A 5′- Regulatory Region and Two Coding Region Polymorphisms Modulate Promoter Activity and Gene Expression of the Growth Suppressor Gene ZBED6 in Cattle

Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. Polymorphisms in its promoter and coding regions are likely to impact ZBED6 transcription and growth traits. In this study, rapid amplification of 5’ cDNA ends (5'-RACE) analysis revealed two transcription start sites (TSS) for the bovine ZBED6 starting within exon 1 of the ZC3H11A gene (TSS-1) and upstream of the translation start codon of the ZBED6 gene (TSS-2). There was one SNP in the promoter and two missense mutations in the coding region of the bovine ZBED6 by sequencing of the pooled DNA samples (Pool-Seq, n = 100). The promoter and coding region are the key regions for gene function; polymorphisms in these regions can alter gene expression. Quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution in cattle and is highly expressed in skeletal muscle. Eleven promoter-detection vectors were constructed, which enabled the cloning of putative promoter sequences and analysis of ZBED6 transcriptional activity by luciferase reporter gene assays. The core region of the basal promoter of bovine ZBED6 is located within region -866 to -556. The activity of WT-826G-pGL3 in driving reporter gene transcription is significantly higher than that of the M-826A-pGL3 construct (P < 0.01). Analysis of gene expression patterns in homozygous full-sibling Chinese Qinchuan cattle showed that the mutant-type Hap-AGG exhibited a lower mRNA level than the wild-type Hap-GCA (P < 0.05) in longissimus dorsi muscle (LDM). Moreover, ZBED6 mRNA expression was low in C2C12 cells overexpressing the mutant-type ZBED6 (pcDNA3.1+-Hap-GG) (P < 0.01). Our results suggest that the polymorphisms in the promoter and coding regions may modulate the promoter activity and gene expression of bovine ZBED6 in the skeletal muscles of these cattle breeds.


Introduction
A quantitative trait locus (QTL) is a genomic region that affects a quantitative trait or traits that vary in degree and can be controlled by multiple loci [1]. Most economically important traits of interest in cattle, such as growth, carcass, fatness and meat quality, have a multifactorial background and are controlled by environmental factors and an unknown number of QTLs. These quantitative traits arise from interactions between two or more genes and their environment and can be mapped to their underlying genes via closely linked stretches of DNA. IGF2 is a secreted peptide hormone that plays an important role in muscle tissues via both endocrine and local autocrine/ paracrine mechanisms primarily by stimulating the IGF1 receptor (IGF1R). The muscle hypoplasia phenotype of IGF1Rnull mice confirms the necessity of IGF signaling in muscle development [2][3][4]. A paternally expressed QTL affecting muscle growth, fat deposition and heart size in pigs maps to the insulin-like growth factor 2 (IGF2) region [5,6]. A single nucleotide substitution in intron 3-G3072A of IGF2 in pigs abrogates a binding site for a repressor and leads to a threefold up-regulation of IGF2 expression in skeletal muscle [7]. This quantitative trait nucleotide (QTN) is one of the rare examples in which a single base substitution underlying a complex trait has been identified and the mechanism of action is partially understood [7].
Zinc finger, BED-type containing 6 (ZBED6) is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle myogenesis and development [8]. ZBED6 has a single exon comprising more than 900 codons and two DNA-binding BED domains. ZBED genes originate from domesticated hAT DNA transposons and encode regulatory proteins of diverse function in vertebrates. The number of BED domains varies among ZBED proteins, and the ability of ZBED6 to interact with chromatin and affect transcriptional regulation is most likely a function derived from the ancestral active transposons. Phylogenetic analyses demonstrate that ZBED6 form a monophyletic group, which is distinct from the ZBED1 gene. The alignments of ZBED-derived BED domains suggest multiple independent duplications of sequences encoding BED domains after ZBED gene domestication [9]. ZBED6 is specific for placental mammals and derived from a domesticated DNA transposon [10]. In addition, ZBED6 is a novel transcription factor that appears to have evolved an essential function in the common ancestor of all placental mammals [11]. The ZBED6 gene is exclusive to placental mammals and highly conserved among species; it is found at the same genomic position and shows near 100% amino acid identity across 26 placental mammals according to the available genome sequence data [8]. An electrophoretic mobility shift assay (EMSA) showed that ZBED6 is a bona fide repressor of IGF2 transcription that interacts with the QTN region of IGF2 [8]. The functional characterization of ZBED6 shows that it has a broad tissue distribution and may affect the expression of approximately 2,500 putative downstream targets, many of which have essential biological functions in placental mammals [8].
Here, we show the promoter activity and gene expression affected by three single nucleotide substitutions in the ZBED6 gene. mRNA expression patterns in eight cattle tissues and organs were obtained using qPCR, and we determined the minimal region required for promoter activity within the upstream bovine ZBED6 gene sequences. Moreover, we assessed whether the variants affect bovine ZBED6 gene transcription and compared bovine ZBED6 expression in longissimus dorsi muscle (LDM) from wild-type and mutanttype haplotype animals to clarify that the effect of the variants on ZBED6 overexpression is associated with IGF2 expression. Thus, we hypothesized that the ZBED6 promoter and coding region polymorphisms may modulate gene expression and thereby influence skeletal muscle myogenesis and development. This hypothesis could provide an insight into the transcriptional regulation of the ZBED6 gene and the potential biological roles of its gene product.

