pituitary-specific expression of GH-N (23, 24, 25, 26). Consistent with its regulatory role in vivo, additional characterization revealed that the chromatin containing the HS I/II region was hyperacetylated in primary human GH-secreting pituitary adenoma cells and the pituitaries of transgenic mice (27). As a part of our study we were able to extend the analysis of chromatin modifications in the human GH/CS locus to normal human pituitaries taken post mortem. Hyperacetylation of the HSI/II region was used to verify the integrity of the pituitary chromatin (data not shown). The use of PCR in our ChIP assays also enabled us to distinguish between the highly homologous GH and CS promoters, which was not possible in previous hybridization approaches (4, 27). As a result, we observed hyperacetylation at the GH but not the CS promoter in pituitary chromatin (Fig. 9
), which is consistent with their expression patterns in vivo (1). Pituitary-specific expression of GH-N is dependent on the transcription factor Pit-1 (25, 26, 28, 29, 30). Pit-1 has been shown to occupy binding sites in the promoters of both transcribed and nontranscribed genes (31) and has the capacity to recruit both the histone acetyltransferase, CREB-binding protein, and the transcriptional corepressor, nuclear receptor corepressor (N-CoR) (32). The presence of one vs. two Pit-1 binding sites in the placental GH/CS promoters and/or their relative distances from the upstream HS I/II may contribute to the differences observed in promoter hyperacetylation compared with GH-N (Fig. 9
). We have also proposed that the presence of P sequences upstream of the placental GH/CS promoters, but not GH-N, plays a role in restricting placental gene expression from the pituitary (3). The absence of hyperacetylation at the CS promoter correlated with both lack of 263P hyperacetylation (Fig. 9
) and association of NF-1 and RFX1 with 263P in pituitary tissue (Fig. 8
). Although both NF-1 and RFX1 are known transcriptional activators, they are also associated with repressor activity (5, 33). In the case of NF-1, down-regulation of Id-1 promoter activity in nonaggressive breast cancer cells was linked to a complex containing both NF-1 and histone deacetylase 1 (HDAC-1) (34). Our observation of NF-1 and RFX1 association with 263P sequences in the pituitary is, to our knowledge, the first example of either of these factors associating with chromatin in human tissue samples. This complex may contribute to a mechanism preventing hyperacetylation of 263P and CS promoter chromatin, thus repressing expression of the placental GH/CS genes in the pituitary.
 |
MATERIALS AND METHODS
|
---|
Oligonucleotide Sequences
Double-stranded DNA elements were generated by synthesizing and annealing sense and antisense oligonucleotides (Invitrogen, Burlington, Ontario, Canada). The sense strands for each element is provided in Table 1
. The sequences of primers used for PCR of 263P, FGF-16 exon 3, and GH and CS promoters are also given in Table 1
.
Preparation and Fractionation of Nuclear Extracts
Nuclear protein extracts from rat anterior pituitary tumor (GC) cells were made according to published protocols (35) and dialyzed as previously described (36). Protein concentration of the extracts was assessed using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada) with BSA as a standard. Extracts were stored as aliquots at -70 C.
Deoxyribonuclease (DNase) 1 Protection Assays
DNase protection assays were performed as previously described (6). Briefly, 25 µg of GC cell nuclear protein were preincubated with double-stranded competitor oligonucleotides (10005000 pmol excess of the probe) for 15 min on ice. Subsequently, 0.5 ng of 263P fragment probe, radiolabeled at the 3'-end, was added to each reaction and incubated for an additional 15 min on ice. DNase digestion proceeded at 26 C for 1 min, and the samples were electrophoresed in a 6% acrylamide gel.
EMSA
EMSA was performed essentially as previously described previously (6). Competitor oligonucleotides (mass excess of the probe), NF-1, RFX1, RFX2, and RFX3 antibodies (1 µl) (6, 9, 14), or normal rabbit serum (1 µl) were preincubated with 5 µg of GC cell nuclear extract and 2 µg poly dIdC in reaction buffer (10 mM Tris, pH 7.5; 50 mM NaCl; 1 mM dithiothreitol; 1 mM EDTA; 5% glycerol) for 10 min at room temperature in a 20 µl final volume. After 10 min, radiolabeled double-stranded oligonucleotide probes were added, and the reactions were incubated an additional 10 min at room temperature before electrophoresis in a 5% acrylamide gel.
