help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2003-0392
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Limesand, S. W.
Right arrow Articles by Anthony, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Limesand, S. W.
Right arrow Articles by Anthony, R. V.
Molecular Endocrinology 18 (2): 447-457
Copyright © 2004 by The Endocrine Society

Pur{alpha}, a Single-Stranded Deoxyribonucleic Acid Binding Protein, Augments Placental Lactogen Gene Transcription

Sean W. Limesand, Kimberly M. Jeckel and Russell V. Anthony

Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523-1683

Address all correspondence and requests for reprints to: Dr. Russell V. Anthony, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1683. E-mail: Russ.Anthony{at}colostate.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Placental lactogen (PL) is thought to alter maternal metabolism to increase the pool of nutrients available for the fetus and to stimulate fetal nutrient uptake. The ovine (o) PL gene is expressed in chorionic binucleate cells (oBNC) and cis-elements located within the proximal promoter (-124 to +16 bp) are capable of trophoblast-specific expression in human (BeWo) and rat (Rcho-1) choriocarcinoma cells. Protein-DNA interactions were identified with oBNC nuclear extracts, and mutational analysis of these regions revealed a previously undefined cis-element from -102/-123 bp that enhances promoter activity in BeWo cells but not Rcho-1 cells. Characterization of this region identified the nucleotide sequence CCAGCA (-105/-110; o110) as the responsible cis-acting element. Southwestern analysis with this element identified a binding protein with an apparent Mr of approximately 41,000. Expression screening of an ovine placental cDNA library identified six homologous cDNAs, which shared identity with human (97%) and mouse (95%) Pur{alpha}, a single-stranded DNA binding protein. The Pur{alpha}-o110 interaction was confirmed by electrophoretic mobility-supershift assays with oBNC and BeWo extracts but was absent with Rcho-1 extracts. Furthermore, overexpression of ovine Pur{alpha} enhanced transactivation of the oPL gene proximal promoter in both choriocarcinoma cell lines through this novel cis-element. This study identified a previously undefined cis-element, which interacts with Pur{alpha} to augment PL gene transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A DEVELOPING FETUS requires a substantial nutrient flux from the mother to the fetus for normal growth and development. Placental lactogen (PL), a member of the GH/prolactin gene family, is thought to alter maternal metabolism such that the pool of nutrients available for the fetus is increased. Additionally, PL is thought to act on fetal tissues to stimulate nutrient uptake (1, 2, 3). Ovine (o) chorionic binucleate cells (oBNC) synthesize oPL and secrete it into both the maternal and fetal vasculature as a nonglycosylated 198 amino acid polypeptide with an apparent Mr of approximately 22,000 (3, 4, 5). The oPL gene has been structurally characterized, and 4.5 kb of 5'-flanking sequence was examined for trophoblast-specific transactivation in heterologous choriocarcinoma cell lines (6). Transient transfection analysis of oPL gene 5'-flanking sequence identified the proximal 383 bp as the promoter region capable of stimulating maximal activity in human (BeWo) and rat (Rcho-1) choriocarcinoma cell lines. In addition, a region from -124/+16 bp (minimal promoter), relative to the transcriptional start site, retained trophoblast-specific activity, albeit reduced from the activity obtained with the -383/+16-bp region (6). Therefore, cis-elements within the oPL minimal promoter region are thought to be crucial for placental-cell transactivation.

Protein-DNA interactions that may regulate gene expression of the oPL minimal promoter were identified by deoxyribonculease I protection using oBNC nuclear extracts (6). Three footprints (FP) at -12/+7 bp (FP1), -74/-48 bp (FP2), and -123/-95 bp (FP3) are located within the minimal promoter, and contain previously recognized cis-acting elements described in trophoblast-cell regulation of other placental genes (6, 7). Two GATA elements at -67 bp (FP2) and -102 bp (FP3) and an activator protein-2 (AP-2) element at -58 bp (FP2) were found to interact with oGATA 2 and oAP-2{alpha}, respectively (6, 7). Functional GATA elements have been described in the human chorionic gonadotropin {alpha}-subunit gene (8), mouse adenosine deaminase gene (9), and mouse PL-I gene (10). AP-2 sites have been demonstrated to enhance placental activity for the mouse adenosine deaminase gene (11), human chorionic gonadotropin {alpha}- and ß-subunit genes (12), and in human PL (hPL) (13) and oPL (7) genes.

The functionality of these previously identified cis-acting elements within the oPL minimal promoter was confirmed by mutational analysis in choriocarcinoma cell lines (6). The mutation disrupting the GATA element at -67 bp exhibited a significant reduction in activity with both choriocarcinoma cell lines, whereas the mutation of the -102-bp GATA element was less detrimental to transactivation (6). However, mutation of both GATA elements reduced activity to basal levels in choriocarcinoma cells, indicating that both GATA elements are functional (6). Because the GATA element in FP3 had less influence on oPL minimal promoter activity and was located within the downstream portion of FP3, an additional mutation was created in FP3 to determine whether the GATA element was the only cis-acting element functioning in this region. A mutation at -102/-109 bp identified a functional cis-acting element, distinct from the GATA element, in BeWo cells, but not Rcho-1 cells. No previously defined elements share similarity with this sequence within FP3. Therefore, the current study characterizes a novel cis-acting element involved in PL gene transactivation and identifies the transacting factor responsible for augmenting transcription of PL genes in hPL and oPL cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A protein-DNA interaction encompassing -123/-95 bp was previously identified (FP3) within the minimal promoter region of oPL, and a functional GATA element (-102 bp) was located at the 3' end of this protected region (6). However, mutational analysis of this GATA element (-124{Delta}100pGL3) did not significantly reduce oPL minimal promoter transactivation in choriocarcinoma cell lines (6). Therefore, an additional mutation in FP3, at -109/-102 bp (-124{Delta}110), was created to identify other potential cis-acting elements distinct from the GATA element that regulates placental-cell transactivation of the oPL gene. These mutations ({Delta}100 and {Delta}110) and the wild type (-124pGL3) were transiently transfected into human (BeWo) and rat (Rcho-1) choriocarcinoma cell lines (Fig. 1Go). BeWo cell transfections with -124{Delta}100 and -124{Delta}110 exhibited different degrees of activity reduction, suggesting additional functional elements within FP3, because the GATA element (-102 bp) is disrupted with both mutations. A reduction of 67% (P = 0.0534) was observed between wild-type and -124{Delta}100 vectors, whereas transfections with the -124{Delta}110 vector had a reduction in activity of 89% (P < 0.01). An additional mutation (-89/-82 bp; {Delta}90), not located within a protected region of the oPL minimal promoter, was transfected to confirm that reduced promoter activity was not a result of the mutation sequence (data not shown). In Rcho-1 cells, both mutations resulted in similar, but nonsignificant reductions in activity suggesting that this element was not critical in all placental cells (Fig. 1Go). Therefore, a protein-DNA interaction located in the center of FP3, distinct from the GATA element (-102 bp), was found to mediate transactivation in human trophoblast cells.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1. FP3 Mutational Analysis

Mutational analysis of FP3 in the oPL gene minimal promoter was tested in BeWo and Rcho-1 choriocarcinoma cell lines. The pGL3 vectors, -124 (wild type), -124{Delta}100 (-99/-92 bp), -124{Delta}110 (-109/-102 bp), transiently transfected into the cells are indicated on the ordinate axis. The bars represent the mean percent activity (n >= 4) with the wild-type designated 100% (labeled on the abscissa), and the error bars represent the SEM. The differences in the means were separated by a Dunnett’s t test; *, P < 0.05.