Ethics Statement
The study protocol was approved by the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, 2004) and approved by the Institutional Animal Care and Use Committee (College of Animal Science and Technology, Northwest A&F University, China). Bovine embryos of slaughtered cows (Chinese Qinchuan) were collected from Tumen Abattoir, a local slaughterhouse in Xi'An, China. Newborn Chinese Qinchuan calf and adult Chinese Qinchuan cattle were obtained from Shannxi Kingbull Animal Husbandry Co., Ltd (Baoji, China). Adult animals were allowed access to feed and water ad libitum under normal conditions and humanely sacrificed as necessary to ameliorate suffering.

Samples and Sequencing
To explore the genetic variation of bovine ZBED6 genes, we sequenced pooled DNA samples (Pool-Seq, n = 100) from four cattle breeds in China: Nanyang cattle (NY), Qinchuan cattle (QC), Jiaxian cattle (JX) and Chinese Holstein (CH). The pooled DNA samples consisted of 25 DNA samples selected randomly from each cattle breed. NY, QC and JX are three important breeds for beef production in China, whereas CH is a dairy breed. These four breeds are the main breeds of China, and they are reared in the provinces of Henan and Shaanxi. Calves were weaned at six months of age on average and raised from weaning to slaughter on a diet of corn and corn silage. The animals of each breed were selected to be unrelated for at least three generations with the aim of having diverse lineages within each breed.
Genomic DNA was isolated from 2% heparin-treated blood samples and stored at -80 °C following standard procedures [12].
The content of DNA was estimated spectrophotometrically, and the genomic DNA was then diluted to 50-100 ng/μl. All DNA samples were stored at -20 °C for subsequent analysis.
Primers used to amplify the bovine ZBED6 gene (NCBI: AC_000173.1) were designed from a published gene sequence. Primers, fragment sizes and annealing temperature (AT) are provided in Table S1.

Tissue and Cell RNA Isolation and Quantitative Real-Time PCR (qPCR)
Samples of eight tissues and organs (heart, liver, spleen, lungs, stomach, intestines, longissimus dorsi muscle (LDM) and adipose tissue) from nine individuals (three individuals per stage) were harvested for RNA isolated within 10 min after slaughter at three key stages of myogenesis and muscle maturation: 90 days at embryo (fetal bovine, FB), 3-day-old (newborn bovine, NB) and 24-month-old (adult bovine, AB).
An analysis of gene expression patterns in six homozygous individuals from full-sibling Chinese Qinchuan cattle (i.e., in which all six animals have the same sire and dam) was conducted. The LDM sample was collected from castrated fullsibling individuals (three wild-type Hap-GCC animals and three mutant-type Hap-AGG animals) of the AB raised by Shannxi Kingbull Animal Husbandry Co., Ltd. (Baoji City, China). The specimens used in this study were collected immediately after slaughter and used for gene expression analysis. Muscle specimens for gene expression analysis were sampled and snap frozen in liquid nitrogen. Total RNA was isolated from 80-100 mg of tissue samples from QC, and RNA from C2C12 cell samples was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. All samples were subjected to reverse transcription using a cDNA high capacity kit (Invitrogen, Carlsbad, CA, USA).
The qPCR analysis using SYBR Green PCR Master Mix (Takara, Dalian, China) was performed on a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA). Data were normalized to the geometric mean of data from bovine GAPDH and ACTB, which were used as endogenous control genes. All primers for validation were designed to cross exon-exon junctions and are shown in Table S2. Relative expression levels of objective mRNAs were calculated using the ∆∆Ct method [13].