Plasmid Constructions
The immediate 5'-flanking region of the CS-A gene (-492/+6) upstream of the firefly luciferase (Luc) gene (CSp.Luc) as well as 263P.CSp.Luc were previously described (6). A two-step PCR approach with 263P primers and appropriate mutant oligonucleotides (Table 1
) was used to introduce mutations into 263P (Taq DNA polymerase; QIAGEN, Mississauga, Ontario, Canada). The final PCR products were inserted as BamHI fragments upstream of the CS-A promoter in CSp.Luc to generate Am2.CSp.Luc, Bm.CSp.Luc, and Am2/Bm.CSp.Luc. All constructs were sequenced. Hemagglutinin (HA) or c-myc (myc)-tagged transcription factors (HA-RFX1, myc-RFX1, myc-NF-1A, myc-NF-1B, myc-NF-1C, and myc-NF-1X) were generated using the epitope-tagged mammalian expression vector set (CLONTECH Laboratories, Inc., Palo Alto, CA). NF-1 cDNAs were isolated from pBSNF-1 vectors (37) and subcloned into XhoI/NotI of pCMV-Myc (CLONTECH Laboratories, Inc.). The RFX1 cDNA was isolated from pSG5RFX1 (38), blunted with Klenow fragment, and subcloned into the (blunted) SalI site in pCMV-Myc and pCMV-HA (CLONTECH Laboratories, Inc.).
Cell Culture and Gene Transfer
GC and JAR 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 of test (firefly luciferase) plasmid and 25 ng/plate of pRL-TkpLuc (renilla luciferase) plasmid (Promega Corp., Madison, WI) as a control for DNA uptake, was done using the calcium phosphate/DNA precipitation method, as previously described (6). The luciferase values were determined using the dual-luciferase assay system (Promega Corp.) according to manufacturers instructions with the exception of cell lysis, which was performed as described previously (39). Values are expressed as a percentage of CSp.Luc activity and are the mean of at least three separate precipitations. Gene transfer with 2 µg of each epitope-tagged plasmid per plate was also performed by calcium phosphate/DNA precipitation as previously described (6). Lysis of cells was carried out in modified RIPA buffer [40 mM Tris, pH 8.0; 150 mM NaCl; 1% Nonidet P-40 (NP-40); 0.25% Na-deoxycholate; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride (PMSF); 1 mM Na3VO4; 1 mM NaF; 1 tablet/10 ml Complete Mini Protease inhibitor (Roche Diagnostics, Laval, Québec, Canada); and 1 µg/ml aprotinin] for 15 min at 4 C. The total cellular lysates were pelleted to clear insoluble material, and protein concentrations were assessed using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.).
Immunoprecipitation and Protein Blotting
Immunoprecipitation was carried out with 0.5 mg of total cellular lysate. Lysates were precleared for 30 min at 4 C using nonspecific monoclonal antibodies or rabbit immunoglobulins, followed by a 1-h incubation with Pansorbin Cells (Calbiochem, La Jolla, CA) prepared according to manufacturers instructions. Specific antibodies to RFX1 (1 µl) (9), c-myc (2 µg) (CLONTECH Laboratories, Inc.) or, as an unrelated control, rat prostate DLP (1 µl) (40) were incubated with precleared supernatants at 4 C for 2 h, and prepared Pansorbin cells were included for overnight incubation. Washes were performed first with 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 finally 10 mM Tris/0.1% NP-40. Pansorbin cell pellets were resuspended in 40 µl of Laemli sample buffer [2% sodium dodecyl sulfate (SDS); 10% glycerol; 100 mM dithiothreitol; 60 mM Tris, pH 6.8; and 0.001% bromophenol blue], boiled for 5 min, and 20 µl was resolved by SDS-PAGE in an 8% gel. Gels were transferred and immobilized on polyvinylidine difluoride membranes (Roche Diagnostics) and blocked. Immunodetection was performed for 1 h (rabbit RFX1 antibodies, 1:10,000) or overnight (mouse c-myc antibodies, 2 µg/ml) at 4 C, and detection of antibody-antigen complexes was performed using BM chemiluminescence blotting substrate (POD, Roche Diagnostics) according to manufacturers instructions. Complexes were visualized on Kodak Biomax film (Amersham Pharmacia Biotech Biosciences, Baie dUrfé, Québec, Canada).
