J. Dairy Sci. 88:2246-2252
© American Dairy Science Association, 2005.
Polymorphic AP-1 Binding Site in Bovine CSN1S1 Shows Quantitative Differences in Protein Binding Associated with Milk Protein Expression
A. W. Kuss,
J. Gogol,
H. Bartenschlager and
H. Geldermann
Department of Animal Breeding and Biotechnology, University of Hohenheim, D-70593 Stuttgart, Germany
Corresponding author: Andreas W. Kuss; e-mail: tzunihoh{at}uni-hohenheim.de.
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ABSTRACT
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Polymorphisms in 5'-flanking regions of milk protein encoding genes can influence the binding activity of the affected response elements and thus have an impact on the expression of the gene products. However, precise quantitative data concerning the binding properties of such variable response elements have so far not been described. In this study we present the results of a quantitative fluorescent electromobility shift assay comparing the allelic variants of a polymorphic activator protein-1 binding site in the promoter region of the bovine 
s1-casein encoding gene (CSN1S1), which is affected by an A
G exchange at –175 bp (CSN1S1–175bp). A supershift assay using a commercial c-jun antibody was carried out to verify the specificity of protein binding. The gel shift analysis revealed specific and significantly reduced protein binding of oligonucleotides containing the G variant of the CSN1S1–175bp binding site. Further investigations comprised genotyping of the variable CSN1S1–175bp activator protein-1 element by an NmuCl restriction fragment length polymorphism in 62 cows of the breed Simmental and 80 cows of the breed German Holstein. Single milk proteins from at least 4 milk samples per cow were quantified by alkaline urea polyacrylamide gel electrophoresis. Homozygotes for CSN1S1–175bp*G were not observed, and the allele frequencies were 0.19 in Simmental and 0.05 in German Holstein. Carriers of CSN1S1–175bp*G showed higher content (%) as well as quantity (g/d) of s1-casein than CSN1S1–175bp*A homozygotes, independent of breed. We assume that the positive association of the CSN1S1–175bp*G variant with CSN1S1 expression is likely to be caused by a reduced affinity of the affected response element to a c-jun-containing CSN1S1 dimer with repressor properties.
Key Words: activator protein-1 • bovine s1-casein encoding gene expression • protein binding
Abbreviation key: AP-1 = activator protein-1, CSN1S1 = s1-CN encoding gene, CSN1S1–175bp= polymorphic AP-1 binding site with an AG exchange at –175 bp of CSN1S1, EMSA = electromobility shift assay, HF = German Holstein, SM = Simmental
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INTRODUCTION
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The term activator protein-1 (AP-1) refers to dimeric leucine zipper transcription factors, comprising combinations of Jun and Fos or activating transcription factor subunits that bind to a common regulatory DNA element, the AP-1 binding site, with the consensus sequence TGAGTCTA (Vogt and Bos, 1990; Angel and Karin, 1991). The AP-1 binding sites mediate gene regulation in response to a variety of extracellular stimuli such as growth factors, cytokines, oncogenes, tumor promoters, and chemical carcinogens (reviewed by Shaulian and Karin, 2001, 2002). In the mammary gland, AP-1 is induced during involution in mice (Marti et al., 1994) and plays a role in glucocorticoid signaling (e.g., Feng et al., 1995; Karin and Chang, 2001). In addition, AP-1 has been shown to be involved in gene regulation of bovine mammary epithelial cells as a response to prolactin (Olazabal et al., 2000).
