Department of Cell Biology and Biochemistry (A.H., E.H., V.H.L., B.S.C.), Texas Tech University Health Sciences Center, Lubbock, Texas 79430; and Department of Cell Biology (M.M.), Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: Dr. Beverly S. Chilton, Department of Cell Biology and Biochemistry, Texas Tech University, Health Sciences Center, 3601 4th Street-Stop 6540, Lubbock, Texas 79430-6540. E-mail: beverly.chilton{at}ttmc.ttuhsc.edu.
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ABSTRACT |
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INTRODUCTION |
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Of the eight Stat family members, Stat5a and Stat5b are important to PRL signal transduction. Lessons from knockout models have taught us that deletion of Stat5a results in the loss of PRL-dependent mammary gland development (3), and deletion of Stat5a/b results in the failure of corpus luteum development (4). Because the Stat5a/b-deficient phenotype mimics the PRL receptor-deficient phenotype, Ihle (5) has tendered the opinion that all physiological functions mediated by PRL require Stat5a/b. However, PRL has more than 300 different biological activities, many of which are achieved in concert with steroid hormones (1). Moreover, very few authentic PRL target genes (ß-casein) have been identified.
RUSH, a member of the Smarca3 family of SWI/SNF-related chromatin remodeling machines, interacts with an inner nuclear membrane protein (6) and binds to the proximal promoter (-170/-85) of the uteroglobin gene (7). Deletion analysis showed this region is essential to PRL signal transduction (8, 9). However, our conclusions were limited by its size and complexity. In addition to a putative RUSH-binding site, the region is characterized by a 7-bp identical inverted repeat (CAGTTTC) that is found in a long terminal repeat (-171/-148) common to all murine leukemia viruses and proviruses (10), and by two hepatocyte nuclear factor 3 (HNF3)-response elements centered at -130 and -95 (11) that completely overlap two octamer transcription factor 1 (Oct-1)-binding sites. In contrast, the absence of Stat5a/b-binding elements is compelling evidence that the primary signaling mechanism is not via the Jak/Stat pathway. The rationale for defining the sequence-selective interactions of RUSH with DNA is supported by these findings.
Initially, the RUSH target search was reduced from -170/-85 to -160/-110 (12). Here we report the use of cyclic amplification and selection of targets (CASTing), a PCR-based immunodetection method that is useful for determining the DNA binding specificity of low-abundance proteins (13), to identify the RUSH-binding site. This method, which is powerful enough to identify a DNA-binding site that occurs only once in the mammalian genome (14), required crude nuclear extract as a source of binding factors and a synthetic oligonucleotide with a sufficient number of internal random bases (26-bp) to accommodate the preferential isolation of multicomponent sites (15) based exclusively on the presence of RUSH. Although the immediate goal was to define the DNA sequence recognized by RUSH, we also show there is a physical interaction between RUSH and GATA-4 that is not required to regulate uteroglobin gene expression in the rabbit uterus. The question of whether or not RUSH, a putative helicase, was transiently affiliated with DNA was eliminated because protein-protein interactions that alter transcriptional activity but do not affect DNA binding are not detected by CASTing (13). Ultimately, we used site-directed mutagenesis to show that the RUSH target sequence is used by PRL to augment progesterone-dependent transcriptional activation of the uteroglobin gene. The identification of an alternative to the Jak/Stat pathway has allowed us to consider a broader role for PRL in modulating steroid-dependent gene transcription.
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RESULTS AND DISCUSSION |
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The RUSH consensus-binding site was determined by CASTing (Table 1). A hexameric core sequence of C/ACT/ATNT/G (MCWTNK), which is at times palindromic (ACATGT), was identified in 38.5% of the sequences after five cycles of selection. Because the fraction of random sequences appeared to be significant, probably because RUSH is a low-abundance protein and because there was some nonspecific binding of the antibody, the number of CASTing cycles was extended to 20. Sequences were cloned and examined at regular intervals to show that 38.5% of a total of 96 sequences contained the consensus-binding site. Despite the inability to enrich beyond this level, the probability [P = 20 ÷ 128 (6!)/2] of such a match occurring in either orientation in sequences with 20 potential hexameric binding sites is 1 in 2304. This 1:2304 probability would occur less than once in 96 sequences, making the presence of this binding site in the examined sequences highly significant.