Nuclei Isolation and ChIP
Postmortem human pituitary tissue was obtained from the Human Pituitary Repository in the Protein and Polypeptide Laboratory at the University of Manitoba, and placentas were from normal term deliveries at the Health Science Centre (Winnipeg, Manitoba). For each ChIP assay, nuclei were isolated from placenta tissue or pooled (3, 4, 5, 6) human pituitary samples as previously described (2) and resuspended in HEPES buffer (10 mM HEPES, 10 mM NaCl, 3 mM MgCl2, 1 mM PMSF) pH 7.5, to an A260 of approximately 20 U/ml. Formaldehyde was added to a final concentration of 1% for 5 min at room temperature, and the cross-linking reaction was quenched by the addition of glycine (final concentration, 0.125 M). Cross-linked nuclei were pelleted, washed in RSB buffer [Complete Mini protease inhibitor cocktail (Roche Diagnostics) with 10 mM Tris, pH 7.5; 10 mM NaCl; 3 mM MgCl2; 1 mM PMSF], and resuspended in lysis buffer (50 mM Tris, pH 8.0; 1% SDS; 10 mM EDTA; 1 mM PMSF) for 10 min at 4 C. Chromatin was sonicated for a total time of 90 sec (pituitary) or 120 sec (placental) at 40% output (Vibra Cell, Sonics and Materials, Inc., Danbury, CT) in 30-sec pulses, and then pelleted to remove insoluble material. To measure DNA content, the A260 of a sample was assessed in 2 M NaCl/5 M urea. For immunoprecipitation, 5-ml samples 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% SDS; 1 mM PMSF) 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 Biosciences) and 25 µg of sheared salmon sperm DNA. Specific antibodies were added for overnight incubation as follows: 25 µl antihyperacetylated (penta) histone H4 (Upstate Biotechnology, Inc., Lake Placid, NY) 50 µl RFX1 (D-19) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and 50 µl NF-1 (H-300) (Santa Cruz Biotechnology, Inc.). The following day, 300 µl of protein A sepharose and 50 µg of sheared salmon sperm DNA were added for 1 h. Samples were washed for 10 min at 4 C in 5 ml of wash buffer as follows: low-salt buffer (20 mM Tris, pH 8; 150 mM NaCl; 2 mM EDTA; 1% Triton X-100; 0.1% SDS), high-salt buffer (20 mM Tris, pH 8; 500 mM NaCl; 2 mM EDTA; 1% Triton X-100; 0.1% SDS), LiCl buffer (10 mM Tris, pH 8; 0.25 M LiCl; 1 mM EDTA; 1% deoxycholic acid Na salt; 1% NP-40), and twice in TE buffer (10 mM Tris, pH 8; 1 mM EDTA). Samples were eluted twice in 1.25 ml elution buffer (1% SDS, 0.1 M NaHCO3) for 15 min at room temperature, and cross-links were reversed for 6 h at 68 C. DNA was isolated using QIAquick columns (QIAGEN, Chatsworth, CA) according to manufacturers instructions. PCR was carried out with 10 ng of input DNA or 5 µl eluted (bound) DNA per PCR reaction (Taq DNA polymerase; QIAGEN) at 55 C annealing temperature for 28 cycles. PCR primer pairs are listed in Table 1
. For amplification of 263P, 263(F) and PSE-Aext(R) were used as primers.
Statistical Analysis
Statistical analysis was done using a two-tailed, unpaired Students t test. A value of P < 0.05 is considered statistically significant. In figures, *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Masayoshi Imagawa for his generous gift of NF-1 cDNAs, Dr. R. P. C. Shiu and Michel Chrétien for human pituitary tissue samples, and Dr. James Davie and Ms. Virginia Spencer for advice in setting up the ChIP assay.
 |
FOOTNOTES
|
---|
This work was supported by Grant 10853 from the Canadian Institutes of Health Research. L.D.N. is the recipient of a Canadian Institutes of Health Research Doctoral Studentship.
Abbreviations: B/I ratio, Bound/input ratio; ChIP, chromatin immunoprecipitation; CS, chorionic somatomammotropin; DLP, dorsolateral protein; DNase, deoxyribonuclease; FGF, fibroblast growth factor; HA, hemagglutinin; hGH, human GH; NF-1, nuclear factor-1; NP-40, Nonidet P-40; PMSF, phenylmethylsulfonyl fluoride; PSE-A and PSE-B, P sequence elements A and B; RFX, regulatory factor X; SDS, sodium dodecyl sulfate.
Received for publication January 22, 2003.
