Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts
Yong-Zhen Huang 1, Liang-Zhi Zhang 1, Xin-Sheng Lai 1, Yu-Jia Sun 1, Cong-Jun Li 2, Xian-Yong Lan 1, Chu-Zhao Lei 1, Chun-Lei Zhang 3, Xin Zhao 1, and
Hong Chen *1
1. College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling Shaanxi,
People’s Republic of China,
2. United States Department of Agriculture–Agricultural Research Service, Bovine Functional Genomics Laboratory, Beltsville, Maryland, United States of
America,
3. Institute of Cellular and Molecular Biology, Jiangsu Normal University, Xuzhou Jiangsu, People’s Republic of China
Table S1.
Primer used in qPCR and ZBED6 overexpression analyses.
Gene
IGF2-SNPs detection
ZBED6-qPCR
IGF2-qPCR
GAPDH-qPCR
ACTINB-qPCR
pcDNA3.1+-ZBED6 1
Ad-ZBED6 1
pDsRed-ZBED6 2
Primer sequences (5’-3’)
Bovine: F: CTCTTCACCAGTGCTTCAACTCTC; R: GCACCGCGCCGCTGCGGAT
Mouse: F: CAAGACATCTGCAGTTTGGAATTT R: TGTCGTTGAAGTGTTGAAGTTCCTA
Bovine: F: GGAACAAGAGCCAAGAC; R: CCAATGGATGGGATGAG
Mouse: F: CGTGGCATCGTGGAAGAGT; R: ACACGTCCCTCTCGGACTTG
Bovine: F: TCTGTGCGGCGGGGAGCTGGT; R: AGTCTCCAGCAGGGCCAGGTCG
Mouse: F: AGACAGCCGCATCTTCTTGT; R: TTCCCATTCTCAGCCTTGAC
Bovine: F: CGACTTCAACAGCGACACTCAC; R: CCCTGTTGCTGTAGCCAAATTC
Mouse: F: CAGAGCAAGAGAGGCATCCTC; R: GTCCAGACGCAGGATGGCATG
Bovine: F: GTCATCACCATCGGCAATGAG; R: AATGCCGCAGGATTCCATG
Bovine: F: CGGggtaccGCCACCATGcaccaccaccaccaccacAGTGTATGTACCTTAAGTGTACC
Bovine: R: GCtctagaTTAAGGTAATATTTCTTTTTCATTGC
Bovine: F: CGGggtaccATGcaccaccaccaccaccacAGTGTATGTACCTTAAGTGTACC
Bovine: R: GCtctagaTTAAGGTAATATTTCTTTTTCATTGC
Bovine: F: CCGgaattcGCCACCATGAGTGTATGTACCTTAAGTGTACC
Bovine: R: GGggtaccGGAGGTAATATTTCTTTTTCATTGC
AT (oC) 3
SAF (bp) 4
59.5
60.0
60.0
60.0
60.0
60.0
60.0
60
60
55.5
578
135
174
94
154
239
118
363
84
2943
55.0
2943
55.0
2943
F: Forward primer; R: Reverse primer.
1
The primer used for constructing vectors pcDNA3.1+; squared nucleotides: which contains Kpn I (forward primer) and Xba I (reverse primer) restriction
sites, as indicated by the lower-case letters; The protective base pairs “CGG” and “GC” were added in order to express in the 5' terminal of primer has also
deliberately added black bold font. Underlined nucleotides: mark recognition site for the His-tag sequences;
2
The primer used for constructing vectors pDsRed-N1; squared nucleotides: which contains EcoR I (forward primer) and Kpn I (reverse primer) restriction
sites, as indicated by the lower-case letters; The protective base pairs “CCG” and “GG” were added in order to express in the 5' terminal of primer has also
deliberately added black bold font.
3
AT=Annealing temperature.
4
SAF=Size of amplification fragment.
Table S2.
Primer pairs used for creation of deletion mutation analyses in bovine ZBED6 gene.