 
Mutational analysis suggested an additional cis-acting element within the -109/-102-bp region of FP3 in human choriocarcinoma cells. Accordingly, the protein-DNA interaction was studied by EMSAs with oBNC nuclear extracts to identify whether a nuclear protein specifically interacted with this sequence. An oligonucleotide encompassing -116/-102 bp (o110) was radiolabeled, and binding of a single protein-DNA complex identified (Fig. 2Go). However, several nonspecific complexes were observed. Therefore, EMSA binding reactions were exposed to various temperatures before adding the radiolabeled o110 DNA, and the data indicate that the resulting protein-DNA complex was heat stable (Fig. 2Go) up to 55 C, but not at temperatures of at least 65 C. The heat stability of this nuclear protein (55 C) was used in all subsequent EMSA binding reactions to reduce nonspecific complex formation.



View larger version (48K):
[in this window]
[in a new window]
  		Fig. 2. o110 Oligonucleotide EMSA and Temperature Sensitivity

An o110 oligonucleotide (-116/-102 bp) derived from the oPL gene minimal promoter bound a nuclear protein in oBNC. The first lane contains 25 µg oBNC nuclear extract with no competitor, and the subsequent lanes with the triangle above them have 10-, 50-, 100-, and 250-fold molar excess of nonradiolabeled homologous competitor (-116/-102 bp). Exposing binding reactions to various temperatures (45, 55, 65, and 75 C) before adding the radiolabeled oligonucleotide indicated the nuclear protein interacting with o110 was heat stable to 55 C and nonspecific interactions were ablated. This procedure was included in subsequent EMSAs to reduce nonspecific interactions.

 
The specificity of this protein-DNA interaction was examined in oBNC and BeWo nuclear extracts in the presence of unlabeled heterologous oligonucleotides (oGATA and oAP2). The protein-DNA complex formed with oBNC nuclear extracts was unaffected by these competitors, whereas addition of unlabeled homologous oligonucleotides reduced complex formation (Fig. 3Go). A BeWo nuclear protein specifically interacted with o110 oligonucleotide, as complex formation with the radiolabeled o110 was reduced with the addition of unlabeled homologous competitor (Fig. 3Go) but not altered with the addition of heterologous competitors (data not shown). No protein-DNA interaction was observed with Rcho-1 nuclear extracts (50 µg), under any binding conditions or with increasing amounts of nuclear protein (data not presented). These data identify a specific protein-DNA interaction within FP3 (-102/-116 bp) with a BeWo and oBNC nuclear protein, which is distinct from the previously identified GATA element (-102 bp). Combined with the mutational analysis results, these data suggest that this protein-DNA interaction functions to augment transactivation of the oPL promoter in hPL and oPL cells, but not in rat trophoblast cells.



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3. Specificity of the o110 Oligonucleotide Binding by EMSA

Binding studies using a radiolabeled o110 oligonucleotide with no, oBNC (25 µg) and BeWo (25 µg) nuclear extracts were analyzed by EMSA. Both oBNC and BeWo nuclear proteins reduced the mobility of the radiolabeled o110 oligonucleotide, when no competitor was added. Subsequent lanes for oBNC nuclear extracts included the addition of homologous (o110) or heterologous (oAP2 or oGATA) unlabeled competitors in increasing amounts (50-, 100-, or 250-fold molar excess), indicated by the triangle above the lane. In addition BeWo cell nuclear extracts were able to interact with the o110 oligonucleotide in a specific fashion.

 
Experiments were designed to identify the nucleotides interacting with the oBNC and BeWo cell nuclear protein. Two base pair transversions across the o110 oligonucleotide were generated and are described in Fig. 4AGo. These mutant oligonucleotides were used in binding reactions as competitors (100-fold molar excess) and their ability to reduce radiolabeled complex formation was analyzed by EMSA in oBNC and BeWo nuclear extracts (Fig. 4BGo). The intensity of each complex with the unlabeled mutated oligonucleotide was expressed as percent binding, relative to the complex formation containing no competitor. The mean percent binding (n >= 4) was statistically compared with the wild-type o110 competitor for oBNC and BeWo nuclear extracts (Fig. 4CGo). Transversions at 110{psi} 4 (CC-> GG) and 110{psi} 6 (CA-> GT) did not compete with the radiolabeled o110 complex formation with oBNC nuclear proteins (Fig. 4BGo). The competition EMSA studies with BeWo cell nuclear extracts demonstrated that 110{psi} 4 and 110{psi} 6 were unable to inhibit complex formation, as well as 110{psi} 5 (AG-> TC; Fig. 4CGo). Therefore, the nucleotides, 5'-CCAGCA-3', located at -110/-105 bp were identified as the putative cis-acting element, which interacts with the nuclear protein in oBNC and BeWo cells.



View larger version (63K):
[in this window]
[in a new window]
  		Fig. 4. EMSA Competition Analysis with Mutant o110 Oligonucleotides

EMSA binding reactions with the o110 radiolabeled oligonucleotide were incubated in the presence of o110 mutant oligonucleotides, which had 2-bp transversions. A, Wild-type sequence of the o110 oligonucleotide and the 2-bp transversions created in the mutant oligonucleotides as labeled. These mutated o110 oligonucleotides were used as unlabeled competitors at 100-fold molar excess in EMSA binding reactions with the radiolabeled wild-type o110 oligonucleotide. Additionally, no oligonucleotide competitor and unlabeled wild-type o110 oligonucleotide at 100-fold molar excess were included in separate binding reactions. B, Representative competitive EMSAs for oBNC and BeWo cell nuclear extracts are shown. The unlabeled competitors that were added to the binding reactions are indicated above their lanes. C, Composite bar graph (n = 4 repetitions) for the competitive EMSA analysis using BeWo cell nuclear extracts is shown in panel B. The binding percentage was calculated by dividing volumetric intensities of the complex formed in lanes containing unlabeled competitor oligonucleotides by the complex intensity from binding reactions with no oligonucleotide competitor. The bar graph summarizes mean percent binding ± SEM for each competitor labeled on the ordinate axis. A Dunnett’s t test was used to separate the mean percent binding of the mutant competitors from the wild-type competitor, and significance was accepted at P < 0.05 (*). The competitive EMSAs identify nucleotides CCAGCA as the cis-acting element interacting with the nuclear protein.