The main component of the protein fraction of bovine milk is s1-CN, comprising 35 to 45%. Its encoding gene (CSN1S1) spans 17.5 kb, includes 19 exons, and is located on Bos taurus autosome 6 as a part of the so-called casein cluster (reviewed by Rijnkels, 2002). Polymorphisms in the open reading frame of CSN1S1, effecting protein variants (Grosclaude et al., 1970, 1972; Mercier et al., 1971; Farrell et al., 2004), as well as DNA variants in noncoding areas such as the 5'-flanking region have been described (Schild and Geldermann, 1996). For a number of CSN1S1 variants, including positions in the 5'-flanking region, associations with milk protein yield and composition have recently been reported (Martin et al., 2002; Prinzenberg et al., 2003; Szymanowska et al., 2004), but their molecular basis remains unclear. Therefore, the objective of this study was to identify and characterize functional differences between variants in DNA sites with putative effects on gene regulation. The investigated AP-1 binding site (CSN1S1–175bp) is affected by an AG exchange (TGA GTCTA/G) at –175 bp of the bovine CSN1S1 promoter (Koczan et al., 1993; Schild and Geldermann, 1996), which reduces its homology with the consensus sequence to about 86%. The protein binding of the variable response element was analyzed by fluorescent electrophoretic mobility shift analysis (EMSA), using an automated DNA sequencer. The EMSA results were combined with distribution of single milk proteins in 2 cattle herds, to allow for a causal interpretation of associations between CSN1S1–175bp variants and expression of the corresponding gene product.
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MATERIALS AND METHODS
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Nuclear Extracts
Udder tissue samples (about 1 cm3) of 6 lactating German Holstein (HF) and 5 lactating Simmental (SM) cows each were collected directly after slaughter and immediately stored in liquid nitrogen. The procedure was based on protocols of Watson et al. (1991). Protein concentrations were determined using the DC protein assay kit (BioRad, Munich, Germany) according to the instructions of the manufacturer.
Gel Shift Analysis
Based on previous reports (Ruscher et al., 2000; Filee et al., 2001), a fluorescence-based EMSA using an automated laser fluorescent sequencer (A.L.F., Pharmacia, Freiburg, Germany) was developed. Double-stranded oligonucleotides were obtained by annealing 5' Cy5-labeled oligonucleotide CSN1S1–175bp*A (TTATAATGA GTCACTTCT) and CSN1S1–175bp*G (TTATAATGAGT CGCTTCT) with their respective unlabeled complements. For competitive assays, unlabeled oligonucleotides were used as competitors. Protein binding was carried out by combining specific oligonucleotides at various concentrations, H2O, 18 µg of nuclear proteins, 300 ng of poly(dI-dC) • poly(dI-dC), and 3 µL of 5-fold binding buffer (50 mM Tris-HCl, pH 7.5; 2.5 mM EDTA; 25 mM dithiothreitol; 20% glycerol; 250 mM NaCl; and 5 mM MgCl2) in a final volume of 29 µL on ice. An unlabeled oligonucleotide containing the consensus sequence (5'-TTCTCGCCCCAGGCTGCA-3') of the response element for activator protein-2 was used for un-specific competition. For supershift assays, 1 µg of rabbit polyclonal antibody against recombinant human c-jun with specified cross-reactivity for bovine proteins of corresponding molecular masses of c-jun, JunD and JunB (Biomol, Hamburg, Germany), was included in the reaction mix. The reaction was then incubated in a thermal cycler for 20 min at 25°C, then 1 µL of Cy5 labeled oligonucleotide (1 ng/µL) was added, and the binding reaction continued at 25°C for 50 min. Electrophoresis of the reaction product was carried out on an automated laser fluorescent sequencer using short (28 cm) nondenaturing 4% acrylamide gels (acrylamide:bi-sacrylamide = 60:1) constantly running with 10 W at 13°C. After an 8- to 10-min prerun, 9 µL of the annealing reaction and 1 µL of 10-fold loading buffer (250 mM Tris-HCl, pH 7.5; 40% glycerol), were quantitatively loaded onto the gel. Runs were stopped after approximately 120 min. The A.L.F.win and Allele Links software (Pharmacia) were used for instrument control and data analysis.
Comparison of Protein Binding Activities
For the shift peaks (peaks representing the bands of protein-bound fluorescent probe), the mean relative peak areas (shift peak area / total peak area) from 2 aliquots per reaction and gel were calculated. The area values were obtained for at least 6 reactions per experiment and used for comparison between experiments using the Student’s t-test. For protein binding assays, extracts of donors from both breeds were used.