Rapid Amplification of 5' cDNA ends (5'-RACE)
The SMARTer TM RACE cDNA Amplification kit (Clontech, California, USA) was used to amplify 5'-cDNA ends of bovine skeletal muscle mRNA. First strand cDNA synthesis was carried out according to the manufacturer's instructions with bovine total RNAs, 5' CDS primer A, SMART II A oligonucleotide and TITANIUM Taq DNA polymerase. The 5'-RACE PCR reaction was performed according to the manufacturer's protocol using universal primer A mix (UPM, forward primer) and two antisense gene specific primers (GSP) to the known exon of the ZBED6 gene (GSP1 and GSP2) using the protocols provided with the RACE kit. To verify the RACE products, nested 5'-RACE PCR was carried out using nested UPM (NUPM) forward primer and the nested specific reverse primers (NGSP) of the ZBED6 gene (NGSP1). The primer sequences are noted in Table S4. Good quality RACE products were separated by 2% agarose gel electrophoresis, and the resulting bands were then extracted from the gel. Each product was ligated into pGEM® T-easy vector (Promega, Madison, WI, USA). All nested RACE products were randomly selected for sequencing. Additional 5'-RACE experiments were carried out using primers closer to the 5' end of the new sequence to confirm the 5' end of the mRNA.

Bioinformatic Analysis of Transcription Factor Binding Sites (TFBSs) in the ZBED6 Promoter Region
The in silico tool prediction of transcription factor binding sites (TFBSs) in promoter sequences was performed using the Genomatix MatInspector software (http://www.genomatix.de/) with standard settings for the highest matrix similarity [14]. This program uses a large library of weight matrices based on known in vivo binding sites to predict TFBS in nucleotide sequences.

Construction of Deletion Mutants of the ZBED6
Promoter. The genomic DNAs were extracted from the longissimus dorsi muscle tissues of the AB using a phenol extraction method [12]. Twelve different primers (listed in Table  S3) designed according to the sequence of the bovine ZBED6 gene were used in the PCR with bovine genomic DNA as a template to obtain the complete bovine ZBED6 promoter (P1) and the truncated promoter fragments (P2 to P11).
Construction of Promoter Haplotypes of ZBED6. Based on genotyping results, genomic DNA isolated from QC with homozygous GG and AA genotypes was used as a template for constructing plasmids of wild-type haplotype WT-836G-pGL3 and mutant-type haplotype M-826A-pGL3, respectively. A DNA fragment that covers the polymorphic sites (SNP1: ZBED6-Promoter -826G>A) ranging from -866 to +25 (relative to initiation codon ATG) of the 5'-regulatory region of the bovine ZBED6 gene was amplified by PCR with PrimeSTAR HS DNA polymerase (2.5 U/μL) (Takara, Dalian, China) and ligated into the pGL3-basic vector (Promega, Madison, WI, USA). The primer used for constructing vectors WT-836G-pGL3 and M-826A-pGL3, which contains Kpn I (forward primer) and Bgl II (reverse primer) restriction sites, is indicated by the lowercase letters in Table S3.
After being digested by restriction enzymes Kpn I and Bgl II, PCR products were separately inserted into the pGL3-base vector (Promega, Madison, WI, USA), which contains the luciferase reporter gene, according to the manufacturer's instructions.
Construction of the pcDNA3.1 + -ZBED6 Expression Vector. The primer pairs (Table S2) designed according to the GenBank sequence database were used in PCR to obtain the CDS sequence of the bovine ZBED6 gene. The restriction enzymes Kpn I (forward primer) and Xba I (reverse primer) were used to digest the amplified bovine ZBED6 CDS and pcDNA3.1 + vector (Invitrogen, Carlsbad, CA, USA). T4 DNA ligase (Takara, Dalian, China) was used to link the products of digestion. Standard molecular cloning techniques [12] were then used to obtain the pcDNA3.1 + -ZBED6 vectors.

Cell Culture and Treatments
Mouse myoblast cell line C2C12 was purchased from American Type Culture Collection. C2C12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin (final concentration) and 100 μg/ml streptomycin. Cells were cultured in a 5% CO 2 humidified incubator at 37 °C.