 
The nucleotides identified within the oPL minimal promoter for the protein-DNA interaction appeared to describe a novel cis-acting element, as no similarity to previously defined cis-acting elements was found. However, an identical 6-bp region was identified in the hPL genes at -147 bp relative to the transcriptional start site. An oligonucleotide encompassing -150/-136 bp (h110) of the hPL-4 gene, containing this cis-acting element was synthesized. No similarities between o110 and h110 oligonucleotides were observed outside of the six identical nucleotides (5'-CCAGCA-3'). Radiolabeled h110 in an EMSA identified a specific protein-DNA interaction with both BeWo and oBNC nuclear extracts, when homologous and heterologous oligonucleotide competitors were included in the binding reactions (Fig. 5Go). Furthermore, the formation of the complex was inhibited with the addition of o110, confirming the interaction was specific for CCAGCA (Fig. 5Go).



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 5. EMSA of h110 in BeWo and oBNC Nuclear Extracts

An oligonucleotide encompassing -150/-136 bp (h110) of the hPL gene contains a CCAGCA sequence (-147 bp) and was tested in EMSA to confirm a specific interaction to the cis-acting element identified in the hPL minimal promoter. The protein-DNA complex with radiolabeled h110 was tested with BeWo cell nuclear extracts (labeled above) and homologous oligonucleotide competitors (h110 and o110) at 50, 100, nd 250-fold molar excess (indicated by triangles) reduced complex formation. However, no change in complex formation was observed with heterologous unlabeled competitors (oAP2 and oGATA) at identical concentrations. Radiolabeled h110 formed a specific complex with oBNC nuclear extracts, which had a similar mobility in comparison to the BeWo protein-DNA complex.

 
The identification and confirmation of this novel cis-acting element in both the oPL and hPL genes prompted studies to identify the transacting factor. Southwestern analysis was used to determine the apparent molecular weight of the transacting factor. A concatamer (3x) of the o110 sequence was used to identify the transacting factor in BeWo cell nuclear extracts, and specificity of the protein-DNA interaction was confirmed with the addition of 50-fold molar excess unlabeled homologous competitor. Southwestern analysis indicates that the o110 concatamer interacts with a nuclear protein that migrates with an apparent Mr of approximately 41,000 (Fig. 6Go).



View larger version (38K):
[in this window]
[in a new window]
  		Fig. 6. Southwestern Analysis of the Nuclear Protein Interacting with o110

Southwestern analysis was performed on BeWo nuclear proteins to identify the apparent molecular weight of the protein interacting with the novel cis-acting element identified in the oPL minimal promoter. Molecular weight standards are indicated to the left of the membranes that had nuclear proteins separated on a 12.5% SDS-PAGE gel transferred to them. A concatamer containing the triplicate o110 binding sites was radiolabeled (-) and used to identify the nuclear protein. In the second lane a 50-fold molar excess (+) of unlabeled concatamer was added to the binding reaction. A specific protein interaction with an apparent Mr of approximately 41,000 was found.

 
The ability to detect the protein by Southwestern analysis allowed the screening of an ovine placental expression library (7) with the o110 concatamer to identify a cDNA encoding the transacting factor. After screening, 1 x 106 plaques of the ovine placental ZAP Express library with both homologous and heterologous probes, 11 positive clones were purified to homogeneity. The cDNA inserts within these clones were radiolabeled and used in Southern hybridizations to verify homology between the isolated cDNAs. Six of 11 positive clones cross-hybridized with one another indicating nucleotide sequence similarity. These cDNA clones were sequenced and found to share identity with one another, as well as with human and mouse Pur{alpha}, a single-stranded (ss) DNA binding protein. Both the sense and antisense strands of clone 925 and 1114 were entirely sequenced and found to contain identical cDNAs (GenBank accession no. AF282406). The ovine Pur{alpha} cDNA insert within clone 925 was 1113 bp, with 15 bp of 5' untranslated region and 130 bp of 3' untranslated region. The open-reading frame encoded 322 amino acids (966 bp) and possessed a preferred translation start site at 16 bp and a polyadenylation signal at 1078 bp. The ovine Pur{alpha} cDNA nucleotide sequence is 97% and 95% identical with human (14) and mouse Pur{alpha} (15) cDNAs, respectively. At the amino acid level, ovine and human Pur{alpha} share an identity of 99%, and only three amino acid variations were found between human and ovine Pur{alpha}: Ser to Gly30, Ser to Gly43, and Gly to Ser42.

EMSAs with oBNC and BeWo nuclear extracts were analyzed in the presence of both double-stranded (ds) and ss unlabeled competitors to determine whether Pur{alpha} interacts with a ss or ds element within the oPL minimal promoter. Both ds and ss competitors influenced the protein-DNA complex formation with the radiolabeled ds o110. The o110 sense strand exhibited a competition equal to the ds o110 unlabeled competitor, whereas the antisense strand exhibited less effect on complex formation (Fig. 7Go). In these binding reactions, only the antisense strand was radiolabeled, suggesting that the protein-DNA complex is associated with ds DNA, because the interaction appears to interact specifically with the sense strand oligonucleotide. These data indicate that the ss DNA binding protein, Pur{alpha}, will interact with ds DNA. Antiserum raised against mouse Pur{alpha} (16) was added to the EMSA binding reaction after the 55 C heat treatment, and a supershift was identified for both oBNC (Fig. 7Go) and BeWo cell (not shown) nuclear extracts but was not identified with the addition of nonimmunized serum. The cis-acting element identified in the oPL minimal promoter was not similar to the putative Pur{alpha} element (GGN)n. Therefore, an oligonucleotide containing the putative Pur{alpha} element (MF0677; Ref. 14) was synthesized and tested by EMSA under the same conditions. The EMSA with BeWo cell (Fig. 8Go) and oBNC nuclear extracts indicated that the ss oligonucleotide, MF0677, specifically interacted with human and ovine Pur{alpha} because unlabeled homologous oligonucleotides reduced complex formation, whereas heterologous competitors had no effect. The addition of Pur{alpha} antiserum further impeded complex mobility. Furthermore, the addition of ds o110 inhibited MF0677 complex formation, confirming Pur{alpha} ’s ability to interact with CCAGCA.



View larger version (48K):
[in this window]
[in a new window]
  		Fig. 7. Ovine Pur{alpha}-o110 Interaction

Binding studies with oBNC nuclear extracts in the presence of ss oligonucleotides or antiserum raised against mouse Pur{alpha} were performed to confirm the ovine Pur{alpha}-o110 interaction. The competitors (ds or ss) at 100-fold molar excess or rabbit serum (antimouse Pur{alpha} or normal rabbit serum; NRS) added to the binding reactions are indicated above the lane. The arrow on the left identifies the protein-DNA complex, and the arrow on the right identifies the supershift complex with the antimouse Pur{alpha}.

 


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 8. EMSA and Supershift with Putative Pur{alpha} Element

The MF0677 ss oligonucleotide was radiolabeled and tested in EMSA for complex formation with BeWo cell nuclear extracts (5 µg). In the first lane, no oligonucleotide competitor was added to the binding reactions, whereas unlabeled homologous (MF0677), heterologous (o99 sense strand) or ds o110 was incubated in the binding reactions, respectively. Additionally rabbit antimouse Pur{alpha} was added to a binding reaction to confirm a Pur{alpha}-MF0677 interaction. These data confirm that the Pur{alpha}-MF0677 interaction can be reduced with the novel cis-acting element identified in the oPL minimal promoter o110.

