Genetically regulated epigenetic transcriptional activation of retrotransposon insertion confers mouse dactylaplasia phenotype

Kano et al. 10.1073/pnas.0705483104.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Figure 6
SI Table 1

SI Figure 5

Fig. 5. Expression of the dactylin gene in dactylaplasia mutants. Total RNA purified from embryonic day 10.5 embryos was reverse transcribed with oligo(dT)₂₀ primer by using SuperScript III according to the protocol of the manufacturer (Invitrogen). Real-time quantitative RT-PCR was performed with SYBR Green PCR Master Mix by using an ABI PRISM 7900HT (Applied Biosystems). The level of dactylin transcript was normalized to Actb. The lack of dactylin transcript in the Dac^2J mutant was independent from Mdac status and the dactylaplasia phenotype. N, normal; D, dactylaplasia.

SI Figure 6

Fig. 6. DNA methylation profile of an unrelated ETnII element on chromosome 17 (NW_001030622) in either the mdac/mdac or Mdac/mdac background. Each LTR of the ETnII was analyzed by bisulfite sequencing. The ETnII was present in the Dac^1J parental strain (mdac/mdac) but was absent in the Dac^2J parental strain and C57BL/6J. F1 hybrids (Mdac/mdac) of Dac^1J parental strain and C57BL/6J demonstrated a slight change in DNA methylation status at its 5' LTR, exhibiting two distinct populations of PCR clones.

Table 1. List of forward (F) and reverse (R) oligonucleotides used for PCR and sequencing

Name	Purpose*	Oligonucleotide sequence
Dac^1JF	Isolation of the Dac^1J insertion	5'-GAGAGATTGATTCACCTGCCTCCTTAGTCCTGGG-3'
Dac^1JR		5'-TACATGCAAGTGAGCTGGATGGGTGAGTGGCAC-3'
Dac^2JF	Isolation of the Dac^2J insertion	5'-CTCATAAAGGCCCCTGTCAGTGTGTCCAAAGG-3'
Dac^2JR		5'-GGCACTGTGTGCAGACCATTAATGGATGTGCTG-3'
Walk.F1	Sequencing of the insertion	5'-GGCCGCAACATTTTGGCG-3'
Walk.F2		5'-CTCTGGGCGCTCGTAAATG-3'
Walk.F3		5'-GGTGCCGCTAGAAAAGGTTG-3'
Walk.F4		5'-TGCAGTGTACGCGCAAATTG-3'
Walk.F5		5'-CCGAGGTTAGACAAACAGG-3'
Walk.F6		5'-TGATCCCTTTGCGCTGGTC-3'
Walk.F7		5'-GTGGACTAATAAAGAAGGGAC-3'
Walk.F8		5'-CCATCCCTTTGCATCCAAAG-3'
Walk.R1		5'-GGAGACTACATCTCCTCCC-3'
Walk.R2		5'-GCCCTTTCAATAGTACGTCTC-3'
Walk.R3		5'-CTGTGCAGCTCCTAAAGGAG-3'
Walk.R4		5'-CAGAACTTCTTTCGGGGAAG-3'
Walk.R5		5'-ACCTTGGGGGATATAGCTG-3'
Walk.R6		5'-ACGTTGGGCCAAAGCAACC-3'
Walk.R7		5'-TGTTAATTCGGCGAGCTGAG-3'
Walk.R8		5'-CGGCAGGGAGTAATCTATG-3'
Dac^1J-5' LTR-F	Bisulfite sequencing of 5' LTRs^†	5'-GAGGTATATTGAATTAGTTTAATTAAAGG-3'
Dac^2J-5' LTR-F		5'-GTGGGAGATTTAGTTTTGATTTTTGTATG-3'
AL773522-5' LTR-F		5'-TAGTTTTAGTTTTTTGTTGTAGATGTAGGG-3'
Chr.17-5' LTR-F		5'-GTTTATGATTTATTTTTTAGTTGTGGTTG-3'
Dac-5' LTR-R		5'-TCTCTACCATTCTTCAAATCCCAATTC-3'
Dac-3' LTR-F	Bisulfite sequencing of 3' LTRs^†	5'-ATAGGGAGGAGATGTAGTTTTTTTT-3'
Dac^1J-3' LTR-R		5'-CCATATCTAACTTTTTCTTAACTCTAC-3'
Dac^2J-3' LTR-R		5'-AAACCACAAAATATATTTCAAAATTTCTC-3'
AL773522-3' LTR-R		5'-CTCAAAAATACACAAACTATAAAAACAAC-3'
Chr.17-3' LTR-R		5'-CCCCTAAACTAAAAAATATCAATAAAAC-3'
Dac^1J-geno-F	Genotyping for Dac^1J	5'-CAGTAGAGATCAACTAGCACTTCC-3'
Dac^1J-geno-R1		5'-GTCTGGCTTTTTCTTAACTCTAC-3'
Dac^1J-geno-R2		5'-GGAGGGGAGACTACAGTTAT-3'
Dac^2J-geno-F1	Genotyping for Dac^2J	5'-CAGCTGACCACTGAAGAAGGG-3'
Dac^2J-geno-F2		5'-CTCTCTTACAGCTCGAGCGG-3'
Dac^2J-geno-R		5'-CACCCTGGCTTCATTGTGCC-3'
Fgf8-F-T7	Probe template of Fgf8	5'-TAATACGACTCACTATAGGGCGATGGTGACGGATCAGCTCAG-3'
Fgf8-R-SP6		5'-ATTTAGGTGACACTATAGAATACAACTCGAAGCGCAGGCTC-3'
MusD-F-T7	Probe template of MusD/ETn	5'-TAATACGACTCACTATAGGGCGAACCGCTTGGTGCAGAGATAC-3'
MusD-R-SP6		5'-ATTTAGGTGACACTATAGAATACGCACTAAGTTGAATTCCTCC-3'
Dac^1JF-ChIP-5'F	ChIP assay and quantitative PCR^‡	5'-CCATAAATGAGGCACATTGAATCAG-3'
Dac^2JF-ChIP-5'F		5'-TCAGAACAGGGCTTTCTTGC-3'
AL773522-ChIP-5'F		5'-CCTAGACGTAGGCCCATAAG-3'
5' LTR-R		5'-GACACCAGTTATGAAGGAAGG-3'
3' LTR-F		5'-CCTTCCTTCATAACTGGTGTC-3'
Dac^1JF-ChIP-3'R		5'-GTCTGGCTTTTTCTTAACTCTAC-3', (the same as Dac^1J-geno-R1)
Dac^2JF-ChIP-3'R		5'-GAGAGAGAGAAAGCCACAGG-3'
AL773522-ChIP-3'R		5'-AGCAACAGAACAGCTGGGAC-3'
Actb-F		5'-GGGAGAGGGGGTAAAAAAATG-3'
Actb-R		5'-GCTGCAAAGAAGCTGTGCTC-3'
Dactylin-RT-PCR-F	RT-PCR	5'-GCTTACACACCATCCAGACCG-3'
Dactylin-RT-PCR-R		5'-GCAGTGTAGAGGGGGACTC-3'
Actb-RT-PCR-F		5'-GGACTCCTATGTGGGTGACG-3'
Actb-RT-PCR-R		5'-GCTGGGGTGTTGAAGGTCTC-3'