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Although CASTing with nuclear extract allowed for the identification of multicomponent interactions, it was also important to determine whether RUSH alone had the ability to specifically bind to the consensus sequence. The hexameric consensus sequence was present in 47% of the sequences after CASTing (five cycles) with recombinant RUSH. As a result of these additional experiments, the consensus shown in Table 2 was derived from the analysis of 59 sequences derived from five cycles of CASTing. However, the frequency of C in position 5 of the RUSH consensus was less than 10%. The fact that the frequency of occurrence was less than 25%, the frequency expected for a random base, was justification for the exclusion of this base from the consensus. Moreover, binding was negligible when sequences with a C in this position were tested in gel shift assays with nuclear extract proteins. Thus, the optimal consensus binding sequence is C/ACT/ATA/T/GT/G (MCWTDK).
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Six of 59 sequences, five from CASTing with nuclear extract and RUSH-1(ß) antibodies and one from CASTing with nuclear extract and the
-specific antibody, contained two or three RUSH-binding sites. The low frequency of expression of multiple binding sites, coupled with the fact that binding affinity was within the range for single sites, suggests that RUSH binding is noncooperative. Negative controls for the gel shift assays included oligonucleotides with flanking sequence but no RUSH-binding site, and oligonucleotides with flanking sequence plus three or four consecutive elements of the RUSH-binding site. Probe 13, a representative of group 3 (ACATWT; Table 1
) was used to further confirm the specificity of RUSH binding. The experiment depicted in Fig. 1
shows that RUSH-specific binding was eliminated when immunodepleted nuclear extract, as verified by Western analysis, was used as the protein source. RUSH-specific binding was eliminated by competition with a 100-fold molar excess of unlabeled probe 13, but not eliminated by competition with a negative control probe that had two partial RUSH sites (four of six bases at each site). The supershift assay with antibody to RUSH-1
(ß) confirmed RUSH-specific binding. Its diffuse appearance supports the suggestion that RUSH is a member of a multiprotein complex.
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Characterization of the RUSH Motif as a Subelement of HNF3 and Oct-1
As shown in Fig. 2A, the RUSH-binding site (-126/-121) is a subelement of the HNF3- and Oct-1-binding sites. Analysis with MatInspector V2.2 (Ref. 18 ; available at http://www.gsf.de/cgi-bin/matsearch2.pl) identified the consensus match for HNF3 as a putative binding site for HNF3ß. However, Braun and Suske (11) used transfection assays to show that both HNF3
and HNF3ß enhanced transcriptional activation of the rabbit uteroglobin promoter. Thus it was important to determine whether HNF3- or Oct-1-binding sites were preferentially isolated with RUSH-binding sites. If confirmed, these results would support the idea that nucleotides outside of the consensus sequence influence the relative binding of RUSH proteins, or that RUSH binding to the -136/-121 region of the promoter is affected by the presence of additional proteins such as HNF3 and/or Oct-1. Evidence that RUSH does not require HNF3 or Oct-1 binding is found in Table 1
. CASTing for multiprotein complexes from nuclear extract, a source of multiple factors, revealed a very low frequency of occurrence of the other binding sites with RUSH. One of 59 sequences had a putative HNF3ß-binding site, and 2 of 59 sequences had putative Oct-1-binding sites.
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RUSH-GATA Interaction
The possible involvement of GATA transcription factors in a DNA-binding complex with RUSH was suggested by CASTing experiments with nuclear extract from uterine endometrium. Table 1 shows the sequence of 39 clones obtained after CASTing with RUSH-1
(ß) antibodies. Thirteen of these sequences contained putative GATA-binding sites according to a search with MatInspector V2.2. Although these sites were not generally within a 3- to 4-bp constant distance (13) from the RUSH site, careful examination of the uteroglobin promoter with the same Genomatix Software revealed a GATA-1 site (-115/-128) on the negative strand in close proximity to the RUSH-binding site (Fig. 2A
). Despite the fact that GATA expression in the uterus has not been documented, three of the six GATA factors, GATA-1, GATA-4, and GATA-6, are expressed in mammalian gonads (for reviews see Refs. 19 and 20). Additional complications included the fact that the nucleotide sequence for mouse GATA-1 is 70% identical with mouse GATA-4 and 7780% identical with rabbit GATA-4. Therefore, the same RT-PCR strategy that was used to obtain the conserved zinc-finger DNA-binding domain of GATA-1 in the mouse testis (21) was used to obtain a 217-bp amplicon from endometrium. A computer search of sequence databases with the BLASTN 2.2.2 program at the National Center for Biotechnology Information revealed that 167 bp of sequence (GenBank accession no. AF467985) that was not forced by PCR priming was 83% identical with the partial coding sequence of GATA-4 (GenBank accession no. 8648976) from rabbit heart, which is 94% identical with the coding sequence of human GATA-4 (GenBank accession no. 508483).