Accepted for publication February 28, 2003.
 |
REFERENCES
|
---|
- Chen EW, Liao Y, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH 1989 The human growth hormone locus nucleotide sequence biology and evolution. Genomics 4:479497[Medline]
- Nickel BE, Cattini PA 1996 Nuclease sensitivity of the human growth hormone-chorionic somatomammotropin locus in pituitary and placenta suggest different mechanisms for tissue-specific regulation. Mol Cell Endocrinol 118:155162[CrossRef][Medline]
- Nachtigal MW, Nickel BE, Cattini PA 1993 Pituitary-specific repression of placental members of the human growth hormone gene family. A possible mechanism for locus regulation. J Biol Chem 268:84738479[Abstract/Free Full Text]
- Elefant F, Su Y, Liebhaber SA, Cooke NE 2000 Patterns of histone acetylation suggest dual pathways for gene activation by a bifunctional locus control region. EMBO J 19:68146822[Abstract/Free Full Text]
- Gronostajski RM 2000 Roles of the NFI/CTF gene family in transcription and development. Gene 249:3145[CrossRef][Medline]
- Norquay LD, Jin Y, Surabhi RM, Gietz RD, Tanese N, Cattini PA 2001 A member of the nuclear factor-1 family is involved in the pituitary repression of the human placental growth hormone genes. Biochem J 354:387395[CrossRef][Medline]
- Wingender E, Chen X, Hehl R, Karas H, Liebich I, Matys V, Meinhardt T, Pruss M, Reuter I, Schacherer F 2000 TRANSFAC: an integrated system for gene expression regulation. Nucleic Acids Res 28:316319[Abstract/Free Full Text]
- Gajiwala KS, Chen H, Cornille F, Roques BP, Reith W, Mach B, Burley SK 2000 Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. Nature 403:916921[CrossRef][Medline]
- Herrero-Sanchez C, Reith W, Silacci P, Mach B 1992 The DNA-binding defect observed in major histocompatibility complex class II regulatory mutants concerns only one member of a family of complexes binding to the X boxes of class II promoters. Mol Cell Biol 12:40764083[Abstract]
- Cornille F, Emery P, Schuler W, Lenoir C, Mach B, Roques BP, Reith W 1998 DNA binding properties of a chemically synthesized DNA binding domain of hRFX1. Nucleic Acids Res 26:21432149[Abstract/Free Full Text]
- Iwama A, Pan J, Zhang P, Reith W, Mach B, Tenen DG, Sun Z 1999 Dimeric RFX proteins contribute to the activity and lineage specificity of the interleukin-5 receptor
promoter through activation and repression domains. Mol Cell Biol 19:39403950[Abstract/Free Full Text]
- Miyake A, Konishi M, Martin FH, Hernday NA, Ozaki K, Yamamoto S, Mikami T, Arakawa T, Itoh N 1998 Structure and expression of a novel member, FGF-16, on the fibroblast growth factor family. Biochem Biophys Res Commun 243:148152[CrossRef][Medline]
- Emery P, Durand B, Mach B, Reith W 1996 RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom. Nucleic Acids Res 24:803807[Abstract/Free Full Text]
- Reith W, Ucla C, Barras E, Gaud A, Durand B, Herrero-Sanchez C, Kobr M, Mach B 1994 RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins. Mol Cell Biol 14:12301244[Abstract]
- Reith W, Herrero-Sanchez C, Kobr M, Silacci P, Berte C, Barras E, Fey S, Mach B 1990 MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain. Genes Dev 4:15281540[Abstract]
- Morotomi-Yano K, Yano K, Saito H, Sun Z, Iwama A, Miki Y 2002 Human regulatory factor X 4 (RFX4) is a testis-specific dimeric DNA-binding protein that cooperates with other human RFX members. J Biol Chem 277:836842[Abstract/Free Full Text]
- Steimle V, Durand B, Barras E, Zufferey M, Hadam MR, Mach B, Reith W 1995 A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome). Genes Dev 9:10211032[Abstract]
- Sáfrány G, Perry RP 1995 The relative contributions of various transcription factors to the overall promoter strength of the mouse ribosomal protein L30 gene. FEBS Lett 230:10661072
- Liu M, Lee BH, Mathews MB 1999 Involvement of RFX1 protein in the regulation of the human proliferating cell nuclear antigen promoter. J Biol Chem 274:1543315439[Abstract/Free Full Text]
- Lemaigre FP, Lafontaine DA, Courtois SJ, Durviaux SM, Rousseau GG 1990 Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter. Mol Cell Biol 10:18111814[Medline]