* See corresponding heading in the Materials and Methods.

^† Each LTR was analyzed by using a primer set consisting of an unique primer (the outside of LTR retrotransposon) and an internal primer (the inside of LTR retrotransposon). Primers used were Dac^1J-5' LTR-F and Dac-5' LTR-R for the 5' LTR of the Dac^1J insertion, Dac-3' LTR-F and Dac^1J-3' LTR-R for 3' LTR of the Dac^1J insertion, Dac^2J-5' LTR-F and Dac-5' LTR-R for the 5' LTR of Dac^2J insertion, Dac-3' LTR-F and Dac^2J-3' LTR-R for the 3' LTR of Dac^2J insertion, AL773522-5' LTR-F and Dac-5' LTR-R for 5' LTR of the MusD element (AL773522), Dac-3' LTR-F and AL773522-3' LTR-R for 3' LTR of the MusD element (AL773522), Chr.17-5' LTR-F and Dac-5' LTR-R for 5' LTR of the ETnII (NW_001030622), and Dac-3' LTR-F and Chr.17-3' LTR-R for 3' LTR of the ETnII (NW_001030622).

^‡ Primers used were Dac^1JF-ChIP-5'F and 5' LTR-R for 5' LTR of the Dac^1J insertion, 3' LTR-F and Dac^1JF-ChIP-3'R for 3' LTR of the Dac^1J insertion, Dac^2JF-ChIP-5'F and 5' LTR-R for 5' LTR of the Dac^2J insertion, 3' LTR-F and Dac^2JF-ChIP-3'R for 3' LTR of the Dac^2J insertion, AL773522-ChIP-5'F and 5' LTR-R for 5' LTR of the MusD element (AL773522), and 3' LTR-F and AL773522-ChIP-3'R for 3' LTR of the MusD element (AL773522).