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. 2016 Jul 5;11(7):e0157445.
doi: 10.1371/journal.pone.0157445. eCollection 2016.

Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene

Anning Li  1 , Yaran Zhang  1 , Zhidong Zhao  1 , Mingming Wang  1 , Linsen Zan  1   2
Affiliations

Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene

Anning Li et al. PLoS One. .

Abstract

The pyruvate dehydrogenase beta subunit (PDHB) is a subunit of pyruvate dehydrogenase (E1), which catalyzes pyruvate into acetyl-CoA and provides a linkage between the tricarboxylic acid cycle (TCA) and the glycolysis pathway. Previous studies demonstrated PDHB to be positively related to the intramuscular fat (IMF) content. However, the transcriptional regulation of PDHB remains unclear. In our present study, the cDNA of bovine PDHB was cloned and the genomic structure was analyzed. The phylogenetic tree showed bovine PDHB to be closely related to goat and sheep, and least related to chicken. Spatial expression pattern analysis revealed the products of bovine PDHB to be widely expressed with the highest level in the fat of testis. To understand the transcriptional regulation of bovine PDHB, 1899 base pairs (bp) of the 5'-regulatory region was cloned. Sequence analysis neither found consensus TATA-box nor CCAAT-box in the 5'-flanking region of bovine PDHB. However, a CpG island was predicted from nucleotides -284 to +117. Serial deletion constructs of the 5'-flanking region, evaluated in dual-luciferase reporter assay, revealed the core promoter to be located 490bp upstream from the transcription initiation site (+1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) in combination with asite-directed mutation experiment indicated both myogenin (MYOG) and the CCAAT/enhancer-binding protein beta (C/EBPß) to be important transcription factors for bovine PDHB in skeletal muscle cells and adipocytes. Our results provide an important basis for further investigation of the bovine PDHB function and regulation in cattle.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structure characteristics of the bovine PDHB gene.
a. Here we show the genomic, mRNA and protein components in detail. 5’- UTR:5’- untranslated region, 3’- UTR:3’- untranslated region, ORF: open reading frame, Transketpyr: transketolase, pyrimidine binding domain. b. 5’-RACE. Lane 1 and 2 are products of the first and second PCR, respectively. Lane M represents the marker of DL2000. c. 5’-regulatory region sequence of bovine PDHB gene. Arrows mark the transcription initiation sites. The cytosine residue is designated as +1. The transcription factor binding sites are boxed. The primers are underlined with the respective names below the line. The CpG island is indicated with red color.
Fig 2
Fig 2. Phylogenetic tree analysis of PDHB.
We calculated 8000 bootstrap replicates to bootstrap confidence values.
Fig 3
Fig 3. Spatial expression analysis of bovine PDHB mRNA.
We normalized the mRNA expression levels of PDHB to those of GAPDH. Error bars represent the standard deviation (SD) (n = 3).
Fig 4
Fig 4. Promoter activity analysis of the bovine PDHB gene.
a. We transferred six serial deletion constructs in pGL3-basic into C2C12 cells. After 5 h we replaced the transfection mixture with DMEM with 5% FBS (myoblasts) or 2% HS (myotubes). b. We transferred the same constructs into 3T3-L1 cells. We normalized relative luciferase activities to Renilla luciferase activity. The transcription factor binding sites of MYOG and C/EBPß are indicated with closed circles and ellipses, respectively. *, P<0.05. Error bars represent the SD (n = 3).
Fig 5
Fig 5. Multi-alignments sequence analysis of the core functional promoter of bovine PDHB in relation toother mammals.
The transcription factor binding sites are marked with boxes. The nucleotide sequence is numbered in 5'-regulatory sequence of the bovine PDHB gene (GenBank No. KJ649747).
Fig 6
Fig 6. Functional analysis of the mutated MYOG and C/EBPß sites.
We transferred the mutated sites MYOG and C/EBPß into C2C12 myotubes (a) and 3T3-L1 cells (b). **, P<0.01. Error bars represent the SD (n = 3).
Fig 7
Fig 7. EMSA involving 5’-biotin labeled MYOG and C/EBPß probes.
a. 5’-biotin labeled MYOG probes and nuclear extracts of C2C12 myotubes. Lane 1: MYOG probes; lane 2: MYOG probes with nuclear extracts of C2C12 myotubes; lane 3: MYOG probes and nuclear extracts with a 125-fold unlabeled MYOG oligonucleotides; lane 4: MYOG probes and nuclear extracts with a 125-fold unlabeled mMYOG oligonucleotides. lane 5: MYOG probes and nuclear extracts with myogenin antibodies. b. 5’-biotin labeled C/EBPß probes and nuclear extracts of 3T3-L1 cells. Lane 1: C/EBPß probes; lane 2: C/EBPß probes with nuclear extracts of 3T3-L1 cells; lane 3: C/EBPß probes and nuclear extracts with 125-fold unlabeled C/EBPß oligonucleotides; lane 4: C/EBPß probes and nuclear extracts with 125-fold unlabeled mC/EBPß oligonucleotides. lane 5: C/EBPß probes and nuclear extracts with C/EBPß antibodies.
Fig 8
Fig 8. ChIP assay of MYOG and C/EBPß binding to PDHB promoter in vivo.
We analyzed immunoprecipitated products for MYOG (a) and C/EBPß (b) antibodies via RT-PCR. We analyzed immunoprecipitated products for MYOG (c) and C/EBPß (d) antibodies via ChIP-QPCR. We used total chromatin from muscle (a and c) and fat (b and d) as the input. We used normal mouse IgG as the negative control antibodies. **, P<0.01. Error bars represent the SD (n = 3).
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Grants and funding

This research was funded by the National Natural Science Foundation of China (Grant No. 31402042), National 863 Program of China (Grant No. 2013AA102505) and the Northwest A & F University Special Funds of Central Colleges Basic Scientific Research Operating Expenses (Grant No. 2014YB009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.