Plasmid Transfection and Dual Luciferase Reporter Assay
When cells reached 90% confluence, the proliferation medium was removed, and the cells were rinsed with phosphate-buffered saline and treated for 2 min with 0.25% trypsin to detach cells. Cells were collected, centrifuged and diluted in a proliferation medium prepared with DMEM without penicillin/streptomycin (pen/strep) and split onto two gelatincoated 24-well plates at a density of ~10 5  , which carries a renilla luciferase gene, was cotransfected with the reporter construct as described above. The experiments were performed in three replicates for each construct.
The relative activities of the full-length promoter and the truncated promoter fragments of the bovine ZBED6 gene were analyzed using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) in accordance with the manufacturer's protocol. Cells were harvested 24 h posttransfection, and firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and a BHP9504 Fluorescent Analytic Instrument (Hamamatsu, Japan). The firefly luciferase activities were normalized by the renilla luciferase activities in each well. The data given in the results section are the average of three replicates. Data are expressed as the mean ± standard error from two independent experiments.

Variants Discovery
We amplified and sequenced the ZBED6 gene from 100 DNA samples selected randomly from the study cattle population. Compared with the published gene sequence from NCBI, there were three SNPs identified in these animals, including one non-coding mutation in the promoter (SNP1: -826G>A) and two coding mutations in the CDS (SNP2: 680C>G and SNP3: 1043A>G) of the ZBED6 gene. The SNP2 and -3 are missense mutations: p. Ala 227 Gly and p. His 348 Arg. The promoter SNP was novel and submitted to the NCBI dbSNP (Table 1).

Expression Profile
To detect the tissue distribution of bovine ZBED6 and IGF2 mRNA, qPCR was carried out with cDNA from eight cattle tissues and organs (heart, liver, spleen, lungs, stomach, intestines, LDM and adipose tissue). As shown in Figure 1, the qPCR analysis showed that ZBED6 ( Figure 1A), similar to IGF2 (Figure 1B), has a broad tissue distribution in cattle tissues and organs, which is consistent with its role as the IGF2 repressor in all examined tissues and organs from nine individuals (three individuals per stage). The expression patterns of the ZBED6 and IGF2 genes were similar; the relative expression levels were decreased in all tissues except liver from day 90 bovine embryos (fetal bovine, FB) to 24-month-old adults (adult bovine, AB), respectively. Specifically, in the fetal stage, ZBED6 and IGF2 had the highest expression levels in the muscle tissues. In newborn stage, ZBED6 and IGF2 had the highest expression levels in the adipose and lung tissues, respectively. In adult stage, ZBED6 and IGF2 had the highest expression levels in the heart and liver, respectively.

Transcription Start Site (TSS) Mapping
To verify the location of the TSS in the ZBED6 gene, we performed 5'-RACE experiments on bovine skeletal muscle total RNA. Three reverse gene-specific primers were designed for the PCR step that follows cDNA synthesis in the 5'-RACE process. Primers GSP1 and GSP2 anneal just downstream of the TSS and the exon of the ZBED6 gene.
The 5'-RACE PCR on bovine total RNA was used as a template together with primers GSP1 and GSP2. The bands that were visible at approximately 2.2 kb and 1.5 kb were close to the expected product size (Figure 2A), and there were some faint bands of low molecular weight (<250 bp) of the crossdimer products between the GSP1 and 5'-RACE UPM primers (as verified by sequencing) (Figure 2A).
To increase specificity and sensitivity, a second round of PCR was performed using nested primers on the products of the 5' -RACE PCR. The expected amplification sizes with the NCBI-predicted TSS were 584 bp and 461 bp for NGSP1 ( Figure 2A). Sequence analysis revealed a partial alignment of the 584 bp (TSS-1) and 495 bp (TSS-2) NGSP1 product, with ZBED6 starting within exon 1 of the ZC3H11A gene (TSS-1) and upstream of the ATG of the ZBED6 gene (TSS-2) ( Figure  2B and 2C). Analysis of these sequences demonstrated that the products corresponded to two distinct transcripts starting at -123 and -32 with respect to the ZBED6 translation start site, which will be referred to as +1 throughout this report ( Figure  2C; 2D). Conventionally, transcriptional initiation is believed to occur from a single focused "TSS" [16]. Previous experiments have suggested that ZBED6 is an intronless gene located in the first intron of ZC3H11A, and the ZBED6 gene is cotranscribed with ZC3H11A by using a common promoter [8].
When the ZC3H11A first intron containing ZBED6 is present in the transcript, only ZBED6 protein is translated because it contains the first translated codon and a termination codon. Transcripts of the ZC3H11A gene will be translated into proteins only when the first intron is spliced out [8,11].