Northern analysis with the 217-bp amplicon was used to evaluate mRNA expression in rabbit endometrium. Figure 3 shows two endometrial transcripts with molecular sizes of 3.2-kDa and 2.5-kDa were detectable in poly(A+) RNA from estrous animals. In a physiologically relevant comparison, steady state levels of both transcripts were increased 10-fold in progesterone-dominated, 5-d pseudopregnant rabbits. The hormone dependency of message expression was further confirmed when transcript levels were elevated 3-fold by the treatment of estrous rabbits with progesterone, and decreased 16-fold by the sequential treatment of estrous animals with progesterone and estradiol benzoate. In situ hybridization (Fig. 4
) showed the spatial expression of putative uterine GATA-4 compares favorably with the Northern analysis. Message was abundantly expressed in the endometrium of 5-d pseudopregnant rabbits compared with estrous rabbits.
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Fine mapping of the -160/-110 region of the uteroglobin promoter revealed that the AT-rich region -132/-123 was critical for transcriptional activation (12). It is now clear that this region (-136/-115) is a composite regulatory element (Fig. 2A) with overlapping binding sites for three proteins capable of initiating chromatin-opening events. Both HNF3 and GATA-4 can bind nucleosome arrays and open compacted chromatin in the absence of ATP-dependent enzymes (23). RUSH proteins are SWI/SNF-related ATPases (24, 25). This site appears uniquely designed to invite chromatin restructuring within the context of many different cell types.
The RUSH Motif Mediates the PRL Effect
To show that the RUSH-binding site mediates the action of PRL, control and hormone-dependent transcriptional activities were measured for the full-length uteroglobin gene, pUG3.1-LUC, and the RUSH mutant, pUG3.1M-LUC. HRE-H9 cells were pretreated with estradiol (10-7 M) and oPRL (10-8 M) for 48 h as previously described (8, 26). After transfection, cells were used as untreated controls, treated with progesterone (10-7 M) alone, or treated with oPRL (10-8 M) ± progesterone. As shown in Fig. 6, oPRL alone failed to stimulate transcription above control levels. Progesterone alone increased (P < 0.05) the transcriptional activity of pUG3.1-LUC. Transcription was further increased (P < 0.05) when cells were treated with oPRL + progesterone. Progesterone alone also increased (P < 0.05) the transcriptional activity of pUG3.1M-LUC. However, mutation of the RUSH-binding site, tested here with pUG3.1M-LUC, was highly deleterious to the PRL effect (Fig. 6
). Using this mutational approach, it was possible to eliminate (P > 0.05) the PRL-mediated increase in uteroglobin gene transcription while retaining a progesterone-dependent increase (P < 0.05) in transcription.
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MATERIALS AND METHODS |
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RUSH-1(ß) antipeptide antibodies (7, 9) recognize both
and ß because they were made to sequences (amino acids 370387) common to both proteins. For this study, antipeptide antibodies were also made to a unique region of RUSH-1
. The C-terminal portion of RUSH-1
consists of 173 amino acids with seven regions that display strong antigenicity as indicated by the PeptideStructure program (Genetics Computer Group, Madison, WI). As a result, antibodies to amino acids 932951 were generated in rabbits by staff at Research Genetics, Inc. (Huntsville, AL). The titer of each antibody was determined by ELISA with free MAP-peptide on the solid phase (1 µg/well), goat antirabbit IgG-horseradish peroxidase conjugate as the secondary antibody, and peroxidase dye. To eliminate complications from nonspecific binding, both antibody preparations were purified by affinity chromatography on columns of immobilized antigen. Antibody-RUSH binding was authenticated by Western analysis. For EMSA and coimmunoprecipitations, small aliquots of affinity-purified antibody (600 µl) were dialyzed against two changes x 2 liters each of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA at 4 C.