 
An expression vector containing the ovine Pur{alpha} cDNA was cotransfected into the choriocarcinoma cell lines with the reporter vector, -124pGL3, to assess its ability to stimulate transactivation. Cotransfections with 2.5 µg of Pur{alpha} clone 925 significantly enhanced activity of the oPL minimal promoter in BeWo cells, whereas a control vector (pBlueScript) did not augment transactivation (Fig. 9Go). A similar enhancement of -124pGL3 transactivation was obtained with 0.5 µg and 2.5 µg of Pur{alpha} expression vector in Rcho-1 cells, which previously appeared to lack Pur{alpha}. Furthermore, the Pur{alpha} enhancement of functional promoter activity in BeWo or Rcho-1 transfections was ablated when the cis-acting element (CCAGCA) was mutated (-124{Delta}110pGL3) (Fig. 9Go). These data indicate that ovine Pur{alpha} was able to stimulate transactivation of the oPL minimal promoter in human and rat choriocarcinoma cell lines by interacting with a novel cis-acting element, CCAGCA.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 9. Functionality of a Pur{alpha} Expression Vector in BeWo Cells

The choriocarcinoma cell lines, BeWo (A) and Rcho-1 (B), were cotransfected with the oPur{alpha} expression vector or a control vector DNA, and either a reporter vector -124pGL3 with the novel cis-acting element or the vector with the mutated site (-124{Delta}110pGL3) to analyze the affect of oPur{alpha}. The bars represent the mean normalized activity ± SEM, and significance was accepted at P < 0.05 (*). The oPur{alpha} expression vector was able to augment oPL minimal promoter expression in both placental cell lines (P < 0.05). A greater amount of expression plasmid was required for BeWo cells (2.5 µg) than Rcho-1 cells (0.5 µg), but similar augmentation in Rcho-1 cells was observed with 2.5 µg of expression vector. Additionally, mutation of the cis-acting element did not allow the oPur{alpha} expression vector to significantly enhance activity in the placental cell lines.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Mutational analysis of FP3 identified a novel cis-acting element (-109/-102 bp) distinct from the previously identified GATA element (-102 bp) within the oPL minimal promoter. A specific protein-DNA interaction was identified by EMSA, and mutant competitors used in binding reactions refined the interaction to CCAGCA (-110/-105 bp). This cis-elements interaction with human (BeWo) and ovine (oBNC) nuclear protein was confirmed with an identical 6-bp element that resides in the promoter sequence of the hPL genes. Expression screening of an ovine placental cDNA library identified Pur{alpha}, a ss DNA binding factor, as the nuclear protein interacting with this cis-acting element. Subsequent binding studies with antiserum raised against mouse Pur{alpha} confirmed the Pur{alpha}:CCAGCA interaction, and represents a new Pur{alpha} cis-acting element.

The PUR element, (GGN)n, was defined as a purine-rich, ss element in the human c-myc gene and hamster dhfr gene at zones of initiation for DNA replication (14). A Pur{alpha} clone encoding a 322-amino acid protein was found to contain three consensus repeat motifs of 23 amino acids (class I) separated by two repeats of a consensus motif of 26 amino acids (class II) (17). The class I repeats contain three conserved aromatic phenylalanine or tyrosine residues, which were proposed to intercalate ss DNA, and possess several basic residues that may interact with DNA (17). Amino- and carboxy-terminal truncation of Pur{alpha} revealed that each of the class I motifs interact with DNA (18). Furthermore, the class II motifs, which are leucine rich, have been found to provide protein-protein interactions with Pur{alpha}, allowing formation of homodimers, as well as with other proteins to form heterodimers (19, 20, 21). Pur{alpha} contains a glutamine/glutamate-rich (Gln282 to Glu322) region at the carboxyl terminus, and these acidic residues have been categorized as activation domains in other transcription factors (22, 23, 24). A psycho domain between residues Pro251 and Met278 interacts with the retinoblastoma protein (25), and this motif was identified in several other viral (simian virus 40 T antigen and the human papilloma virus E7 protein) and mammalian proteins (Erg1 and Erg2, of the Ets family) (15). The final motif identified in this protein includes a glycine-rich region at the amino terminus of Pur{alpha}, which has been identified in helix destabilizing proteins and RNA-binding proteins (26, 27, 28, 29). However, the helix destabilizing properties of Pur{alpha} has been shown to require amino acid residues 72–274, which does not include the glycine-rich region but the DNA binding domain (30). The identification of these regions suggested that Pur{alpha}, originally described as an initiator of replication, may possess many different functions.

One important function of Pur{alpha} was its ability to stimulate transcription by interacting with a specific DNA element in eukaryotic cells. Transactivation by Pur{alpha} has been shown in JC virus early and late promoters (19), myelin basic protein (31, 32), neuron-specific Fe65 promoter (33), HIV-1 long terminal repeat (21), neuropeptide Y (34), the avian clusterin gene (35), Schistosoma mansoni p14 (eggshell precursor) gene (36), and now with oPL minimal promoter. Additionally, Pur{alpha} enhances transcription of a neural gene, BC1, which is transcribed by RNA polymerase III (37). Transcriptional enhancement for both RNA polymerase II and III products requires cis-acting elements located within the gene’s 5'-flanking sequences.

Another action of Pur{alpha} was its ability to form protein-protein interactions with other transcription factors or intercellular signaling factors to promote transactivation. Associated proteins of Pur{alpha} appear to add an additional hierarchy of enhancement to the transactivator, by stimulating DNA interaction or facilitating transcriptional stimulation. Subsequent studies have identified or purified activator proteins associated with Pur{alpha} transcriptional enhancement (19, 34, 38, 39). One of the activator proteins purified was calmodulin, which may indicate that intracellular signaling mechanisms are able to mediate Pur{alpha} transactivation (34). Pur{alpha} has been found to associate with phosphorylated Sp1 during myelination of the mouse brain, synergistically stimulating transactivation (39). However, the effects of Pur{alpha} were not always stimulatory, because retinoblastoma protein (25) and E2F-1 (40) association negatively affected transcription either by inhibiting Pur{alpha}-DNA interactions or E2F-1-DNA interactions.

The previously defined cis-acting element (GGN)n, which interacts with Pur{alpha} has very little identity to the cis-acting element described in the oPL gene. Binding studies suggest that Pur{alpha} interacts with a ds sequence of CCAGCA (Fig. 4Go), but addition of the o110 sense strand competed for binding (Fig. 7Go). Additionally, the ds o110 oligonucleotide was able to displace binding of the previously defined PUR element in the MF0677 oligonucleotide (Fig. 8Go). However, the binding affinities of these interactions may vary. Protein-DNA interaction with o110 required 25 µg of BeWo or oBNC nuclear extracts, whereas the binding studies with radiolabeled MF0677 were performed with 5 µg of nuclear extract. Therefore, Pur{alpha} may have a lower affinity for the CCAGCA binding site requiring a greater concentration of Pur{alpha} to distinguish the interaction. Different affinities for the two elements seems plausible, in light of another low-affinity interaction proposed for Pur{alpha} found when a ds cAMP response element (TGACGTCA) was used as a competitor (34, 41). Moreover, the preferred binding site of Schistosoma mansoni Pur{alpha} was a sequence of alternating pyrinidines (36), further indicating that Pur{alpha} may in fact interact with several DNA sequences to initiate transactivation. However, differences in Pur{alpha} sequence affinity could also result from posttranslational modifications of Pur{alpha}, and might account for the doublet observed in Southwestern analysis (Fig. 6Go).