Primer name
439-G-SNP-pGL3
439-A-SNP-pGL3
396-G-SNP-pGL3
396-A-SNP-pGL3
232-G-SNP-pGL3
232-A-SNP-pGL3
Primer sequence (5’-3’) 1
Location 2
F: CGGggtaccGGGCCGCGGCTTCGCCTAGGCTCGCA
R:CCCaagcttGGATGGAGAGCTTCGTTTGGGA
F: CGGggtaccGGGCCGCGGCTTCGCCTAGGCTCACA
R:CCCaagcttGGATGGAGAGCTTCGTTTGGGA
F: CGGggtaccGGGCCGCGGCTTCGCCTAGGCTCGCA
R: CCCaagcttGCACCGCGCCGCTGCGGAT
nt-3083~nt-3108
nt-3522~nt-3544
nt-3083~nt-3108
nt-3522~nt-3544
nt-3083~nt-3108
nt-3479~nt-3498
F: CGGggtaccGGGCCGCGGCTTCGCCTAGGCTCACA
R: CCCaagcttGCACCGCGCCGCTGCGGAT
F: CGGggtaccGGGCCGCGGCTTCGCCTAGGCTCGCA
R:CCCaagcttACGTCGCTTCTCCCGATTTTG
F: CGGggtaccGGGCCGCGGCTTCGCCTAGGCTCACA
R:CCCaagcttACGTCGCTTCTCCCGATTTTG
nt-3083~nt-3108n
t-3479~nt-3498
nt-3083~nt-3108n
t-3315~nt-3336
nt-3083~nt-3108
nt-3315~nt-3336
AT (oC) 3
Size (bp) 4
60.0
439
60.0
439
60.0
396
60.0
396
60.0
232
60.0
232
F: Forward primer; R: Reverse primer.
1
The primer used for constructing vectors pGL3-P (1-6), squared nucleotides: which contains Kpn I (forward primer) and Bgl II (reverse primer) restriction
sites, as indicated by the lower-case letters. The protective base pairs “CGG” and “GGA” were added in order to express in the 5' terminal of primer has also
deliberately added black bold font.
2
The location and size of each deletion fragment is indicated to the left of each bar relative to the intron 3 start codon; nt: nucleotide(s).
AT=Annealing temperature.
4
SAF=Size of amplification fragment.
3
Table S3.
Characteristics of shRNA used in bovine ZBED6 gene.
shRNA 1
shRNA-18-sense
shRNA-18-antisense
shRNA-906-sense
shRNA-906-antisense
shRNA-1698-sense
shRNA-1698-antisense
shRNA-2819-sense
shRNA-2819-antisense
shRNA-NC-sense
shRNA-NC-sense
1
Sequence (5’-3’)
GATCCGTGTACCAGTTTCCTCACTgAGTACTgAGTGAGGAAACTGGTACACTTTTTTC
TCGAGAAAAAAGTGTACCAGTTTCCTCACTcAGTACTcAGTGAGGAAACTGGTACACG
GATCCCTTCAACACTTCAACGACAgAGTACTgTGTCGTTGAAGTGTTGAAGTTTTTTC
TCGAGAAAAAACTTCAACACTTCAACGACAcAGTACTcTGTCGTTGAAGTGTTGAAGG
GATCCGTTCTTCTAATGTAGTACAgAGTACTgTGTACTACATTAGAAGAACTTTTTTC
TCGAGAAAAAAGTTCTTCTAATGTAGTACAcAGTACTcTGTACTACATTAGAAGAACG
GATCCGAACAGCTGATGTTTCTGAgAGTACTgTCAGAAACATCAGCTGTTCTTTTTTC
TCGAGAAAAAAGAACAGCTGATGTTTCTGAcAGTACTcTCAGAAACATCAGCTGTTCG
GATCCTTCTCCGAACGTGGCACGAgAGTACTgTCGTGCCACGTTCGGAGAATTTTTTC
TCGAGAAAAAATTCTCCGAACGTGGCACGAcAGTACTcTCGTGCCACGTTCGGAGAAG
Four shRNAs (numbers stand for their position in cDNA) were designed, and each shRNA was added with restriction sites BamH I and Xho I. The loop
domain (between the lower-case nucleotides) contained a Scal I site.