Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene

Ethics Statement

All animal procedures were performed according to guidelines laid down by the China Council on Animal Care, and the protocols were approved by the Experimental Animal Manage Committee (EAMC) of Northwest A&F University.

Molecular cloning and sequence analysis

The complete PDHB coding sequence (CDS) with primers (PDHB-CDSF/R, Table 1) and longissimus dorsi muscle cDNA was cloned. To obtain the 5’-regulatory region, we searched the PDHB gene in the UCSC genome database (http://genome.ucsc.edu/cgi-bin/hgGateway). We isolated a bovine genomic sequence (bosTau4_refGene_NM_001035435 range = chr22:43767058–43774471) and cloned 1899 bp of 5’-regulatory region sequence with the primers (PDHB-PF/R, Table 1). We used genomic DNA extracted from Qinchuan cattle blood as template and applied 2×GC Buffer I (TaKaRa, Biotechnology Co. Ltd, Dalian, P.R.C.) to amplify 5’-regulatory region sequence. The potential transcription factor binding sites were analyzed using the Genomatix suite (http://www.genomatix.de/), TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess?RQ=WELCOME) and TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html). CpG islands were predicted using Meth Primer (http://www.urogene.org/methprimer/).

We obtained the 4kb5'-regulatory region of PDHB in cattle (gi|258513345:43457246–43461246), sheep (gi|417531901:42930424–42934424), goat (gi|541128965:42734750–42738750), porcine (gi|347618781:c44131745-44127745), dog (gi|357579611:32181538–32185538), human (gi|568815595:c58437807-58433807), mouse (gi|372099096:c8176930-8172930), rat (gi|666183559:18537242–18541242) and chicken (gi|358485500:c11816992-11812992) from the genome database at the National Center for Biotechnology Information (NCBI). We performed multi-alignments of the 5'-regulatory region among the nine species using ClustalX 2.0.

Structural and phylogenetic tree analysis

We used the SMART database (http://smart.embl-heidelberg.de/) to predict putative domains. We constructed the phylogenetic tree as previously described [21].

5’-Rapid amplification of cDNA ends (5’-RACE)

To identify the transcription initiation sites, 5’-RACE from total RNA of subcutaneous fat was performed according to the manufacturer’s protocol using the SMART^TM RACE Kit (Clontech Inc, Palo Alto, CA, USA). We applied the nest PCR primers (PDHB-GSP1 and PDHB-GSP2, Table 1) to obtain the 5’-end of the PDHB gene and used touchdown PCR in the first PCR with conditions as previously described [21]. The second PCR template was a 20-fold dilution of the first PCR products.

Real-time PCR analysis of spatial expression pattern

We obtained sixteen tissue samples (longissimus dorsi muscle, kidney, heart, biceps femoris, liver, subcutaneous fat, spleen, lung, cecum, rumen, reticulum, omasum, abomasums, testis fat, large intestine and small intestine) from three adult Chinese indigenous Qinchuan cattle and selected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous reference. We used the primers of PDHB-RT-F/R (Table 1) and GAPDH-F/R (Table 1) [22] for this assay. The cycling conditions were according to SYBR Premix Ex Taq^TMⅡ (TaKaRa, Biotechnology Co. Ltd, Dalian, P.R.C.), using the ABI 7500 RT-PCR system (Applied Biosystems, USA). We used the 2^-△△Ct method for this assay.

Cell culture, transfection and dual-luciferase reporter assay

We cultured the mouse myoblast cell line (C2C12) and the 3T3-L1 cell line in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO-Invitrogen) under humidified air containing 5% CO2 at 37°C. 5’-nested PCR primers (PDHB-P1~P6, Table 1) and common 3’ primer (PDHB-R, Table 1) were used to amplify six serial deletion fragments of the 5’-flanking region. Then these fragments were cloned into the KpnI-SmaI site of the pGL3-basic vector (Promega Corp.). After sequencing verification, we extracted the plasmids with an Endo-free Plasmid Mini Kit (OMEGA, USA). We co-transfected the plasmids (0.8 μg) and the lipofectamine 2000 (2μl) into C2C12 or 3T3-L1 cells grown in 24-well plates with 10 ng pRL-TK (Promega Corp.). At 5 h after transfection, we replaced the media with DMEM with 2% horse serum (HS) (GIBCO-Invitrogen) and incubated for 40 h to induce differentiation of the C2C12 myoblasts into myotubes. We performed all remaining steps as previously described [21].