A 50:50 slurry (wt/vol) of Protein A-Sepharose was prepared by equilibration in Dignams (27) buffer D [20 mM HEPES (pH 7.9), 20% (vol/vol) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride, 0.5 mM dithiothreitol] with 0.1% Nonidet P-40. Modified ESB buffer (16) consists of 20 mM HEPES (pH 7.9), 40 mM NaCl, 6 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 0.1% Nonidet P-40, 10% glycerol, 2% Ficoll, BSA (3 mg/ml), and sonicated salmon sperm DNA (50 µg/ml).
Isolation of Nuclear Proteins
All studies with virgin, adult New Zealand white rabbits were conducted according to the NIH Guidelines for the Care and Use of Laboratory Animals, as reviewed and approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center. Optimal hormone treatments (28) included sc injections of progesterone (3 mg/kg·d), oPRL (2 mg/d), and estradiol benzoate (50 µg/animal·d). Animals were killed 24 h after the last injection.
For CASTing, EMSA, and coimmunoprecipitation, one rabbit received sc injections of progesterone for 5 d. Nuclear extract proteins were prepared from the endometrium as described by Kleis-SanFrancisco et al. (28). To reduce nonspecific binding during immunoprecipitation, small aliquots of nuclear extract (200 µl) were precleared by gentle mixing with protein A-Sepharose slurry (10 µl) for 2 h at room temperature. The mixture was centrifuged (200 x g) for 1 min and the supernatant was stored at 4 C.
For chromatin immunoprecipitation, one rabbit received sc injections of oPRL for 5 d followed by progesterone for 5 d. Nuclei were isolated from the endometrium as described by Kleis-SanFrancisco et al. (28).
For Northern analysis and/or in situ hybridization, six rabbits were divided into two groups. Group 1 animals (n = 3) were used as estrous controls, and group 2 animals (n = 3) were made pseudopregnant by iv injection of 1520 IU human chorionic gonadotropin and cervical stimulation. Group 2 rabbits were killed on d 5 of pseudopregnancy, and ovulation was confirmed by the presence of corpora lutea at necropsy. In addition, three rabbits were treated with progesterone for 5 d, and two rabbits were treated sequentially with progesterone (5 d) and estradiol benzoate (5 d).
Recombinant RUSH
Recombinant protein was obtained from the 15A cDNA (amino acids 40542) in the phagemid pBluescript II SK(+) by coupled transcription/translation (TNT lysate). The predicted 56-kDa recombinant protein was dialyzed against 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and substituted (0.61 µg/reaction) for nuclear extract in binding site selection assays.
Double-Stranded Oligonucleotide Preparation
The random sequence R76 (5'-CAG GTC AGT TCA GCG GAT CCT GTC G (N)26GAG GCG AAT TCA GTG CAA CTG CAG C-3') begins with 25 nucleotides that match the forward primer, followed by a stretch of 26 totally random bases, and ends with 25 nucleotides that match the reverse primer. This oligonucleotide was made double stranded by synthesis with the reverse primer (5'-GCT GCA GTT GCA CTG AAT TCG CCT C-3'), the Klenow fragment of DNA polymerase I, and [32P]dCTP. The final specific activity was 58 x 106 cpm/µg.
Binding Site Selection
For the initial reaction, 44.2 µg of precleared nuclear extract protein were mixed (tumbled) with 30 µg of dialyzed RUSH-1(ß) antibodies for 1 h at 4 C. Next, 20 µl of the protein A-Sepharose slurry were added. After gentle mixing (tumbling) for 1 h at 4 C, immune complexes were washed three times with 250 µl ice-cold Dignams (27) buffer D with acetylated BSA (1 mg/ml) and 0.1% Nonidet P-40. Immune complexes were then washed twice with 250 µl modified ESB. Double-stranded degenerate oligonucleotides were added (50 µl, 171 pmol DNA), and the mixture was incubated for 30 min at room temperature. Protein-DNA complexes were recovered by centrifugation and washed three times with modified ESB (250 µl each wash).