- Nickel BE, Nachtigal MW, Bock ME, Cattini PA 1991 Differential binding of rat pituitary-specific nuclear factors to the 5'-flanking region of pituitary and placental members of the human growth hormone gene family. Mol Cell Biochem 106:181187[Medline]
- Reith W, Mach B 2001 The bare lymphocyte syndrome and the regulation of MHC expression. Annu Rev Immunol 19:331373[CrossRef][Medline]
- Jones BK, Monks BR, Liebhaber SA, Cooke NE 1995 The human growth hormone gene is regulated by a multicomponent locus control region. Mol Cell Biol 15:70107021[Abstract]
- Bennani-Baiti IM, Asa SL, Song D, Iratni R, Liebhaber SA, Cooke NE 1998 DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression. Proc Natl Acad Sci USA 95:1065510660[Abstract/Free Full Text]
- Jin Y, Surabhi RM, Fresnoza A, Lytras A, Cattini PA 1999 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. Mol Endocrinol 13:12491266[Abstract/Free Full Text]
- Shewchuk BM, Asa SL, Cooke NE, Liebhaber SA 1999 Pit-1 binding sites at the somatotrope-specific DNase I hypersensitive sites I, II of the human growth hormone locus control region are essential for in vivo hGH-N gene activation. J Biol Chem 274:3572535733[Abstract/Free Full Text]
- Elefant F, Cooke NE, Liebhaber SA 2000 Targeted recruitment of histone acetyltransferase activity to a locus control region. J Biol Chem 275:1382713834[Abstract/Free Full Text]
- Pfaffle RW, DiMattia GE, Parks JS, Brown MR, Wit JM, Jansen M, Van der Nat H, Van den Brande JL, Rosenfeld MG, Ingraham HA 1992 Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 257:11181121[Medline]
- Ingraham HA, Chen R, Mangalam HJ, Elsholtz HP, Flynn SE, Lin CR, Simmons DM, Swanson L, Rosenfeld MG 1988 A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519529[Medline]
- Castrillo JL, Bodner M, Karin M 1989 Purification of growth hormone-specific transcription factor GHF-1 containing homeobox. Science 243:814817[Medline]
- Scully KM, Jacobson, EM, Jepsen K, Lunyak V, Viadiu H, Carrière, Rose DW, Hooshmand F, Aggarwal AK, Rosenfeld MG 2000 Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification. Science 290:11271131[Abstract/Free Full Text]
- Xu L, Lavinsky RM, Dasen JS, Flynn SE, McInerney EM, Mullen TM, Heinzel T, Szeto D, Korzus E, Kurokawa R, Aggarwal AK, Rose DW, Glass CK, Rosenfeld MG 1998 Signal-specific co-activator requirements for Pit-1 activation. Nature 395:301306[CrossRef][Medline]
- Katan Y, Agami R, Shaul Y 1997 The transcriptional activation and repression domains of RFX1, a context-dependent regulator, can mutually neutralize their activities. Nucleic Acids Res 25:36213628[Abstract/Free Full Text]
- Singh J, Murata K, Itahana Y, Desprez PY 2002 Constitutive expression of the Id-1 promoter in human metastatic breast cancer cells is linked with the loss of NF-1/Rb/HDAC-1 transcription repression complex. Oncogene 21:1222
- Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]
- Lytras A, Surabhi RM, Zhang JF, Jin Y, Cattini PA 1996 "Repair" of the chorionic somatomammotropin-A "enhancer" region reveals a novel functional element in the chorionic somatomammotropin-B enhancer. Mol Cell Endocrinol 119:110[CrossRef][Medline]
- Osada S, Matsubara T, Daimon S, Terazu Y, Xu M, Nishihara T, Imagawa M 1999 Expression, DNA-binding specificity and transcriptional regulation of nuclear factor 1 family proteins from rat. Biochem J 342:189198[CrossRef][Medline]
- Siegrist CA, Durand B, Emery P, David E, Hearing P, Mach B, Reith W 1993 RFX1 is identical to enhancer factor C and functions as a transactivator of the hepatitis B virus enhancer. Mol Cell Biol 13:63756384[Abstract]
- Nickel BE, Kardami E, Cattini PA 1990 Differential expression of human placental growth-hormone variant and chorionic somatomammotropin in culture. Biochem J 267:653658[Medline]
- Donjacour AA, Rosales A, Higgins SJ, Cunha GR 1990 Characterization of antibodies to androgen-dependent secretory proteins of the mouse dorsolateral prostate. Endocrinology 126:13431354[Abstract]