Another interesting finding was the location of this element in the hPL and GH genes. The proximal 500 bp of promoter region for these human genes are described to share greater than 90% identity (42). Additionally, the location of this element in the human genes (-147 bp) was closely related to its location in the oPL gene (-110 bp), in respect to the transcriptional start site. Fitzpatrick et al. (43) analyzed the promoter region of the hPL genes and indicated that a region between -152/-142 bp contributed to the transactivation of the hPL gene. A subsequent deletion from -142/-129 bp had a much more dramatic effect on transactivation, and was suggested to contain a noncanonical Sp1 site (43). The functionality of the hPL promoter elements in human trophoblast cells was postulated to provide a basal region to nucleate the preinitiation complex because elements that reside about 2.2 kb 3' of the hPL/hGH gene locus stimulate trophoblast-specific transactivation (44, 45, 46, 47). Therefore, general transcription factors such as Sp1 at -137 bp has been shown to play a critical role in enhancing hPL gene transcription by recruiting the TATA-binding protein. A Pur{alpha}-Sp1 interaction has been shown to synergistically stimulate expression of the myelin basic protein gene (39), and addition of Sp1 to purified Pur{alpha} increased Pur{alpha} binding to DNA. Furthermore, Pur{alpha} and Sp1 have been shown to interact in the absence of DNA, which may indicate that a heterodimer may form at either of these sites to augment transactivation.

Mutation of the Pur{alpha} element in the oPL minimal promoter significantly reduces transactivation in BeWo cells, and overexpression of Pur{alpha} in BeWo or Rcho-1 cells augments the expression of a reporter vector containing the -109/-102-bp region (Figs. 1Go and 9Go). Therefore, Pur{alpha} ’s function is mediated through a protein-DNA interaction at CCAGCA, but binding of Pur{alpha} to this element appears to be at a lower affinity than the previously identified site. The data of Sp1 stimulating a Pur{alpha}-DNA interaction to the myelin basic protein gene (39) may indicate that other transacting factors facilitate Pur{alpha}-DNA interactions. Calmodulin has been shown to enhance Pur{alpha}-DNA interaction to a ss cAMP response element, as well as the myelin basic protein (MB1) site in the myelin basic protein (34). Other transacting factors that interact with Pur{alpha} include T-antigens from polyomaviruses JC virus (48); E2F-1 (40); and Y box-binding protein-1 (YB-1) (19). Possible Pur{alpha} interactions with GATA-2 and AP-2{alpha}, which augment oPL minimal promoter activity in BeWo cells (6, 7), may provide other factors that interact with Pur{alpha}. The proximity of the GATA element (-102/-99) and Pur{alpha} element (-110/-105) in FP3 may imply that these two factors cooperate to augment transcription, but no interactions with these transacting factors have been investigated.

In conclusion we have identified a novel cis-acting element, CCAGCA that was required for transactivation of the oPL minimal promoter in human choriocarcinoma cells, but not in rat choriocarcinoma cells. The transacting factor associated with this element, as well as an identical element in the hPL genes, was found to be ovine Pur{alpha}. The recent report that Pur{alpha}(-/-) mice possess a normal phenotype at birth (49) after an uncomplicated pregnancy, may further suggest that Pur{alpha} is active as a transcription factor in human and ovine placenta, but not in rodent placenta. Although the protein-DNA interaction at this cis-acting element exhibited a lower affinity for Pur{alpha} than a previously described cis-acting element, the interaction was specific. Ovine Pur{alpha} proved to enhance transactivation of the oPL gene minimal promoter in placental cells, thus confirming its role as a transactivator for the oPL gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals
Mature cross-bred ewes were mated at behavioral estrus (d 0) and hysterectomized by midventral laparotomy after administration of pentobarbital and exsanguinations at 100 d post coitus. Animal care was provided by Colorado State University Laboratory Animal Resources, under an Institutional Animal Care and Use Committee approved protocol (99-024A).

Plasmid Construction and Purification
Mutations in the minimal promoter of the oPL gene (-124/+16 bp) were generated by site directed mutangenesis, as previously described by this laboratory (6, 7). Overlapping synthetic oligonucleotides used for PCR are: {Delta}90F, 5'-AGG CGG CCG CAA AAG AGA AGC AGT GAT AGC-3'; {Delta}90R 5'-TTT GCG GCC GCC TTC TCT CTT ATC AAT GCT-3' {Delta}100F, 5'-ATG CGG CCG CAG AAT GCG GTA AAA GAG AAG-3'; {Delta}100R, 5'-TCT GCG GCC GCA TCA ATG CTG GTG CCT-3'; {Delta}110F, 5'-ACG CGG CCG CAT AAG AGA GAA GAA TGC GGT-3'; and {Delta}110R, 5'-TAT GCG GCC GCG TGC CTG CAT AAG AGC-3' (Macromolecular Resources, Colorado State University, Fort Collins, CO). The mutated minimal promoter products were digested with KpnI and HindIII restriction endonucleases (New England Biolabs, Inc., Beverly, MA), ligated into the pGL3 Basic vector (Promega, Madison, WI) and confirmed by dideoxy-nucleotide sequencing (50). Positive plasmid DNAs were prepared for each construct using an alkaline-lysis procedure (51), and purified by cesium chloride equilibrium gradient centrifugation.

Cell Culture and Transient Transfection
The BeWo cell line, a human choriocarcinoma cell line, was obtained from American Type Culture Collection (Manassas, VA). BeWo cells were maintained in Waymouth medium MB (GIBCO Invitrogen Corp., Carlsbad, CA) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma, St. Louis, MO) and 15% heat-inactivated fetal bovine serum (Gemini Bio-Products, Inc., Calabasas, CA). The Rcho-1 cell line was a gift from Dr. M. J. Soares (University of Kansas, Kansas City, KS), and cultured in NCTC-135 (Sigma) supplemented with 20% fetal bovine serum, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained in monolayer cultures at 37 C in 5% CO2, 95% air with 100% humidity. Transient transfections were performed and harvested as previously described by Liang et al. (6). Luciferase activity was normalized for intraassay variation by ß-galactosidase activity (RSVß gal vector) for each sample. Data are presented as the mean percent activity ± SEM determined from replicated transient transfection experiments (n >= 4). The transfection data were analyzed by least square analysis of variance using Statistical Analysis Systems (52). In general linear model procedures (53), differences in the means between wild-type vector and mutant vector were separated with a Dunnett’s t test (P < 0.05).