Amplification by PCR
The entire immunoprecipitated DNA sample was amplified in a 50 µl PCR mix containing ExTaq buffer (1x), TaKaRa ExTaq DNA polymerase (2.5U/50 µl), TaqStart antibody (0.55 µg/50 µl), deoxynucleoside triphosphates (dNTPs) (0.2 mM each), and primers (0.2 mM each). The forward primer (5'-CAG GTC AGT TCA GCG GAT CCT GTC G-3') had a unique BamHI site (bold) and the reverse primer (5'-GCT GCA GTT GCA CTG AAT TCG CCT C-3') had a unique EcoRI site (bold). A hot start PCR was performed with the following conditions: 30 sec at 94 C, followed by 15 cycles of 94 C, 5 sec; 62 C, 60 sec; and 72 C, 60 sec. Samples were rapidly cooled to 4 C. Reaction products were fractionated by electrophoresis on a 2% NuSieve GTG agarose gel. The 76-bp DNA products were extracted with QIAEX II according to the manufacturers instructions. Purified products were either subjected to additional rounds of binding site selection or cloned. For binding site selection, purified products were resuspended in 50 µl modified ESB buffer, and the entire sample was used in the reaction. For cloning, products were ligated to pCRII-TOPO and sequenced in both directions by the dideoxy chain termination method.
Alternative Approaches To Binding Site Selection
Because antibodies against a specific epitope can disrupt critical protein-protein or protein-DNA interactions, RUSH-1 antibodies were substituted for RUSH-1
(ß) antibodies. To determine whether RUSH requires multicomponent interactions, recombinant protein was substituted for nuclear extract. In the initial reactions with either of the described substitutions, the amount of DNA was reduced from 170 pmol to 34 pmol.
Partial Cloning of Rabbit Endometrial GATA-4
Poly(A+)RNA samples from rabbit endometrium and mouse testis (positive control) were reverse transcribed at 42 C for 25 min in a thermal cycler (Minicycler, MJ Research, Inc., Watertown, MA) with reagents from Applera Co. Maloney murine leukemia virus reverse transcriptase, and random hexamers. After the reaction had been heated to 99 C and cooled to 5 C, 40 µl of a PCR mix containing Amplitaq DNA polymerase and forward (5'-GAC AGG TCA CTA CCT GTG CAA-3') and reverse (5'-CAC CTG ATG GAG CTT GAA ATA GAG GCC GC-3') primers (0.25 µM/each) were added to each tube. A four-step PCR was performed with the following conditions: 30 sec at 95 C, followed by five cycles of 94 C, 5 sec; 62 C, 60 sec; 72 C, 60 sec; five cycles of 94 C, 5 sec; 61.5 C, 60 sec; 72 C, 60 sec; five cycles of 94 C, 5 sec; 61 C, 60 sec; 72 C, 60 sec; 15 cycles of 94 C, 5 sec; 60 C, 60 sec; 72 C, 60 sec; and a final extension for 5 min at 72 C. Samples were rapidly cooled to 4 C. A single 217-bp amplicon from each sample was cloned into pCRII-TOPO and sequenced in both directions by the dideoxy chain termination method.
Gel Shift Assays
To test the RUSH-binding site within the context of the flanking sequence, DNA fragments (190-bp) were excised from the cloning cassette of pCRII-TOPO with the restriction enzymes XbaI and HindIII. To further test selected RUSH-binding sites in the absence of flanking sequence, DNA fragments (42-bp) were excised from the flanking sequence with the restriction enzymes BamHI and EcoRI. DNA fragments were separated by electrophoresis on 4% polyacrylamide (acrylamide:bis-acrylamide, 30:0.8) gels, in Tris-borate buffer (45 mM Tris; 45 mM boric acid; 1 mM EDTA, pH 8.3), and recovered by electroelution. The 190-bp DNA fragments were radiolabeled on both ends with [-32P]dATP (5'-end) and [
-32P]dCTP (3'-end) using the Klenow fragment of DNA polymerase I. The 42-bp fragments were radiolabeled on both ends with [
-32P]dGTP (5'-end) and [
-32P]dATP (3'-end). The specific activities for the probes averaged 1 x 107 cpm/µg. GATA oligonucleotides were 32P labeled with (
32P)ATP using polynucleotide kinase (50,000 cpm/ng). UG200 was 3'-end-labeled on the coding strand at an XbaI site with [
-32P]dCTP using the Klenow fragment of DNA polymerase I. The specific activity for UG200 was 12 x 107 cpm/µg. Binding reactions were performed as previously described (28).
