Fig 2: Structural characteristics of the bovine SIX1 gene. (a) The detailed genomic, mRNA and protein components containing the 5'/3'-untranslated region (5'/3'-UTR), and the open reading frame (ORF). (b) 5' Regulatory region sequence of the bovine SIX1 gene. Arrows mark the transcription initiation sites. The translational start site (ATG) is shown in red letters. The transcription factor binding sites are boxed, and primer sequences are underlined with the respective names shown below the line. The CpG island is indicated by a blue box. (c) Schematic representation of the relative loci of the E-box motif, MyoD, CEBP, SIX4, CREB, PAX7, MyoD, MEF2, TEAD4 and MEF3 motif binding sites in the SIX1 promoter. Arrows mark the motifs and transcription factors recognition sites. Dashed lines indicate the GC percentage as represented on the y-axis and the x-axis denotes the bp position in the 5' untranslated region. Coordinates are given relative to the translational start site (shown as +1).

Fig 3: Multi-alignment sequence analysis of the MyoD, PAX7, MEF3 and CREB transcription factor binding sites in the promoter region of the SIX1 gene in cattle, pigs, goats, humans, sheep, and mice.

Fig 4: Analysis of MyoD, PAX7, MEF3, CREB and MyoG binding sites by site-directed mutagenesis and siRNA interference of the bovine SIX1 promoter constructs in two cell lines. (a) A series of plasmids containing 5' unidirectional deletions of the promoter region of the SIX1 gene (pGL3-P1, pGL3-P2, pGL3-P3, pGL3-P4 and pGL3-basic) fused in-frame to the luciferase gene were transfected into 3T3-L1 and C2C12 cells. After 5 h, we replaced the transfection mixture with DMEM with 2% HS (myotubes). (b) Site-directed mutagenesis was carried out in the construct pGL-689/+170. The different constructs were transiently transfected into C2C12 and 3T3-L1 cells. (c) MyoD, PAX7, CREB and MyoG knockdown by siRNA co-transfected with pGL-689/+170 in C2C12 or 3T3-L1 cells. The NC siRNA was used as a negative control. MyoG overexpression using the constructed pcDNA3.1 (+) expression plasmid. After 48 h, the cells were harvested for the luciferase assays. The results are expressed as the means ± SD in arbitrary units based on the firefly luciferase activity normalized against the Renilla luciferase activity for triplicate transfections. The error bars denote the standard deviation. Paired Student’s t-test was used to detect significant differences. *P < 0.05 and **P < 0.01. Data are shown as the means ± SD (n = 3).

Fig 5: EMSAs showing direct binding of MyoD, PAX7, CREB and MyoG to the SIX1 promoter in vitro. Nuclear protein extracts were incubated with 5'-biotin labelled probe containing the MyoD, PAX7, CREB or MyoG binding site in the presence or absence of competitor (lane 2), 50× mutation probe (lane 3) and 50× unlabelled probes (lane 4). The super-shift assay was conducted using 10 µg of anti-MyoD, anti-PAX7, anti-CREB or anti-MyoG antibodies (lane 5). The arrows mark the main complexes. Muscle NE, QCMC nuclear protein extracts (a,b,c and d). Adipocytes NE, adipocyte nuclear protein extracts (e).