Animals and Samples for Association Analysis
At least 4 milk samples and 2 EDTA-stabilized blood samples per cow as well as monthly performance data [SCC, total protein (% and kg/d), and milk yield (kg/d)] were collected between April 1997 and March 1999, from 80 cows of an HF herd and 62 cows of an SM herd, both from experimental stations of the Hohenheim University (for pedigree information of the animals, see Kuss et al., 2003). Milk samples were collected at 8-wk intervals. Fat was removed after centrifugation (10 min at 4550 x g and 4°C), and the defatted samples were stored at –80°C. Genomic DNA was isolated from blood by means of the NucleoSpin Blood Quick Pure kit (Macherey Nagel, Düren, Germany; www.macherey-nagel.com) according to the instructions of the manufacturer.
Quantification of Milk Proteins
Milk proteins were separated by vertical urea poly-acrylamide gel electrophoresis (8 M urea, 8.4% acrylamide; Ehrmann et al., 1997). The proteins were then stained (staining solution = 8% wt/vol TCA, 26.7% vol/ vol methanol, 9.3% vol/vol acetic acid, 0.033% wt/vol Coomassie Brilliant Blue R250) overnight with agitation. After standard destaining (29% vol/vol methanol, 5% vol/vol acetic acid; 4 h with agitation), single milk protein fractions were quantified using a densitometer (ATH Elscript 400, Hirschmann, Germany) using the manufacturer’s software.
Genotyping of DNA Variants
A 192-bp fragment of the bovine CSN1S1 5'-flanking region between –317 and –125 bp was amplified by PCR from genomic DNA (forward primer: 5'-GAATTTTTCAAAGGTTACAAAGGAA-3'; reverse primer: 5'-TGTTCAGTTCCTCTCCCAAGA-3'). The amplicon was digested with NmuCl (MBI Fermentas, St. Leon-Rot, Germany) and subsequently submitted to agarose gel electrophoresis for RFLP determination of the polymorphic CSN1S1–175bp site. Genotype nomenclature refers to the variable nucleotides A or G at position –175 bp. Restriction fragment lengths were 138 and 54 bp for allele A and 192 bp for allele G. Heterozygous animals were double-tested to ensure correct typing, and to exclude errors caused by incomplete restriction.
Statistical Analysis of Associations Between Genotypes and Trait Values
The GLM procedure of SAS, version 8 (SAS Institute Inc., Cary, NC), was applied within breed, using the following model:
where Yijklmn = trait value [content (%) and amount (g/d or kg/d) of S1-CN and total protein]; µ = overall mean; MFLi = fixed effect of genotype i ; Aij = fixed effect of animal j within genotype i; Lk = fixed effect of lactation number with k = 1 (first lactation), 2 (second lactation), 3 (third and later lactation); LSl = fixed effect of lactation stage with l = 1 (1 to 25 d), 2 (25 to 60 d), 3 (61 to 90 d), 4 (91 to 120 d), 5 (121 to 210 d), 6 (211 to 240 d), 7 (241 to 300 d), and 8 (>300 d); Sm = fixed effect of season with m = 1 (Jan–Apr), 2 (May–Aug), 3 (Sep–Dec); b1, b2 = regression coefficients on cell count, linear 2 and square; Cijklmn, Cijklmn = cell count and square cell count; 
,2 = overall mean of cell count and square mean of cell count; and eijklmn = residual error.
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RESULTS AND DISCUSSION
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Gel Shift Analysis of CSN1S1–175bp
Both variants of the CSN1S1–175bp element showed protein binding properties, independent of breed or individual protein extract (not shown). Figure 1
displays an example curve view of the fluorescence-based EMSA. The free labeled probe passed the detector after approximately 60 min and the shifted band was detected with an approximate retardation of 35 min. The peak size of the shifted band was strongly reduced when using an unlabeled specific competitor. As shown in Figure 2, the mean relative shiftpeak area observed for the G variant was more than 40% lower than that of the A variant. This difference was highly significant (P < 0.01) and indicates a reduced protein binding capacity of the G variant. A 100-fold excess of unspecific competitor did not significantly affect the protein binding of the respective CSN1S1–175bp probes. In contrast, for both CSN1S1–175bp variants, the addition of 100-fold specific competitor to the reaction reduced the protein binding of the labeled probes by about 70%.