Nuclear Protein Isolation and EMSA
Cotyledonary tissue was collected from pregnant sheep at 100 d of gestation, and the binucleate cells were isolated in the same manner as described previously (6, 7). BeWo cells were expanded in culture and used for nuclear extract isolation (18). The nuclear proteins obtained were precipitated with ammonium sulfate (one third to two thirds fraction), and dialyzed (10,000 molecular weight cutoff) against 500 times the volume of Dignam D buffer [20 mM HEPES (pH 7.9) at 4 C, 20% glycerol (vol/vol), 100 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.25 mM dithiothreitol] (54). Nuclear protein concentrations were assessed, snap frozen and stored at -70 C. EMSAs were performed as described in Liang et al. (6) with synthetic oligonucleotides (o110, 5'-CAG GCA CCA GCA TTG-3'; h110, ATC CCA GCA TGT GTG-3'; oAP2, GCT CCA CCC CAG GGC ATG-3'; o99, 5'-AAG AGA GAA GAA TGC GGT A-3'; and oGATA, 5'-CTT CCT GAT AAA ACC ACT GG-3'; sense strands represented). Briefly, binding reactions with 20 µg of nuclear proteins contained 20 mM HEPES (pH 7.9), 20% glycerol 100 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.25 mM dithiothreitol, 25 µg/ml BSA, 1 mM spermidine, 1 µg poly(deoxyinosine-deoxycytosine), and 5 fmol of radiolabeled oligonucleotide. To examine heat stability of the protein-DNA interaction, the binding reactions were exposed to various temperatures (45, 55, 65, or 75 C) before adding the radiolabeled oligonucleotide. In addition, synthetic oligonucleotides possessing 2 bp transversions across o110 (-116 to -102) were generated and used as competitor oligonucleotides in binding reactions.

Southwestern Analysis
SDS-PAGE (12.5%) was used to separate 100 µg of BeWo nuclear extract protein (51, 55). The size-fractionated proteins were transferred to a 0.22 µm nitrocellulose membrane (Micron Separation, Inc., Westborough, MA) and renatured in 10 mM HEPES (pH 7.9), 50 mM KCl, 10% glycerol, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, and 0.5 mM spermidine for 16 h (4 C). The membrane was blocked for 3 h at 4 C with 20 mM HEPES (pH 7.9), 100 mM KCl, 10% glycerol, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 1 mM spermidine, 5 µg/ml sonicated herring sperm DNA, 25 µg/ml yeast tRNA, 100 µg/ml BSA, and 5% nonfat dry milk. The binding reaction contained 20 mM HEPES (pH 7.9), 100 mM KCl, 10% glycerol, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 1 mM spermidine, 5 µg/ml sonicated herring sperm DNA, 2.5 µg/ml yeast tRNA, 10 µg/ml BSA, 0.5% nonfat dry milk, and 1 x 106 cpm/ml radiolabeled ds oligonucleotide (Concat o110, 5'-GATCT TAATG CTGGT GAATG CTGGT GAATG CTGGT GGTAC-3'), which was end-labeled with T4 polynucleotide kinase and [{gamma}-32P] ATP. Binding reactions were carried out for 2 h (16 C). After the binding reaction, the membranes were washed in ice cold 10 mM HEPES (pH 7.9), 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, and 1 mM spermidine three times for 2 min and exposed to x-ray film. Specificity of this interaction was assessed by competition with 50-fold molar excess of unlabeled Concat o110.

Expression Library and Screening
An ovine cotyledonary cDNA library (6.9 x 107 pfu/µg) constructed in ZAP Express EcoRI/XhoI vector (Stratagene, La Jolla, CA), previously described by this laboratory (7), was screened for cDNAs encoding transcription factors with Concat o110. Bacteriophage infection, plating, and protein expression was performed in accordance with Young and Davis (56), as modified by Vinson et al. (57). Briefly, plates were cultured for 3 h at 42 C, isopropyl ß-D-thiogalactopyranoside impregnated nitrocellulose filters (Micron Separation, Inc.) were overlayed and plates were incubated at 37 C for 6–8 h. Overlaying a second isopropyl ß-D-thiogalactopyranoside impregnated filter for an additional 2 h generated a duplicate filter. After the filters were removed, they were allowed to dry slightly on Whatman paper, submerged into ice-cold Dignam D buffer (54) containing 6 M guanidine hydrochloride and gently agitated at 4 C for 10 min to denature the proteins. The buffer was changed once and the process repeated. After the second incubation, the filters were washed twice for 5 min at 4 C with Dignam D buffer containing 3 M guanidine hydrochloride. This process was repeated with Dignam D buffer containing 1.5, 0.75, 0.375, and 0 M guanidine hydrochloride. After denaturation, the proteins on the filters were renatured, blocked and probed in the same manner described for Southwestern analysis. Positive plaques were purified to homogeneity. To confirm that the protein expressed specifically interacts with Concat o110, a radiolabeled heterologous oligonucleotide (oAP2; 6) was tested on duplicate filters at the quaternary screen. Single plaques were isolated, and the helper phage ExAssist (Stratagene) was used to in vivo excise the pBK-CMV plasmids. Restriction endonuclease digestion and Southern hybridization, using multiprime radiolabled cDNA inserts from the isolated plaques were used to confirm sequence homology between purified cDNAs. Six independent clones cross-hybridized to one another. The 5' and 3' ends of each of these cDNAs were sequenced (Davis Sequencing, Davis, CA) with T3 and T7 oligonucleotide primers and found to contain identical cDNA inserts. Additionally, internal synthetic oligonucleotides were designed (925 F1, 5'-CTA CAT GGA TCT CAA GGA G-3'; and 925 R1, 5'-CTA GTC GTC GAT GAG CTT G-3') to sequence the entire sense and antisense strands of the cDNA inserts.

Pur{alpha} Binding Studies
Binding reactions were performed after heat exposure as previously described, but radiolabeled MF0677 ss oligonucleotide (5'-GGA GGT GGT GGA GGG AGA GAA AAG-3'; 21) or o110 oligonucleotide were used. The MF0677 oligonucleotide possesses the preferred binding site for human Pur{alpha} (17). Additionally, only 5 µg of nuclear extracts were used in the presence of radiolabeled MF0677, and a heterologous competitor o99 (5'-AAG AGA GAA GAA TGC GGT A-3'; 6) was included to detect nonspecific interactions. Antiserum raised against mouse Pur{alpha} was obtained from Dr. N. Miki (Osaka University, Osaka, Japan) (16), and supershift assays with the MF0677 and o110 oligonucleotides were performed with the addition of 1 µl of the antiserum raised against mouse Pur{alpha}, or 1 µl of normal rabbit serum (negative), to the binding reaction containing BeWo cell or oBNC nuclear extracts.

Cotransfection of Pur{alpha} and Reporter Constructs
Functionality of ovine Pur{alpha} was tested in both choriocarcinoma cell lines by cotransfecting 5 µg reporter vector (124pGL3 or {Delta}110pGL3) with 0.5µg (Rcho-1) or 2.5 µg (BeWo) of ovine Pur{alpha} (clone 925 pBK-CMV vector) or a control vector (pBlueScript; Stratagene). Transfections were carried out in the same manner as with the mutation constructs, but only the intraassay control was used to normalize the luciferase activity. The data were analyzed by general linear model procedures (52) and Tukey’s (53) was used as a means separation test (P < 0.05).