Northern Analysis and in Situ Hybridization
Total RNA was isolated from endometrium using TriReagent according to the manufacturers instructions. The integrity of each RNA sample was confirmed by electrophoretic fractionation through formaldehyde-containing agarose gels (1.2%) and ethidium bromide staining. Poly(A+)RNA was isolated with the NucleoTrap Acid Purification Kit. RNA concentrations were determined spectrophotometrically (A260). The A260/280 ratio for each sample was greater than 1.8. Poly(A+) RNA samples (1 µg) were subjected to Northern analysis (7) with random-prime-32P-labeled rabbit GATA-4 (specific activity 1 x 109 cpm/µg). Relative intensities of the resulting mRNA autoradiograms were quantified with a computer-assisted image analysis system (Bio-Image Visage 2000). In situ hybridization was performed as previously described (29).
Mutagenesis and in Vitro Transfection Assays
The hexameric sequence ACTTAT in the uteroglobin promoter (-126/-121) was changed to a sequence (ACTTGG) that is not recognized by RUSH (Table 1). For mutagenesis, 50 ng of pUG3.1-LUC, a 3.1-kb SacI/HindIII PCR fragment (-3090/+37) of the rabbit uteroglobin gene in the luciferase reporter plasmid pGL3-Basic (8), was used as template. Each 50-µl reaction mix contained reaction buffer (1x), Pfu Turbo DNA polymerase (2.5 U/50 µl), dNTPs (0.2 mM each), and primers (125 ng each). The mutagenesis site is indicated in bold for both the forward (5'-GAG AAA AGG GAA TAT TTA CTT GGC CCA CCA AGT CAA TGC CC-3') and the reverse (5'-GGG CAT TGA CTT GGT GGG CCA AGT AAA TAT TCC CTT TTC TC-3') primers. Cycle parameters were as follows: 30 sec at 95 C, followed by 15 cycles of 95 C, 30 sec; 55 C, 60 sec; 68 C, 11 min. Samples were rapidly cooled to 4 C. The parental (nonmutated) DNA template was digested for 2 h at 37 C with 1 µl of DpnI (10 U/µl). The authenticity of the mutated site was confirmed by sequencing. Nonmutated (pUG3.1-LUC) and mutated (pUG3.1M-LUC) constructs were used in standard transformation reactions.
HRE-H9 cells were grown in -modified MEM plus 4% charcoal-stripped fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin (Life Technologies) (250 ng/ml) in a humidified atmosphere of 95% air-5% CO2 at 33 C (8, 30). As verified by RIA, charcoal stripping reduced estradiol levels to less than 0.6 pg/ml. Cells were seeded in 24-well tissue culture plates and transfected at 5060% confluency with 100 ng of pUG-LUC DNA and 200 pg of pRL-TK-LUC (transfection efficiency control) in 5 µl PLUS reagent with 2 µl LipofectAMINE reagent. Transfections were performed in quadruplicate, and pUG-LUC activity was normalized to pRL-TK-LUC activity. Ratio data were ranked (31), and the ranks between the groups were analyzed by ANOVA, followed by Student-Newman-Keuls multiple range test (P < 0.05 significance level) using the SAS Statistics package (32).
Chromatin Immunoprecipitation and PCR
Chromatin immunoprecipitation was performed according to Gould et al. (33) as modified by Phelps and Dressler (34). Briefly, nuclei (1 x 108/experiment) were collected by centrifugation (1000 x g for 5 min at 4 C) and resuspended in buffer (10 mM HEPES, pH 7.9; 10 mM KCl; 2 mM MgCl2; 0.5 mM dithiothreitol) which contained the following protease inhibitors: leupeptin (1 µg/ml), antipain (2 µg/ml), benzamidine (10 µg/ml), chymostatin (10 µg/ml), pepstatin (10 µg/ml), and phenylmethylsulfonylfluoride (2 mM). Chromatin was digested in situ with 250 U/ml Sau3A I for 30 min at 37 C. Nuclei were collected by centrifugation (750 x g for 2 min at 4 C), resuspended in 1 ml of buffer (12 mM Tris, pH 7.5; 3 mM EDTA plus the cocktail of protease inhibitors described above), and mixed (tumbled) at 4 C for 3060 min. Nuclear debris was collected by centrifugation (750 x g for 2 min at 4 C). The supernatant containing soluble chromatin was transferred to a new tube. NaCl and BSA were added to final concentrations of 100 mM and 1 mg/ml, respectively, and the tube was incubated for 5 min at 4 C. Residual nuclear debris was collected by centrifugation (12,000 x g for 5 min at 4 C).
