Vincent et al.Revised ONC-2006-01229(EBM)R

Supplemental materials and methods

Identification of the 5’-end of MUC6 gene - At the time we initiated this work, the promoter of MUC6 was not known. To identify the genomic sequence of the human MUC6 promoter, we blasted the human draft sequence ( using the 514bp genomic sequence upstream of the ATG initiator of the mouse Muc6 gene. A unique sequence was found (clone AC139749; SuperContig NT_035113.5) that mapped to the chromosome 11 in the cluster of mucin genes and that did not belong to MUC2, MUC5AC or MUC5B. Further analysis of the contig showed that the genomic sequence we identified was located upstream of the MUC6 coding region (Toribara et al., 1993). Alignment of part of the human contig nucleotide sequence with the mouse Muc6 gene (Desseyn and Laine, 2003) sequence allowed to predict the first three exons. RT-PCR was carried out as follows using the MUC6 forward primer 5'-TGCTGCGGAGCCCTGCTCAGC-3' located within the predicted exon 1 and the reverse MUC6 primer 5'-CTTGACTGAGATGATGGCTTC-3' located within the predicted exon 3 with the Taq DNA Polymerase (2.5units, Roche Diagnostics, Meylan, France). These two primers flanked the 4808 genomic nucleotide sequence submitted to the GenBank with the accession number AY500284. The sequence of the first three exons was submitted to GenBank with the accession number AY458429. Cycling conditions were as follows (i) denaturation, 94°C, 2min for one cycle; (ii) denaturation, 94°C, 30s; annealing, 60°C, 30s; and extension, 72°C, 1min for 30 cycles and (iii) final extension, 72°C, 10min. PCR products (10µl) were separated on a 1.5% agarose gel containing ethidium bromide.

5’ RACE-PCR - Prediction of the transcription start site was carried out using the software. Using a score cut off set at 0.80, the adenine residue located 23 bases downstream of the TATA box was predicted to be the transcription start site (position 2023 in the AY370683 sequence). From there, two primers were designed to perform 5’ RACE-PCR experiments (5’/3’ RACE kit, Roche Molecular Biochemicals) according to the manufacturer’s instructions. Briefly, cDNA was synthesized using RNA (1µg) from the LS174T cell line, which expresses MUC6, with the following primer: 5’-TGGAGGCCTGGGCTGGTGTAG-3’ located in exon 2 (nucleotides 3570-3590 in AY500284 GenBank sequence). cDNA was then purified with the Wizard® DNA Clean-Up System (Promega) and 19µl of purified cDNA were used to add a homopolymeric A-tail to the 3’-end using 80 units of the recombinant Terminal Transferase. PCR amplification of dA-tailed cDNA (5µl) was performed in a 50µl final mixture using the specific reverse primer:5’-AGCGCTGAGCAGGGCTCCGCAGCAGGACAGCAGCAGCCACCGCTGGAC-3’ (12.5µM) located in exon 1. Cycling conditions were as follows: (i) denaturation, 94°C, 2min for one cycle; (ii) denaturation, 94°C, 45s; annealing, 55°C, 1min; and extension, 72°C, 1min for 35 cycles and (iii) final extension, 72°C, 10min. PCR products (15µl) were separated on a 1.5 % agarose gel and then cloned into the pCR2®.1 vector (Invitrogen, Cergy-Pontoise, France). Ligation products were used to transform INVαF’ competent cells (Invitrogen) and positive clones were selected for plasmid extraction using QIaprep 8 Miniprep Kit (Qiagen) and sequenced on both strands on an infra-red based 4000L LI-COR sequencer (ScienceTech, Les Ulis, France) using T7 and RM13 universal primers.

Analysis of transcription factor expression by RT-PCR - Total RNA was prepared from LS174T PC as described in Material and Methods section. PCR was performed on cDNA (5µl), using specific pairs of primers for Sp1: 5’-CCATACCCCTTAACCCCG-3’ and 5’-GAATTTTCACTAATGTTTCCCACC-3’ and c-fos: 5’-TAGTTAGTAGCATGTTGAGCCAGG-3’ and 5’-ACCACCTCAACAATGCATGA-3’. The ribosomal RNA 28S subunit was used as the internal control. PCR products (10µl) were separated on a 1.5% agarose gel containing ethidium bromide run in 1X TBE. The transcription factor/28S gene ratio was calculated after scanning DNA bands with GelAnalyst-GelSmart software (Claravision).