Site-directed mutagenesis

We mutated the transcription factor-binding sites for MYOG and C/EBPß with the corresponding primers (mMYOG-F/R, mC/EBPß-F/R, Table 1) using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). PCR was carried out as previously described [21].

Electrophoretic mobility shift assay (EMSA)

After differentiation of the C2C12 myoblasts into myotubes for 7 days, we prepared nuclear protein extracts as previously described [21,23]. We incubated probes of a 200 fmol of 5'-biotin labeled MYOG (or C/EBPß) with 10μg nuclear extracts of C2C12 myotubes (or 10μg3T3-L1nuclear extracts), 2μl 10×binding buffer, 1μl 50% Glycerol, 1μl MgCl2 and 1μl poly (dI.dC) in a volume of 20 μl. We incubated 5μg of antibodies myogenin (sc-12732×, Santa Cruz Biotechology, CA) (or C/EBPß (sc-150×, Santa Cruz Biotechology, CA)) with the nuclear extracts before adding the labeled probes. We used 6% polyacrylamide gels to separate DNA-protein complexes. Several steps followed according to the Light Shift® Chemiluminescent EMSA Kit (Pierce Corp., Rockford, IL, USA) Manufacturer’s Instructions.

Chromatin immunoprecipitation (ChIP) assay

We isolated longissimus dorsi muscle and inguinal fat from bulls at 3 days after birth (n = 3). We conducted the ChIP assay using the EZ-ChIP™ Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s protocol. We cross-linked the DNA-protein complexes with 37% formaldehyde and neutralized with glycine. After sonication, we added about 200–1000 bp of fragmented chromatin into the ChIP dilution buffer. We immunoprecipitated an equal amount of chromatin overnight at 4°C with 4μg of the myogenin (or C/EBPß) antibodies and normal mouse IgG. We then collected the immunoprecipitated products with Protein A+G coated magnetic beads. We eluted the bound chromatin in ChIP Elution Buffer and digested with proteinase K, then purified the DNA for PCR analysis. We used the primers (ChIP-MYOG-F/R and ChIP-C/EBPß-F/R, Table 1) and the negative control primers (ChIP-control-F/R, Table 1) in the RT-PCR and ChIP-QPCR. % Input = 2^{(-△Ct (Ct [ChIP]—(Ct [Input]—Log2 (Input Dilution Factor))))} [24]. As a negative control, we used the immunoprecipitate products from the normal mouse IgG group.

Statistical analysis

All values are expressed as mean±standard deviation (SD). We analyzed the differences between groups with a Student’s two-tailed T-test. * P<0.05. ** P<0.01. n = 3.

Molecular cloning and sequence analysis

Based on the bovine PDHBc DNA sequence (GenBank No. NM_001035435.3), we obtained a1576 bp cDNA. It contained an open reading frame (ORF) of 1080 bp, which had 359 amino acids (aa) encoded with a calculated molecular mass of 39.13 kDa and an isoelectric point (pI) of 6.466. Our cDNA consisted of a 5’-UTR of 91 bp and a 3’-UTR of 405 bp with a consensus AATAAA polyadenylation signal 16 bp upstream of the poly (A) stretch. The bovine PDHB gene spanned approximately 5.49 kb on the genome, containing 10 exons and 9 introns (Fig 1A). In addition, the bovine PDHB protein contains two putative domains: the transketolase, pyrimidine binding domain (Transketpyr) resides in 33-208 amino acid residues and the transketolaseC domain resides in 226–350 aa (Fig 1A).

The bovine PDHB amino acid sequence shared a high similarity with other mammalian, with the following levels of identity: sheep PDHB1 (99%), goat PDHB1 (99%), porcine (97%), dog PDHB1 (96%), sheep PDHB2 (94%), goat PDHB2 (94%), mouse (94%), rat (94%), human PDHB1 (94%), human PDHB2 (89%), dog PDHB2 (86%) and chicken (82%). The phylogenetic tree indicated the bovine PDHB to be closest related to goat and sheep, and the lowest relatedness to chicken of all nine species evaluated for this study (Fig 2).

Transcription initiation site of the bovine PDHB gene

In the first and second PCR, we cloned 682 bp and 400 bp fragments, respectively (Fig 1B). Sequencing eleven positive clones identified seven different 5’ ends with 91 bp, 31 bp, 26 bp, 16 bp, 14 bp, 12 bp and 11 bp upstream from the translational start site, respectively (Fig 1C). We designated the cytosine residue (C) as +1 and found it to be located 91 bp upstream from the translational start site.

Characterization of the bovine PDHB gene 5’-regulatory region

We cloned a 1899 bp fragment of the bovine PDHB gene 5’-regulatory region spanning nucleotides from nucleotides -1724 to +175 and submitted it to GenBank (GenBank No. KJ649747). Several transcription factor-binding sites were recognized via sequence analysis of the 5’-regulatory region, including the Sp1 transcription factor (Sp1), the transcription factor Yin Yang-1 (YY1), the CCAAT/enhancer-binding protein ɑ (C/EBPɑ), myogenin (MYOG), the CCAAT/enhancer-binding protein ß (C/EBPß) and the cAMP responsive element-binding protein (CREB) (Fig 1C). Interestingly, we did not find a consensus TATA-box or CCAAT-box in the 5’-flanking region. However, using Meth Primer, a CpG island with a length of 401 bp between nucleotides -284 and +117 was predicted (Fig 1C).

Compared to the bovine PDHB gene 5’-regulatory region (GenBank No. KJ649747), the 5'-flanking sequences of cattle, goat and sheep shared 97%, 94% and 93% sequence similarity. However, it had no significant sequence similarity with the 5'-flanking sequences of porcine, dog, human, rat, mouse or chicken PDHB.

Spatial expression pattern of the bovine PDHB gene

In order to understand the role of the bovine PDHB gene products in various tissues, it was necessary to provide mRNA expression profiles via real-time PCR analysis. The results showed the highest expression in testis fat, followed by lung, cecum, reticulum, rumen, kidney, spleen, liver, large intestine, heart, small intestine, omasum, subcutaneous fat, abomasums, longissimus dorsi and biceps femoris (Fig 3). This result indicates the PDHB mRNA to be widely expressed with the highest level in testis fat, revealing the PDHB gene to be a housekeeping gene in multiple tissue types.

Transcriptional regulation of the bovine PDHB gene

To identify the transcriptional activity of the bovine PDHB gene 5’-regulatory region, we generated six serial deletion constructs in pGL3-basic containing -1725/+119, -1354/+119, -1054/+119, -790/+119, -490/+119 and -190/+119. After we transfected the construct plasmids into C2C12 cells, the transcriptional activity of the construct -490/+119 was about 25-fold higher than the construct -190/+119 (Fig 4A). The activities of all other constructs were higher than the construct -190/+119 and lower than that of the construct -490/+119. Inducing the C2C12 cells via HS after transfection, significantly increased the transcriptional activities of the six constructs and the activity of the construct -490/+119 was highest in the six constructs. After transferring the construct plasmids into 3T3-L1 cells, the transcriptional activity of the construct -490/+119 was also highest in the six constructs and the activity of the construct -190/+119 was increased compared to that of the C2C12 cells (Fig 4B). These results suggest a core functional promoter to be present in the upstream region of 490 bp from the transcription initiation site.

To identify important positive regulatory elements among the nine species, the region from nucleotides -490 to +92 was predicted by transcription factor binding sites prediction software. The result of multi-alignments revealed MYOG, C/EBPß SP1 and AP1 to be conserved in this region for domestic animals such as cattle, sheep, goat, porcine and dog (Fig 5). Mutation of the MYOG site at position -388 to -382 led to a sharp decrease in activity of 70%, while mutation of the C/EBPß site at position -331 to -325 had no effect on the promoter activity of C2C12 myotubes (Fig 6A). In contrast, mutation of the C/EBPß site at position -331 to -325 led to a decrease in activity of 36%, while mutation of the MYOG site at position -388 to -382 had no significant effect on promoter activity in 3T3-L1 cells (Fig 6B).

In order to validate interaction between the transcription factors MYOG and C/EBPß with the 5’-regulatory region of PDHB, we carried out EMSA and ChIP assays both in vitro and vivo. The EMSA results revealed that 5’-biotin labeled MYOG probes with nuclear extracts of C2C12 myotubes formed two upshifted bands (Fig 7A, lane 2). When 5’-biotin labeled MYOG probes were added with unlabeled MYOG oligonucleotides, the upshifted bands disappeared (Fig 7A, lane 3). The shifted MYOG complexes did not compete with unlabeled mutated MYOG oligonucleotides (Fig 7A, lane 4). Adding the MYOG antibody strongly diminished the upshifted bands (Fig 7A, lane 5). C/EBPß site and 3T3-L1 cells nuclear extracts exhibited similar results with MYOG site and C2C12 myotubes nuclear extracts (Fig 7B). The ChIP results revealed MYOG interacted with the MYOG binding site (Fig 8A and 8C) and C/EBPß interacted with the C/EBPß binding site in the PDHB promoter (Fig 8B and 8D).