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Figure 1. Example curve views of electromobility shift assay (EMSA) results: a) 1 ng of Cy5-labeled oligonucleotide CSN1S1–175bp*A without competitor, b) 1 ng of Cy5-labeled oligonucleotide CSN1S1–175bp*A with 100 ng of unlabeled oligonucleotide CSN1S1–175bp*A.
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Figure 2. Relative shiftpeak areas (shiftpeak area/total peak area) without (control) and with 100-fold specific (SC = corresponding unlabeled oligonucleotide) and unspecific (UC = unlabeled oligonucleotide containing the activator protein-2 consensus sequence) competitors. The results for CSN1S1–175bp*A and CSN1S1–175bp*G probes are represented by light and dark grey columns respectively, whiskers represent the standard error of the mean based on n  6 observations.
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The specificity of the CSN1S1–175bp probes was tested by adding 1 µg of an antibody against recombinant human c-jun protein to the protein binding reaction, which caused a significant (P < 0.01) decrease of the shiftpeak area (Figure 3a), but did not produce a supershift band. As a control for the specificity of the antibody effect, a parallel experiment was carried out using 1 µg of porcine immunoglobulin G, which did not cause a significant shiftpeak reduction (Figure 3b). These results indicate that specific AP-1 protein binding was observed, because it can be surmised that the epitope recognized by the antibody was probably in a region which is either critical for dimerization or close to the DNA-binding domain of the bovine c-jun. Thus, instead of forming a detectable complex with DNA-bound c-jun dimers, the antibody either prevented c-jun dimerization, which has been previously suggested to impair DNA-binding (Zhou et al., 1999), or blocked the efficient binding of the transcription factor to the DNA element. Alternatively, the DNA/protein complex may have been too large to migrate to the detector.
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Figure 3. Relative shiftpeak area (shiftpeak area/total peak area) using labeled oligonucleotide CSN1S1–175bp*G. Whiskers represent the standard error of the mean based on n 6 independent observations. a) Protein binding without (control) and with 100-fold specific (SC = corresponding unlabeled oligonucleotide) and unspecific (UC = unlabeled oligonucleotide containing the activator protein-2 consensus sequence) competitors as well as 1 µg of recombinant antihuman c-jun antibody (anti-c-jun), b) protein binding without (control; n = 4) and with the presence of 1 µg of porcine immunoglobulin G (IgG; n = 6), and c) protein binding in the presence of specific competitors (oligonucleotides CSN1S1–175bp*A and *G, unlabeled) in different concentrations.
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Using labeled probes for the G variant, we compared the differences in protein binding capacities between the CSN1S1–175bp variants. For this purpose, various amounts of unlabeled probes for either variant were added to the binding reactions. As shown in Figure 3c
, about 3 times more of the G variant lead to a similar reduction in shiftpeak area as of the A variant. For 30-and 100-fold excesses of unlabeled oligonucleotide, the differences were significant (P < 0.05). Significance was lost when using the competitors at 300-fold excess, probably because of the extreme overabundance of unlabeled oligonucleotides.
These findings strongly indicate that the reduced identity of the CSN1S1–175bp*G variant with the AP-1 binding consensus sequence leads to impaired protein binding efficiency. This is in keeping with earlier findings for a polymorphic activator protein-2 binding site in bovine ß-LG, where semiquantitative EMSA showed differences in protein affinity between DNA variants, but without verification of the binding specificity by supershift assays (Lum et al., 1997). Similar observations were made by Martin et al. (2002) for a number of variants in the promoter regions of bovine casein encoding genes (identical results were presented by Szymanowska et al., 2004), including 2 CSN1S1 variants upstream (–728 and –733 bp) of the DNA element investigated here. However, the authors did not determine the precise identity of the involved transcription factors and the significance of differences in protein binding was not conclusively established. Our study describes an EMSA method that represents a refined tool for the comparative assessment of protein binding, allowing the determination of significant differences in the protein binding activities of the allelic variants of a polymorphic response element. We also present evidence for the identity of the involved transcription factor.