   ACKNOWLEDGMENTS
 
We thank Dr. N. Miki (Osaka University, Osaka, Japan) for the generous gift of mouse Pur{alpha} antiserum.


   FOOTNOTES
 
This work was supported by the United States Department of Agriculture Grant 99-35203-7817 from the National Research Initiative. The ovine Pur{alpha} nucleotide sequence has been submitted to DDBJ/EMBL/GenBank under accession no. AF282406.

Present address for S.W.L.: Perinatal Research Center, Department of Pediatrics, University of Colorado Health Sciences Center, Aurora, Colorado 80010.

Abbreviations: AP, Activator protein; BeWo, human choriocarcinoma cells; BNC, chorionic binucleate cells; ds, double-stranded; FP, footprint; (GGN)n, putative Pur{alpha} element; hPL, human PL; o, ovine; PL, placental lactogen; Rcho-1, rat choriocarcinoma cells; ss, single-stranded.

Received for publication October 9, 2003. Accepted for publication November 17, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Talamantes F, Ogren L 1988 The placenta as an endocrine organ: polypeptides. In Knobil E, Neill J, eds. The physiology of reproduction. New York: Raven Press; 2093–2144
  2. Handwerger S 1991 Clinical counterpoints: the physiology of placental lactogen in human pregnancy. Endocr Rev 12:329–336[Abstract/Free Full Text]
  3. Anthony RV, Limesand SW, Fanning MD, Liang R 1998 Placental lactogens and growth hormone regulation and action. In: Bazer FW, ed. The endocrinology of pregnancy. Totowa, NJ: Humana Press Inc.; 461–490
  4. Anthony RV, Liang R, Kayl EP, Pratt SL 1995 The growth hormone/prolactin gene family in ruminants placenta. J Reprod Fertil 49:83–95
  5. Anthony RV, Pratt SL, Liang R, Holland MD 1995 Placental-fetal hormonal interactions: impact on fetal growth. J Anim Sci 73:1861–1871[Abstract]
  6. Liang R, Limesand SW, Anthony RV 1999 Structure and transcriptional regulation of the ovine placental lactogen gene. Eur J Biochem 265:883–895[Medline]
  7. Limesand SW, Anthony RV 2001 Novel activator protein-2 splice-variants function as transactivators of the ovine placental lactogen gene. Eur J Biochem 268:2390–2401[Medline]
  8. Steger DJ, Hecht JH, Mellon PL 1994 GATA-binding proteins regulate the human gonadotropin {alpha}-subunit gene in the placenta and pituitary gland. Mol Cell Biol 14:5592–5602[Abstract/Free Full Text]
  9. Shi D, Winston JH, Blackburn MR, Datta SK, Hanten G, Kellems RE 1997 Diverse genetic regulatory motifs required for murine adenosine deaminase gene expression in the placenta. J Biol Chem 272:2334–2341[Abstract/Free Full Text]
  10. Ng Y-K, George KM, Engel JD, Linzer DIH 1994 GATA factor activity is required for the trophoblast-specific transcriptional regulation of the mouse placental lactogen I gene. Development 120:3257–3266[Abstract]
  11. Shi D, Kellems RE 1998 Transcription factor AP-2 {gamma} regulates murine adenosine deaminase gene expression during placental development. J Biol Chem 273:27331–27338[Abstract/Free Full Text]
  12. Johnson W, Albanese C, Handwerger S, Williams T, Pestell RG, Jameson JL 1997 Regulation of the human chorionic gonadotropin {alpha}- and ß-subunit promoters by AP-2. J Biol Chem 272:15405–15412[Abstract/Free Full Text]
  13. Richardson BD, Langland RA, Bachurski CJ, Richards RG, Kessler CA, Cheng Y, Handwerger S 2000 Activator protein-2 regulates human placental lactogen gene expression. Mol Cell Endocrinol 160:183–192[CrossRef][Medline]
  14. Bergemann AD, Johnson EM 1992 The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication. Mol Cell Biol 12:1257–1265[Abstract/Free Full Text]
  15. Ma ZW, Bergemann AD, Johnson EM 1995 Conservation in human and mouse Pur {alpha} of a motif common to several proteins involved in initiation of DNA replication. Gene 149:311–314
  16. Osugi T, Ding Y, Tanaka H, Kuo CH, Do E, Irie Y, Miki N 1996 Involvement of a single-stranded DNA binding protein, ssCRE-BP/Pur {alpha}, in morphine dependence. FEBS Lett 391:11–16[CrossRef][Medline]
  17. Bergemann AD, Ma ZW, Johnson EM 1993 Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein. Mol Cell Biol 12:5673–5682
  18. Chen NN, Khalili K 1995 Transcriptional regulation of human JC polyomavirus promoters by cellular proteins YB-1 and Pur {alpha} in glial cells. J Virol 69:5843–5848[Abstract]
  19. Safak M, Gallia GL, Khalili K 1999 Reciprocal interaction between two cellular proteins, Pur{alpha} and YB-1, modulates transcriptional activity of JCVCY in glial cells. Mol Cell Biol 19:2712–2723[Abstract/Free Full Text]
  20. Gallia GL, Darbinian N, Johnson EM, Khalili K 1999 Self-association of Pur {alpha} is mediated by RNA. J Cell Biochem 74:334–348[CrossRef][Medline]
  21. Chepenik LG, Tretiakova AP, Krachmarov CP, Johnson EM, Khalili K 1998 The single-stranded DNA binding protein, Pur-{alpha}, binds HIV-1 TAR RNA and activates HIV-1 transcription. Gene 210:37–44[CrossRef][Medline]
  22. Courey AJ, Holtzman DA, Jackson SP, Tjian R 1990 Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell 59:827–836
  23. Darnell JJ 1982 Variety in the level of gene control in eukaryotic cells. Nature 297:365–371[CrossRef][Medline]
  24. Ptashne M 1989 How eukaryotic transcriptional activators work. Nature 335:683–689
  25. Johnson EM, Chen PL, Krachmarov CP, Barr SM, Kanovsky M, Ma ZW, Lee WH 1996 Association of human Pur {alpha} with the retinoblastoma protein, Rb, regulates binding to the single-stranded DNA Pur {alpha} recognition element. J Biol Chem 270:24352–24360
  26. Haynes SR, Rebbert ML, Mozer BA, Forquignon F, Dawid IB 1987 pen repeat sequences are GGN clusters and encode a glycine-rich domain in a Drosophila cDNA homologous to the rat helix destabilizing protein. Proc Natl Acad Sci USA 84:1819–1823[Abstract/Free Full Text]
  27. Haynes SR, Raychaudhuri G, Beyer AL 1990 The Drosophila Hrb98DE locus encodes four protein isoforms homologous to the A1 protein of mammalian heterogeneous nuclear ribonucleoprotein complexes. Mol Cell Biol 10:316–323[Abstract/Free Full Text]
  28. Kelm RJ, Elder PK, Getz MJ 1999 The single-stranded DNA-binding proteins, Pur {alpha}, Pur ß, and MSY1 specifically interact with an exon 3-derived mouse vascular smooth muscle {alpha}-actin messenger RNA sequence. J Biol Chem 274:38268–38275[Abstract/Free Full Text]