Bioinformatic Analyses Predict the Allele-Dependent Presence of Different TFBS in the ZBED6 Promoter Region
Bioinformatic analyses using the Genomatix software predicted transcription factors with the highest core matrix similarities and revealed 481 cis-acting elements in the 1.5 kb region (5' relative to the translation start site) of the wild-type (Table S5) and mutant-type (Table S6) ZBED6 promoter region. Accordingly, a total of 82 acting elements were scored on both strands of the promoter core sequence (-866 to -556), of which 44 were located in the plus strand and 38 in the minus strand. Several binding sites for transcription factors were predicted for the region containing SNP1 (-826G>A); the homeobox gene family -1 (HOMF), paired box gene 2 (PAX2) and promyelocytic leukemia zinc finger (PLZF) directly span the 826 position. Interestingly, the PLZF binding site is present only when the ZBED6 promoter sequences contain the 826 major "A" allele, because the 826 major "A" allele is part of the binding site core sequence of PLZF ( Figure 3A, B and C).

Promoter Analysis of the ZBED6
We next sought to determine the minimal region required for promoter activity within the upstream bovine ZBED6 gene sequences. A series of deletion constructs from the 5'-flank region of this gene was generated via PCR-based approaches ( Figure 4A). These deletion constructs were transiently transfected into C2C12 cells, and luciferase activity was detected. The results showed that deletion constructs P1 to P6 displayed elevated promoter activity relative to P0 ( Figure 4A). They indicated a potential enhancer located between nucleotides -101 and +25 because this region was adjacent to the translation initiation codon (ATG) and far away from the TSS [15]. Moreover, deletion construct P8 (nucleotides -866 to +25) had significantly elevated promoter activity relative to others, leading us to conclude that the core region of the basal promoter of bovine ZBED6 is located within the -866 to -556 sequence upstream of ZBED6, which is a region in the vicinity of the TSS1 ( Figure 4A).
By generating a series of deletion constructs of the bovine ZBED6 upstream region, the optimal promoter was defined to be the 311 bp (-866 to -556) region upstream of the initiation codon. Sequence analysis of this region showed that it lacked a TATA box, which is known to recruit transcriptional machinery needed for efficient expression of a gene [16]. Recent bioinformatics studies have suggested that the majority of mammalian gene promoters lack a "TATA box", have multiple TSSs and are highly GC-rich [17][18][19][20]. Our 5'-RACE analysis demonstrated that the ZBED6 upstream region contained two transcriptional start sites (Figure 2A) and had a significantly high GC content in the three CpG islands (51.96%, 54.17% and 55.70%.) ( Figure 2E).

Comparison of the Promoter Region of ZBED6 Harboring Different Haplotypes in Driving Transcription
To assess whether the haplotypes affect bovine ZBED6 gene transcription, the 896 bp fragment spanning the 5'flanking region from -1481 to +25 (relative to initiation codon ATG) was cloned and linked to a luciferase reporter construct. Two constructs differing only at promoter -826 were obtained, both of which harbor the identified haplotypes, namely the wildtype haplotype WT-826G-pGL3 and the mutant-type haplotype   Sequence that was recognized by Genomatix MatInspector to contain a TFBS and also the "A" allele of the SNP1 (B) or the "G" allele of the SNP1 (C). Nucleotides in capitals denote the core sequence used by MatInspector. Square box nucleotides highlight the position of the respective polymorphism. The plus-strand indicates that a TFBS sequence was found for the positive strand, and the minus-strand indicates that a TFBS sequence was found for the opposite strand.   Figure 4B, the activity of WT-826G-pGL3 in driving reporter gene transcription is significantly higher than that of the M-826A-pGL3 construct (P < 0.01). Of the two haplotypes identified in C2C12 cells, WT-826G-pGL3 has the highest activity, and M-826A-pGL3 has the lowest activity.

Comparison of mRNA Expression in LDM from Homozygous Wild-Type and Mutant-Type Animals
As described above, an in vitro study indicated that compared with WT-826G-pGL3, M-826A-pGL3 has lower activity in driving reporter gene transcription. The bovine ZBED6 and IGF2 mRNA expression in LDM was compared between full-sibling cattle (share the same sire and dam) carrying the homozygous wild-type haplotype GCA (Hap-GCA, n = 3) and the homozygous mutant-type haplotype AGG (Hap-AGG, n = 3). The results indicated that the bovine ZBED6 mRNA level was lower in mutant-type Hap-AGG (0.8123±0.041) than in wild-type Hap-GCA (1.0000±0.072) in LDM (P < 0.05); however, the mutant-type Hap-AGG (1.2058±0.135) exhibited a higher IGF2 mRNA level than the wild-type Hap-GCA (1.0000±0.052) (P < 0.05) ( Figure 5A). This result suggested that ZBED6 acts as a loss-of-function mutation in the homozygous mutant-type individuals and that the ZBED6 mutation alleles lead to up-regulation of IGF2 expression in the skeletal muscle of cattle.

Over-expression of ZBED6 Is Associated with IGF2 Expression
An interesting avenue of research involves identifying the mechanism for the association between the two coding region mutations (SNP2 and 3) and growth traits in beef cattle. The coding region is the key region for gene function; the polymorphisms in this region can alter gene expression. To further confirm the effect of ZBED6 overexpression on the regulation of IGF2 expression, we used qPCR to detect the amount of ZBED6 and IGF2 mRNA expression in the C2C12 cells from overexpressed wild-type Hap-CA and mutant-type Hap-GG ZBED6. As shown in Figure 5B, the ZBED6 mRNA expression level was 2.8284±0.289 in the wild-type (pcDNA3.1 + -Hap-CA) and 1.8532±0.248 in the mutant-type (pcDNA3.1 + -Hap-GG), and the ZBED6 levels were low in mutant-type ZBED6-overexpression cells (P < 0.05). The expression of IGF2 mRNA was significantly upregulated by mutant-type (8.4561±0.386) and wild-type (4.6589±0.239) cells compared with the vector control.

Discussion Expression Profile
In the present study, the expression profile results indicated that ZBED6 and IGF2 had the lowest expression level at FB and gradually increased in abundance during the three different developmental stages of the liver. In contrast, the highest expression levels occurred at FB and gradually decreased in abundance during the three different developmental stages in the other tissues and organs. However, the expression levels in the spleen did not change between 90 days FB and 3-day-old NB but significantly decreased in the 24-month-old AB spleen organ. The expression patterns and levels of the ZBED6 and IGF2 genes in all tested bovine tissues and organs suggests that these two genes may play an important role during development, cell proliferation and growth in placental mammals. The functional characterization of ZBED6 shows that it may affect the expression of thousands of other genes in addition to IGF2 that control fundamental biological processes [8]. Therefore, further research is needed to discover their regulatory functions.

Bioinformatic Analyses Predict the Allele-Dependent Presence of Different TFBSs
Despite the importance of ZBED6 in muscle development, little is known about its regulation. To identify the possible TFBS in the bovine ZBED6 promoter, we analyzed the promoter sequence using web-based software. The results showed that there were many potential factors; several binding sites for transcription factors were predicted for the region containing the myocyte enhancer factor-2 (MEF2), POU domain, class 1, transcription factor 1 (PIT1) and MYOD. MEF2 transcription factors control muscle-specific and growth factorinducible genes [21]. MEF2 was originally identified as a transcription factor complex through promoter analysis of the muscle creatine kinase (mck) gene to identify nuclear factors interacting with the mck enhancer region during muscle differentiation [22]. PIT1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals and the regulation of many genes, including the growth hormone gene [23]. PIT1 was shown to be associated with the economically relevant traits of many livestock, such as porcine carcass traits [24], bovine milk yield and conformation traits [25] and milk performance traits [26]. MyoD is a muscle-specific transcription factor that belongs to a family of proteins known as myogenic regulatory factors (MRFs); it is a protein with a key role in regulating muscle differentiation. MyoD can activate downstream myogenic structural genes and myogenic conversion in many different cell types. MyoD also activates its own transcription and the transcription of myogenin, another member of the MyoD gene family [27]. Recent studies have demonstrated that MyoD initiates a feedforward regulation of skeletal muscle gene expression and predict that MyoD binds directly to many genes expressed during differentiation. MyoD binds on a genomewide basis and has the ability to broadly alter the epigenome in myoblasts and myotubes [28].
The presence of all of the described transcription factor binding sites in the bovine ZBED6 promoter indicates that it is under a high level of transcriptional control by several transcription factors. Future research should address the interaction between all of these sites to provide a better understanding of ZBED6 regulation and the myogenic process.

Promoter Analysis of the ZBED6 Gene
We carried out the deletion mutant analysis to demonstrate the effect of the transcription factors on the activity of the promoter. The results of a dual-luciferase reporter assay ( Figure 4) showed that the full-length promoter fragment (P1) has a strong promoter activity (the empty reporter vector pGL3base as control), indicating that the obtained promoter region was correct. P2 to P11 had promoter activity similar to or higher than the full-length promoter, showing that these regions contain transcription factor binding sites mediating positive regulation. Among them, the activity of P8 was significantly higher than that of other promoter fragments.
The ZBED6 gene is located in the first intron of zinc finger CCCH type containing 11A (ZC3H11A), but the ZBED6 protein has no significant sequence similarity to the ZC3H11A protein.
Chromatin immunoprecipitation (ChIP) followed by highthroughput DNA sequencing (ChIP-Seq) revealed approximately 2,500 genomic regions bound by ZBED6, including the IGF2 locus, and approximately 1,200 genes had at least one putative ZBED6 binding site occurring within 5 kb of the TSS in C2C12 cells. The gene ontology analysis results suggest that ZBED6 is an important regulator of development, cell proliferation and growth [8].
SNP1 is located in the 5' flanking region of the ZBED6 gene. Normally, transcription factors and cis-acting elements are located in this region, and these elements are the recognition sequences for transcription factors. Transcription factors regulate the start and efficiency of gene transcription by binding to cis-acting elements. Therefore, we speculated that mutations in the recognition sequences can change the binding between the transcription factors and cis-acting elements, thus regulating gene transcription and affecting the related signaling pathways. To test this inference, a previous study performed an association analysis between the genotype and bovine growth traits, and the results revealed that the cattle with genotype SNP1-AA could be selected to obtain greater growth traits [29]. We hypothesize that the mutations in the flanking region of the ZBED6 gene may change the cis-acting elements associated with this gene and thus the transcription of this gene. The mutations in the promoter of the bovine lactoferrin (LTF) gene lead to different basal transcriptional activities that are associated with the lactoferrin levels in milk [30].
In the current study, through conjoint analysis of the data in Table S5 and S6, we found that the mutant-type individuals (-826A) (Figure 3) with the recognition sequences of the promyelocytic leukemia zinc-finger (PLZF) transcription factors (Table S6, Figure 3) exhibited better performance than the individuals with wild-type SNP1-G. The PLZF belongs to the POK (POZ and Krüppel) family of transcriptional repressors. This domain, common to several zinc finger-containing transcription factors, mediates protein-protein interactions and allows POZ domain proteins to participate in various differentiation pathways, including hematopoiesis, adipogenesis, osteoclastogenesis and muscle differentiation [31]. Therefore, we predict that individuals with mutant-type (-826A) in the recognition sequences of cis-acting elements may have better growth performance.

Overexpression of ZBED6 Is Associated with IGF2 Expression
The results suggest that overexpression of mutant-type ZBED6 significantly inhibited the expression level of the ZBED6 gene. In contrast, the IGF2 mRNA expression level was significantly up-regulated in C2C12 cells. This result is similar to the increased IGF2 expression in skeletal muscle of pigs carrying the mutation at the ZBED6 target site in IGF2 gene [7]. ZBED6 acts as a repressor of IGF2 transcription in skeletal muscle myogenesis and development [8]. These studies suggested that ZBED6 was a major regulator of IGF2 expression in skeletal muscle but a repressor of the constitutive expression of IGF2.

Conclusions
Three SNPs of the ZBED6 gene exist in cattle population; these SNPs have some effects on ZBED6 transcription activity and mRNA expression. Results obtained with the promoter analysis and gene expression data suggest that the mutanttype M-826A-pGL3 has the lowest promoter activity, the mutant-type Hap-AGG and pcDNA3.1 + -Hap-GG significantly inhibited ZBED6 expression and up-regulated IGF2 mRNA and ZBED6 negatively regulated the expression of the IGF2 gene and thus may affect IGF2 transcription in skeletal muscle myogenesis and development. However, we conclude that further research and validation of the various allelic effects, functional mechanisms and bioactivity are needed in an independent sample. These results may provide important insights into genes that are associated with the adaptation and specialization of beef cattle breeds in China.