Association Between CSN1S1–175bp and Lactation Traits
Genotyping of the CSN1S1–175bp response element yielded A homozygotes and heterozygotes, whereas G homozygotes were not observed. The frequency of CSN1S1–175bp*G (Table 1) was more than 3 times lower in HF (0.05) than in SM (0.16), which may be the result of differing breeding objectives concerning milk performance. In view of earlier results for ß-LG (Kuss et al., 2003) and according to our findings concerning the protein binding activity of the CSN1S1–175bp variants, differential gene activity in animals carrying the G allele can be expected. This is in agreement with the associations of CSN1S1–175bp*G with quantity (g/d) as well as content (%) of s1-CN in bovine milk, as shown in Table 1. For both breeds, a positive association of CSN1S1–175bp*G with s1-CN content and yield was found. Therefore, because CSN1S1–175bp*G showed lower protein binding affinity in our gel shift analysis, it could be assumed that the involved AP-1 dimer might function as a repressor for CSN1S1. This would also be in agreement with the observation that c-jun acts on tumor suppressor genes through repression (Shaulian and Karin, 2001).
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Table 1. Associations between genotypes of the CSN1S1–175bp activator protein-1 binding site and S1-CN as well as total milk protein traits (LS means ± SE).
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For the SM breed, significant superiority of CSN1S1–175bp*G was observed solely for the casein fraction. In HF, associations with the whey proteins -LA and ß-LG were significant (not shown). Interestingly, in HF, the carriers of CSN1S1–175bp*G showed an approximately 35% higher yield of -LA (g/d) which was accompanied by 17.5% higher milk yield (not shown). However, -LA is known to enhance the concentration of lactose, which is the major osmole in milk (Ramakrishnan et al., 2001), and thus, its elevated production is likely to cause a higher ratio of water in the milk. This could be due to a coincidental effect of breeding, focused on higher milk performance in HF, and would explain the significantly reduced total protein percentage in the milk of HF CSN1S1–175bp*G carriers, who showed a concurrent increase in total protein yield. Associations with total protein were also described by Prinzenberg et al. (2003) between carriers of single strand conformational polymorphism genotypes of a 655-bp fragment in the CSN1S1 5'-flanking region and protein percentage in 8 Holstein half-sib families. The results are in agreement with the allocation of QTL for total protein percentage and yield to a chromosome interval containing CSN1S1 in Finnish Ayrshire cattle (MARC97: 82.6 cM; Maki-Tanila et al., 1998; Ikonen et al., 1999). However, both studies in half-sib families have a low resolution (>10 cM) and can reflect the influences of several loci.
Taken together, the coincidence of superior values of CSN1S1–175bp*G carriers both for s1-CN percentage and yield in 2 breeds is a further indication that this variant might be more directly involved in the regulation of CSN1S1 expression.
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CONCLUSIONS
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For the first time, using the method presented in this paper, the significance of differences in protein affinities between the allelic variants of a polymorphic protein binding DNA element could be established. The observed positive association for carriers of the variant CSN1S1–175bp*G with s1-CN quantity (g/d) and content (%) in the milk could be caused by a reduced affinity of the affected response element to a c-jun-containing AP-1 dimer with repressor properties. However, because the regulation of gene expression is under multifactorial control, and with regard to further polymorphisms that affect other putative response elements (Geldermann et al., 1996), the possibility remains that additional polymorphic binding sites have contributed to the observed effect. Future studies, for example, reporter gene analyses in transgenic cells including variable sections of the CSN1S1 5'-flanking region, will be needed to verify our findings and obtain additional evidence.
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ACKNOWLEDGEMENTS
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We gratefully acknowledge the technical assistance of Adelheid Tanzer-Schink. Financial support was received from the Deutsche Forschungsgemeinschaft.
Received for publication July 15, 2004.
Accepted for publication January 18, 2005.
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ABSTRACT
INTRODUCTION