  29. Herault Y, Chatelain G, Brun G, Michel D 1995 RNA-dependent DNA binding activity of the Pur factor, potentially involved in DNA replication and gene transcription. Gene Expr 4:85–93[Medline]
  30. Darbinian, N, Gallia GL, Khalili K 2001 Helix-destabilizing properties of the human single-stranded DNA- and RNA-binding protein Pur {alpha}. J Cell Biochem 80:589–595[CrossRef][Medline]
  31. Morasso MI, Grinberg A, Robinson G, Sargent TD, Mahon KA 1999 Placental failure in mice lacking the homeobox gene Dlx3. Proc Natl Acad Sci USA 96:162–167[Abstract/Free Full Text]
  32. Haas S, Thatikunta P, Steplewski A, Johnson EM, Khalili K, Amini S 1995 A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells J Cell Biol 130:1171–1179[Abstract/Free Full Text]
  33. Zambrano N, De RS, Minopoli G, Faraonio R, Donini V, Scaloni A, Cimino F, Russo T 1998 DNA-binding protein Pur {alpha} and transcription factor YY1 function as transcription activators of the neuron-specific FE65 gene promoter Biochem J 328:293–300
  34. Kuo CH, Nishikawa E, Ichikawa H, Sadakata T, Niu SY, Miki N 1999 Calmodulin functions as an activator of Pur {alpha} binding to single-stranded purine-rich DNA elements (PUR elements). Biochem Biophys Res Commun 255:406–411[CrossRef][Medline]
  35. Herault Y, Chatelain G, Brun G, Michel D 1995 The PUR element stimulates transcription and is a target for single strand-specific binding factors conserved among vertebrate classes. Cell Mol Biol Res 39:717–725
  36. Fantappie M. R, Osman A, Niles EG, LoVerde PT 2000 Identification and functional characterization of a member of the PUR-{alpha} family from Schistosoma mansoni. Mol Biochem Parasitol 110:373–390[CrossRef][Medline]
  37. Kobayashi S, Agui K, Kamo S, Li Y, Anzai K 2000 Neural BC1 RNA associates with pur {alpha}, a single-stranded DNA and RNA binding protein, which is involved in the transcription of the BC1 RNA gene. Biochem Biophys Res Commun 277:341–347[CrossRef][Medline]
  38. Ding Y, Osugi T, Kuo CH, Tanaka H, Do E, Irie Y, Miki N 1997 Characterization of a nuclear factor that enhances DNA binding activity of SSCRE-BP/PUR {alpha}, a single-stranded DNA binding protein. Neurochem Int 31:45–54[CrossRef][Medline]
  39. Tretiakova A, Steplewski A, Johnson EM, Khalili K, Amini S 1999 Regulation of myelin basic protein gene transcription by Sp1 and Pur {alpha}: evidence for association of Sp1 and Pur {alpha} in brain. J Cell Physiol 181:160–168[CrossRef][Medline]
  40. Darbinian N, Gallia GL, Kundu M, Shcherbik N, Tretiakova A, Giordano A, Khalili K 2000 Association of Pur {alpha} and E2F-1 suppresses transcriptional activity of E2F-1. Oncogene 18:6398–6402
  41. Osugi T, Ikemoto M, Tanaka H, Wang, XB, Miki N 1994 Modulation by chronic morphine administration of single-stranded cAMP response element (ssCRE) binding proteins in the mouse cerebellum. Brain Res Mol Brain Res 21:256–262[Medline]
  42. Walker WH, Fitzpatrick SL, Barrera-Saldana HA, Resendez-Perez D, Saunders GF 1991 The human placental lactogen genes: structure, function, evolution and transcriptional regulation. Endocr Rev 12:316–328[Abstract/Free Full Text]
  43. Fitzpatrick SL, Walker WH, Saunders GF 1990 DNA sequence involved in the transcriptional activation of a human placental lactogen gene. Mol Endocrinol 4:1815–1826[Abstract/Free Full Text]
  44. Rogers BL, Sobnosky MG, Saunders GF 1986 Transcriptional enhancer within the human placental lactogen and growth hormone multigene cluster. Nucleic Acids Res 14:7647–7659[Abstract/Free Full Text]
  45. Lytras A, Cattini PA 1994 Human chorionic somatamammotropin gene enhancer activity is dependent on the blockade of a repressor mechanism. Mol Endocrinol 8:478–489[Abstract/Free Full Text]
  46. Jiang S-W, Eberhardt NL 1994 The human chorionic somatomammotropin gene enhancer is composed of multiple DNA elements that are homologous to several SV40 enhansons. J Biol Chem 269:10384–10392[Abstract/Free Full Text]
  47. Jacquemin P, Oury C, Peers B, Morin A, Belayew A, Martial JA 1994 Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen. Mol Cell Endocrinol 14:93–103
  48. Gallia GL, Safak M, Khalili, K 1999 Interaction of the single-stranded DNA-binding protein Pur{alpha} with the human polyomavirus JC virus early protein T-antigen. J Biol Chem 273:32662–32669
  49. Khalili K, Del Valle L, Muralidharan V, Gault WJ, Darbinian N, Otte J, Meier E, Johnson EM, Daniel DC, Kinoshita Y, Amini S, Gordon J 2003 Pur{alpha} is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse. Mol Cell Biol 23:6857–6875[Abstract/Free Full Text]
  50. Sanger F, Niklen S, Coulson AR 1977 DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract/Free Full Text]
  51. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K 1995 Current protocols in molecular biology. New York: John Wiley and Sons, Inc.
  52. SAS Institute 1993 SAS/STAT user’s guide. 4th ed. Cary, NC: SAS Institute Inc.
  53. Snedecor GW, Cochran WC 1976 Statistical methods. Ames, IA: Iowa State University Press
  54. Dignam JD, Lebovitz RM, Reoder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract/Free Full Text]
  55. Warren WC, Liang R, Krivi GG, Siegel NR, Anthony RV 1990 Purification and structural characterization of ovine placental lactogen. J Endocrinol 126:141–149[Abstract/Free Full Text]
  56. Young RA, Davis RW 1983 Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci USA 80:1194–1198[Abstract/Free Full Text]
  57. Vinson CR, LaMarco KL, Johnson PF, Landschulz WH, McKnight SL 1988 In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev 2:801–806[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. M. Knapp, J. E. Ramsey, S.-X. Wang, K. E. Godburn, A. R. Strauch, and R. J. Kelm Jr.
Nucleoprotein Interactions Governing Cell Type-dependent Repression of the Mouse Smooth Muscle {alpha}-Actin Promoter by Single-stranded DNA-binding Proteins Pur{alpha} and Purbeta
J. Biol. Chem., March 24, 2006; 281(12): 7907 - 7918.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Limesand, S. W.
Right arrow Articles by Anthony, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Limesand, S. W.
Right arrow Articles by Anthony, R. V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals