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Physiol. Genomics 25: 116-120, 2006; doi:10.1152/physiolgenomics.00145.2005
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Received 22 June 2005; accepted in final form 22 December 2005.
Physiological Genomics 25:116-120 (2006)
American Physiological Society © 2006 American Physiological Society

Alleles of the bovine DGAT1 variable number of tandem repeat associated with a milk fat QTL at chromosome 14 can stimulate gene expression

Rainer Fürbass1, Andreas Winter2, Ruedi Fries2 and Christa Kühn1

1 Research Unit Molecular Biology, Research Institute for the Biology of Farm Animals, Dummerstorf
2 Lehrstuhl für Tierzucht der Technischen Universität München, Freising, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A quantitative trait locus (QTL) affecting milk fat percentage has been mapped to the centromeric end of the bovine chromosome 14 (BTA14). This genomic area includes the DGAT1 gene, which encodes acyl-CoA:diacylglycerol acyltransferase 1, the key enzyme of triglyceride biosynthesis. Genetic and biochemical studies led to the identification of the nonconservative DGAT1-K232A polymorphism as a causal mutation for the QTL. In addition to this, another polymorphism in the 5'-regulatory region of this gene, the DGAT1 variable number of tandem repeat (VNTR), also showed a strong association with milk fat percentage. This promoter VNTR polymorphism affects the number of potential Sp1 binding sites and therefore might have an impact on DGAT1 expression and also milk fat content. Hence, the DGAT1 VNTR polymorphism might be another causal mutation for the BTA14 QTL. However, evidence for Sp1 binding to this polymorphic site and for the capability of DGAT1 VNTR alleles to stimulate gene expression was lacking. In the current work Sp1-VNTR interactions were analyzed by EMSA. In addition, effects of DGAT1 VNTR alleles on gene expression were measured with reporter gene analyses. Conclusions from the results are that 1) the DGAT1 VNTR sequence is indeed a target for Sp1 binding; 2) DGAT1 VNTR alleles can stimulate gene expression in vitro and probably in vivo as well; and 3) although the stimulating effects of the different DGAT1 VNTR alleles did not show significant differences in vitro, their effects on transcription might be different in the chromatin context existing in vivo.

DGAT1 promoter polymorphism; causal mutation; transcription factor Sp1; reporter gene expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A QUANTITATIVE TRAIT LOCUS (QTL) with a strong effect on milk fat content has been mapped to the centromeric region of the bovine chromosome 14 (BTA14) by Coppieters et al. (7). Using a positional candidate approach, Grisart et al. (12) cloned this QTL region, which included the DGAT1 gene. Its product, acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20), catalyzes the final step of triglyceride biosynthesis (6). DGAT1 became also a functional candidate gene because of the observation that DGAT1-deficient mice generated by targeted gene disruption did not lactate (28), most likely because of impaired triglyceride biosynthesis in the mammary glands. Nonconservative mutations in the DGAT1 gene resulting in a lysine to alanine substitution at position 232 (K232A) of the DGAT1 enzyme had a major effect on milk fat percentage, with the K-encoding allele being associated with a higher milk fat content (12, 32). Direct evidence for the causality of the K232A polymorphism was provided by biochemical data that revealed that the K-encoding allele is characterized by a higher Vmax in producing triglycerides than the A allele (13). The QTL effect of the K232A polymorphism was confirmed by several studies (29, 30); however, apparent differences in the effect sizes observed between families and across populations could not be fully explained by this diallelic polymorphism alone. Indeed, Bennewitz et al. (3) and Kühn et al. (20) reported that genetic variation additional to the DGAT1 K232A mutation affecting milk fat content should be present in the same QTL. Alleles of the DGAT1 promoter region, which comprise a variable number of tandem repeats (VNTR) (32), were considered as likely candidates for several reasons. First, genetic analysis revealed that DGAT1 VNTR alleles were associated with variation in milk fat content in homozygous DGAT1 232A/232A animals. The sequence of one grandsire with the genotype 232A/232A, which was heterozygous at the QTL for milk fat percentage as determined by granddaughter design linkage analysis, has been examined at all polymorphic DGAT1 sites detected by Winter et al. (32). Interestingly, all these sites were homozygous in this grandsire except for the DGAT1 VNTR (20). Of the five DGAT1 VNTR alleles 1–5 containing three to seven repeats, respectively (32), allele 5 showed the strongest QTL effect and its direction paralleled that of the 232K-encoding allele (20). Furthermore, because a potential Sp1 binding motif is present in the VNTR element a variation in its number might well affect gene expression. Finally, in humans the DGAT1 promoter mutation T79 reducing transcriptional activity was associated with a lower body mass index in females (22), which represents an example of DGAT1 promoter effects. Therefore, it has been suggested that the DGAT1 promoter VNTR polymorphism might be another causal mutation for the QTL at BTA14. However, neither binding of the transcription factor Sp1 nor the functional significance of DGAT1 VNTR alleles with regard to gene expression has been studied so far. These issues were addressed in the present work by EMSA and reporter gene analyses. Our data show that the DGAT1 VNTR sequence is a target for the transcription factor Sp1. Furthermore, DGAT1 VNTR alleles can stimulate gene expression in vitro. However, in vitro analysis did not reveal a significant difference between alleles in stimulating gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Electrophoretic mobility shift assays.
Nuclear extracts were prepared from frozen mammary gland (MG) samples obtained from lactating cows and from cultured HeLa cells essentially as described in Refs. 31 and 1, respectively. Tissue samples were dissected from lactating MG of freshly slaughtered cows in the local abattoir. No living animals were used during our study. The protein content of nuclear extracts was determined by the method of Bradford (4). The VNTR-GC probe was prepared by annealing the oligonucleotides 5'-CCGGGGAGGGCGGGGCCTACTAACAGTGTT-3' (GC box motif is underlined) and 5'-AACACTGTTAGTAG-3' and labeling with [{alpha}-32P]dCTP and Klenow polymerase (26). Binding reactions contained 35 fmol of labeled DNA and 2 µg of nuclear extract in a total volume of 10 µl of binding buffer [in mM: 10 Tris·HCl pH 7.5, 50 KCl, 1 MgCl2, 0.5 EDTA, and 0.5 DTT, with 0.01 mg/ml poly(dI-dC) and 5% glycerol]. To demonstrate specificity of DNA-protein complexes a 100-fold excess of unlabeled competitor oligonucleotides were included in the reaction mixes [VNTR-GC; VNTR-GCm, 5'-CCGGGGAGGGAGGGGCCTACTAACAGTGTT-3', (mutated GC box is underlined)]. For supershift assays 4 µg of a polyclonal Sp1 antibody (sc-59X, Santa Cruz) was added to the binding reaction. After incubation at 20°C for 20 min, samples were subjected to electrophoresis through 6% native polyacrylamide gels in 0.5x Tris-borate-EDTA. Gels were analyzed on a STORM 840 PhosphorImager with Image Quant software (Molecular Dynamics, Krefeld, Germany).

Detection of DGAT1 transcripts (RNA isolation, reverse transcription, PCR).
DGAT1 transcripts were assessed in the bovine MG samples used for EMSA experiments and in murine mammary epithelial HC11 cells (2) used during reporter gene studies. RNA was prepared with the RNeasy Mini Kit (Qiagen, Hilden, Germany) as recommended by the supplier. For cDNA synthesis 1.0 µg of total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Promega, Mannheim, Germany) and DGAT1 primers [bovine specific (5'-GAACAGCTTGAGGAAGAGGATGG-3') or mouse specific (5'-GAATAAAGCTTGAGGAACATGATGG-3')]. Generated cDNA samples were purified with the High Pure PCR Product Purification Kit (Roche, Mannheim, Germany). PCR was performed with the DGAT1 cDNAs as templates and bovine- or mouse-specific primer pairs (bovine DGAT1, 5'-GTGGCATCCTGAATTGGTGTGTG-3'/5'-GCCACAATACAGCAAGAG-3'; mouse DGAT1, 5'-CAGCGTGGGCGACGGCTACT-3'/5'-TGGGGCAGGCCAGCTGTAGG-3').Resulting amplimeres of 162 and 232 bp, respectively, were analyzed in a 4% agarose gel stained with ethidium bromide. The identity of amplimeres was confirmed by sequence analysis with an ABI PRISM 310 instrument and an ABI PRISM BigDye Kit (PE Biosystems, Weiterstadt, Germany).

Plasmids and reporter gene constructs.
DNA sequences encompassing DGAT1 VNTR alleles were amplified by PCR using genomic DNA samples from cows with known VNTR genotypes as templates. Primers, VNTR forward, 5'-GTGGTACCTCAGGATCCAGAGGTACCAG-3' and VNTR reverse, 5'-GTGAATTCGGGGTCCAAGGTTGATACAG-3', included additional KpnI and EcoRI restriction sites (underlined), respectively, facilitating directional cloning. Amplimeres were cloned into the pGEM-T vector (Promega). VNTR alleles were then fused to the minimal promoter of the herpes simplex virus thymidine kinase gene (referred to as PTK here) by transferring the KpnI-EcoRI inserts of the respective pGEM-T clones into the pGL3 Enhancer (Promega) derivate p756 (kindly provided by H.-M. Seyfert, Research Unit Molecular Biology, Research Institute for the Biology of Farm Animals, Dummerstorf, Germany). The PTK of plasmid p756 is identical to the 117-bp EcoRI-HindIII restriction fragment of plasmid phRG-TK (Promega; EMBL no. AF362551, positions 684 to 800). Finally, to get rid of the SV40 enhancer sequence of pGL3 Enhancer, the KpnI-HindIII restriction fragments with VNTR plus PTK sequences were transferred into pGL3 Basic (Promega) to yield the reporter gene plasmids pVNTR1-, pVNTR4-, and pVNTR5-PTK-luc, respectively. The control plasmid pPTK-luc contains a synthetic KpnI-EcoRI linker (sense 5'-CAGATCTG-3', antisense 5'-AATTCAGATCTGGTAC-3') instead of a KpnI-EcoRI VNTR fragment. All reporter gene constructs were confirmed by sequence analysis using a luciferase antisense sequencing primer, 5'-GCGCCGGGCCTTTCTTTAT-3'. Plasmid CMV-lacZ (18) served as a control for transfection efficiency during reporter gene experiments.

Cell culture, transient DNA transfection, and reporter gene assay.
Murine mammary epithelial HC11 cells (2) were grown in RPMI 1640 medium containing 10% fetal calf serum, 5 µg/ml insulin, 10 ng/ml EGF, 2 mM L-glutamine, and 50 µg/ml gentamycin (Biochrom, Berlin, Germany). For DNA transfection a mix of DNA (2 µg reporter gene plasmid and 0.5 µg CMV-lacZ vector), Lipofectamine transfection reagent, and Plus reagent (Invitrogen, Karlsruhe, Germany) was added to 1 x 106 HC11 cells. Activities of the luciferase and lacZ reporter genes were measured 24 h after transfection with the Dual Light System (PE Biosystems), according to the supplier's instructions, and a luminometer instrument (Lumat LB9501, Berthold, Wildbad, Germany). To normalize for transfection efficiency, the quotient of the luciferase and ß-galactosidase activity measurements was calculated for each sample. The transcriptional activities of the VNTR-PTK-reporter gene constructs and of the pPTK-luc control were presented as values relative to that of the promoterless pGL3 Basic. Data are expressed as means ± SE of values from three or more independent experiments. Statistical analyses were done with SIGMA STAT software (SPSS Science Software, Erkrath, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DGAT1 VNTR sequence is binding target for transcription factor Sp1.
The DGAT1 VNTR sequence comprises three to seven repeats of a GC-rich 18-mer element (32) including the GC-box motif (CCCGCCC), which is a potential binding site for the ubiquitous transcription activating factor Sp1 (16). First of all we analyzed whether Sp1 indeed could bind to the VNTR sequence, as a prerequisite for its possible role in stimulating expression of the DGAT1 gene. To this end, EMSA experiments were performed with a 32P-labeled probe carrying the GC-box element and nuclear extracts from bovine lactating MG tissue samples, which were expressing DGAT1 transcripts as revealed by reverse transcription PCR (not shown). A typical autoradiograph representing a binding experiment is presented in Fig. 1. As depicted in Fig. 1, left, two complexes formed consistently, a major, slower-migrating complex (I) and a minor, faster-migrating complex (II), both of which could be efficiently competed by the unlabeled probe. On the other hand, an oligonucleotide with identical flanking sequences but a mutated GC-box motif no longer competed for binding. Hence, two MG-derived factors specifically bound to the DGAT1 VNTR GC-box, most likely Sp1 and Sp3 (see DISCUSSION). To further demonstrate the presence of the transcription factor Sp1, supershift experiments were performed with commercial Sp1 antibodies, which were raised against the human factor. However, HeLa nuclear extracts, which are known to contain Sp1 abundantly, were used instead of bovine MG extracts, because in previous Western blot analyses the Sp1 antibodies failed to bind to the bovine Sp1 and recognized only the human Sp1 protein (not shown). Indeed, supershift experiments revealed several DNA-protein complexes, of which the slowest-migrating complex, corresponding to complex I, was completely abolished by the Sp1 antibodies (Fig. 1, right). Together, EMSA experiments have shown that Sp1 can actually bind to the DGAT1 promoter VNTR.


Figure 1
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  		Fig. 1. Binding of the transcription factor Sp1 to the DGAT1 promoter variable number of tandem repeats (VNTR) sequence. EMSA experiments with a radiolabeled probe representing the 18-mer VNTR element are shown. Left: 2 binding complexes (I and II) formed with nuclear proteins from bovine lactating mammary gland. Specificity of both complexes was demonstrated by competition with a 100-fold excess of either the unlabeled wild type probe (w) or the mutated probe (m). Right: demonstration of Sp1 binding by supershift EMSA with antibodies raised against Sp1 ({alpha}-Sp1) and HeLa nuclear proteins. {alpha}-Sp1 completely abolished formation of the binding complex I indicating that Sp1 was present in complex I; + and – indicate presence and absence of {alpha}-Sp1, respectively.

 
Functional significance of DGAT1 VNTR alleles.
To assess stimulating effects of the DGAT1 promoter VNTR polymorphism exclusively, separated from other sequences of the QTL region, murine mammary epithelial cells (HC11) were transiently transfected with luciferase reporter gene constructs containing three, six, or seven DGAT1 VNTR repeat elements, representing VNTR alleles 1, 4, and 5, respectively. HC11 cells synthesized endogenous DGAT1 transcripts under the experimental conditions (not shown). Therefore, HC11 cells were considered to meet the physiological requirements also for bovine DGAT1 VNTR-enhanced reporter gene expression. Luciferase activity was measured 24 h after transfection with a luminometer instrument. The results are summarized in Fig. 2. Each of the DGAT1 VNTR alleles was capable of stimulating gene expression. Measurements revealed stimulation factors of 11.8 ± 1.3 (allele 1), 11.1 ± 1.0 (allele 4), and 12.4 ± 1.5 (allele 5) compared with the promoterless pGL3 Basic reporter gene vector. In contrast, stimulation of gene expression by the PTK control alone was only 1.6 ± 0.4-fold. The observed differences between the DGAT1 VNTR constructs and the PTK control were statistically significant (P = 0.0079 by Mann-Whitney rank sum test). Thus isolated DGAT1 VNTR alleles were shown to be functional transcriptional enhancers in vitro. However, we were not able to show significant differences between the DGAT1 promoter VNTR alleles regarding stimulation of gene expression in our reporter gene system.


Figure 2
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  		Fig. 2. Stimulation of gene expression by DGAT1 VNTR alleles. HC11 mammary gland epithelial cells were transiently transfected with the indicated reporter gene constructs (control is without a DGAT1 VNTR sequence; alleles 1, 4, and 5 contain 3, 6, and 7 repeats of the VNTR 18-mer element, respectively, as indicated by the arrowheads marked GC). PTK, minimal promoter of the herpes simplex virus thymidine kinase gene; luc, firefly luciferase gene of pGL3 Basic. Reporter gene activities were measured 24 h after transfection. Stimulation activities of the reporter gene constructs are expressed relative to that of the promoterless reporter gene vector pGL3 Basic. Means ± SE of 3 or more independent experiments are shown. Results demonstrate that DGAT1 VNTR sequences are capable of stimulating gene expression. However, no significant functional differences among VNTR alleles 1, 4, and 5 could be detected in vitro.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Possible causality of DGAT1 VNTR polymorphism for milk fat QTL on BTA14.
Genetic and biochemical data strongly suggest that the nonconservative K232A mutation of the DGAT1 enzyme is the background for the QTL on BTA14 affecting milk fat content (12, 13, 32). Besides this, however, association studies in the German Holstein population revealed that the VNTR polymorphism of the 5'-regulatory region of the DGAT1 gene (32) had an additional, significant effect on milk fat content (20). Complex traits (like milk fat content) may often result from noncoding sequence variation (see Ref. 11 for review). However, the relationship among noncoding sequence variation, gene expression level, and QTL phenotype is not well understood. Therefore, it may be difficult to establish causal sequence variants, and final proof of causality in most cases may not be feasible. Glazier et al. (11) propose criteria of circumstantial corroborating pieces of evidence (e.g., appropriate tissue expression pattern, strong mechanistic support, reporter gene assays) that together might be convincing. The DGAT1 VNTR polymorphism results in a variation in the number of a potential binding motif for the transcription factor Sp1. In eukaryotes, this cis-acting DNA motif is common to promoters of many inducible genes. Also, DGAT1 expression needs substantial induction in mammary epithelial cells dependent on the stage of lactation. Sp1 action can be modulated by a variety of posttranslational modifications, among which is activation by glucose-induced phosphorylation (reviewed in Ref. 27). Interestingly, glucose also enhanced DGAT1 mRNA expression (25), possibly via functional Sp1 binding sites present in the DGAT1 promoter sequence. It is well known that Sp1 can stimulate transcription in a synergistic manner when multiple binding motifs are present in a juxtaposed or separate arrangement (8, 9). Hence, variation in the number of Sp1 binding sites might well have an impact on DGAT1 expression. Furthermore, DGAT1 is the key enzyme of triglyceride biosynthesis (6), and its enzymatic activity is controlled mainly at the transcriptional and posttranscriptional levels (33). Consequently, possible effects of the VNTR polymorphism on DGAT1 expression could directly affect the milk fat content. Hence, the DGAT1 VNTR polymorphism might be another causal mutation underlying the BTA14 QTL. This hypothesis was supported by recent association studies (20). In particular, VNTR allele 5 had a significant effect on milk fat percentage in individuals with the homozygous genotype 232A/232A, the direction of the effect being identical to that of the 232K-encoding allele.

Sp1 binding to DGAT1 VNTR sequence.
First of all, binding of the transcription-activating factor Sp1 to the DGAT1 promoter VNTR GC-box motif had to be demonstrated, because this was a prerequisite for a possible role of the VNTR alleles in gene expression. Besides Sp1, related proteins Sp3 and Sp4 can also bind to the same sequence motif (14, 19), but only Sp1 and Sp3 are expressed in a broad array of tissues, whereas Sp4 is restricted to neuronal tissues (14). It has been observed in the context of other promoters that Sp1 and Sp3 act in an antagonistic manner (15, 21). A variation in the Sp1-to-Sp3 ratio could be a means for fine-tuning the expression of target genes. During EMSA experiments with the VNTR probe and bovine lactating MG extract two specific binding complexes formed. Using commercial antibodies, we could detect Sp1 in the slower-migrating complex I. Although we did not attempt to identify the factor present within the faster-migrating complex II, it was most likely Sp3, because it is the only ubiquitous factor binding to GC-box elements beside Sp1.

Functional significance of DGAT1 VNTR alleles.
After it had been established that Sp1 could indeed bind to the DGAT1 VNTR sequence, the functional significance of the VNTR polymorphism for transcription had to be demonstrated. To this end we studied transcription stimulation capabilities of DGAT1 VNTR alleles 1, 4, and 5 in transiently transformed murine mammary epithelial HC11 cells, which are also widely used by others studying expression of genes from various species, including bovine (e.g., Ref. 24). An advantage of our in vitro reporter gene system over genetic studies in vivo is its ability to measure the stimulating effects of only the isolated VNTR alleles, avoiding overlapping effects of flanking sequences, which may occur in vivo. We have chosen the shortest (3 repeats) and the longest (6 or 7 repeats) of the VNTR alleles previously analyzed by association studies in dairy cattle populations, the latter alleles showing the strongest phenotypic effects compared with all other VNTR alleles (20), because they were the most likely alleles to show any functional difference. On one hand, as demonstrated by the enhanced stimulation effects of promoters with VNTR alleles compared with the control, DGAT1 VNTR sequences indeed can act as transcriptional enhancers in vitro. However, on the other hand, stimulation activities of alleles 1, 4, and 5 were not significantly different, regardless of the number of repeats/Sp1 binding sites. Hence, this observation could not prove the promoter VNTR polymorphism to be a causal mutation for the BTA14 QTL effect. One possible reason could be the use of heterologous mammary epithelial cells. Although they did show physiological potential for transcribing their endogenous DGAT1 gene, nevertheless all requirements for VNTR-activated transcription of the reporter gene may not have been fulfilled. However, aside from recruiting the transcription machinery to promoters, Sp1 can also prevent CpG islands located nearby from being methylated (5, 23). DNA methylation/demethylation is another means to control gene expression by affecting the chromatin structure (10, 17). Interestingly, the bovine DGAT1 gene also possesses a large CpG island of ~700 bp spanning the proximal promoter sequence and part of the first exon, as revealed by computer analysis of the DGAT1 sequence available in the EMBL data bank (accession no. AJ318490; software: EMBOSS-CpGPlot). Hence, Sp1 binding to (different) VNTR alleles could well (differently) affect DNA methylation, DGAT1 expression, and also milk fat content. However, because reporter gene constructs are not as organized as chromatin, studying this aspect of gene regulation is beyond the scope of reporter gene experiments. Therefore, further experiments are necessary to elucidate this possible functional aspect of the DGAT1 promoter VNTR.


   ACKNOWLEDGMENTS
 
We appreciate the excellent technical assistance of M. Sundt, V. Schreiter, and M. Anders.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: R. Fürbass, Research Unit Molecular Biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany (e-mail: fuerbass{at}fbn-dummerstorf.de).


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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