The chromatin supernatant was preabsorbed with Protein A/G PLUS-Sepharose beads for 60 min at 4 C. Beads were collected by centrifugation (1000 x g for 5 min at 4 C). Supernatant was divided into two aliquots which were incubated ± 2 µg RUSH-1(ß) antibodies with continuous mixing (tumbling) for 60 min at 4 C. Protein A/G PLUS-Sepharose beads were added with continuous mixing (tumbling) for 60 min at 4 C. Sepharose was collected by centrifugation (3000 x g for 30 sec) and washed three times with PBS containing 1 mM MgCl2 and 1 mM CaCl2. Sepharose was resuspended in buffer (50 mM Tris, pH 8.0; 1% sodium dodecyl sulfate; 100 mM NaCl) containing Pronase (1 mg/ml) and incubated for 1 h at 50 C. The DNA was extracted with phenol:chloroform (1:1) and precipitated with ethanol. DNA (50 ng) aliquots were amplified in 50-µl PCR containing LA PCR buffer (1x), TaKaRa ExTaq DNA polymerase (2.5 U/50 µl), TaqStart antibody (0.55 µg/50 µl), dNTPs (0.2 mM each), and primers (0.2 mM each). The forward (5'-GTG TGA GTT CAG TTT CAA TAG GG-3') and reverse (5'-GGG GCT CCA CTT ACT TGA CTG-3') primers flank a 110-bp region (-180/-70) of the uteroglobin promoter. A five-step PCR was performed with the following conditions: 30 sec at 95 C, followed by five cycles of 94 C, 5 sec; 50 C, 60 sec; five cycles of 94 C, 5 sec; 51.5 C, 60 sec; five cycles of 94 C, 5 sec; 53 C, 60 sec; five cycles of 94 C, 5 sec; 54.5 C, 60 sec; 15 cycles of 94 C, 5 sec; 56 C, 60 sec; and a final extension for 10 min at 68 C. Samples were rapidly cooled to 4 C. Reaction products were fractionated by electrophoresis on a 3% NuSieve GTG agarose gel. The 110-bp amplicons were extracted from a single band with QIAEX II according to the manufacturers instructions, ligated to pCRII-TOPO, and sequenced in both directions by the dideoxy chain termination method.
Coimmunoprecipitation Assays
Aliquots (30 µg) of precleared nuclear extract protein were incubated overnight at 4 C with affinity-purified, RUSH-1(ß) antibodies, followed by incubation for 2 h at 4 C with a 50% slurry of protein A-Sepharose. Proteins were fractionated by SDS-PAGE on 10% gels and transferred to nitrocellulose membrane. Membranes were processed for Western analysis as previously described (6, 7). Briefly, membranes were blocked in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.6) with 0.1% Tween 20 (TBST) and 2% powdered milk. Membranes were incubated overnight at 4 C in the same buffer containing either rabbit antimouse Jak2 antibodies (1:5000) or rabbit anti-RUSH-1
(ß) antibodies (1:100). Membranes were washed 3 x 30 min in TBST, and incubated in TBST with horseradish peroxidase-conjugated donkey antirabbit IgG (1:5000) for 90 min at room temperature. Membranes were subsequently washed 3 x 30 min in TBST. Specific signals were detected by chemiluminescence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CASTing, Cyclic amplification and selection of targets; dNTPs, deoxynucleoside triphosphates; HNF3, hepatocyte nuclear factor 3; Jak2, Janus kinase 2; Oct-1, octamer transcription factor 1; oPRL, ovine prolactin; PRL, prolactin; Stat, signal transducer and activator of transcription; TBST, Tris-buffered saline with 0.1% Tween 20.
Received for publication February 8, 2002. Accepted for publication May 13, 2002.
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REFERENCES |
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