Primer
Pair / Methylated set (5’-3’)
forward/reverse / Unmethylated set (5’-3’)
forward/reverse / Primer position
(product size) / Position of the mapped cytosines / Annealing temperature (°C)
MUC2 / GGAGTTATAAAGAGATGATTTTCGA
ACGATATAAATTACGCCCGA / GGAGTTATAAAGAGATGATTTTTGA
AAAAACAATATAAATTACACCCAAA / M, pos.-182/+13 (195bp)
U, pos.-182/+17 (199bp) / -160/-6 / 55
MUC6 (I) / TGTTTGAAGGGGTTAGGAGTATATC
ACCCTAAAAACCTAAAAAACTACCG / GTTTGAAGGGGTTAGGAGTATATTG
CCTAAAAACCTAAAAAACTACCATA / M, pos. -830/-627 (204bp)
U, pos. -829/-629 (201bp) / -806/-651 / 60

Supplemental table 1. Sequences of the pairs of primers used for MS-PCR studies. Sizes of the PCR products (bp) and primer position (pos.) referring to the proximal transcription start site (+1) are indicated. Modified nucleotides to discriminate between methylated (M) and unmethylated (U) CpG sites are bold and underlined.

Primer Pair / Forward Primer (5’-3’) / Reverse Primer
(5’-3’) / Primer position
(Product size) / Annealing temperature (°C)
MUC2(a) / GGGATATGGAAAAATGGTTTTAGAT / CCTACTTTCCTAAAAAACACTCCAC / pos.-3368/-3172 (197bp) / 55
MUC2(b) / AGGTATTGGTTATATGGGGAGTGT / AAAAAAAACCTTAATCCTCCAAAAA / pos.-3140/-2951 (190bp) / 50
MUC2(c) / AAGATTGGAGTTATGGTTAGATAGGT / TACTAAAAAAACCAAAAACAACAAATAC / pos.-2853/-2753 (101bp) / 50
MUC2(d) / TTTTTTTTGTAGTTATTATTGTAAATTTTA / CATATCAAACTACCCCAACCTC / pos.-2555/-2302 (254bp) / 50
MUC2(e) / GGGGTTATATTTGGATTAATATAGGA / AAAAAACTAAACCCCATTCCTAAC / pos.-2030/-1767 (264bp) / 55
MUC5B(a) / GGGGGTTAGTAGGGGAGATATTAG / ACCTACTCCAAACCAAACTAAACAA / pos.-2553/-2305 (249bp) / 50
MUC5B(b) / GGTTTTAGTTTTGTTATGGAGAAAA / CCTACTAACCCCCACTACCTATC / pos.-2710/-2541 (170bp) / 50
MUC5B(c) / TGGGGTTTGGGTGTAGTTATAGTTA / AAATCTCCAAACTCTCTTTCACATC / pos.-2278/-2113 (166bp) / 50
MUC5B(d) / GGGTTTTTGGAAATAGAGTTTTTTT / ATAATCAACCAACCTACCTCACACT / pos.-523/-313 (211bp) / 50
MUC5B(e) / AGTGTGAGGTAGGTTGGTTGATTAT / AACTCTATACCCTAAAACCCAAAAC / pos.-337/-161 (177bp) / 50
MUC5B(f) / TGGGAGTATTTGAGGTGTAGGTTATA / ACCTCCTCATTAACCCTAACAAAAT / pos.-1002/-732 (271bp) / 55
MUC5AC(a) / TTTGGGTTAAGATAGGATATGGG / TATAACTAAACTCTCCCTCCCAAAC / pos.-1362/-1062 (301bp) / 50
MUC5AC(b) / AGGAATTTATAGGTTGTTGGGTATG / TTTTATAACCCCAAAACTAACTCCA / pos.-288/-24 (265bp) / 50
MUC6(a) / GAGGTTTTAAGATTTTTGTTTTTTAT / AACCTTAACTATCCCTTCTTAACAC / pos.-1792/-1593 (200bp) / 50
MUC6(b) / AAGAAGGGATAGTTAAGGTTGGATA / CCCACACCAAACATTCTAAATATTC / pos.-1612/-1416 (197bp) / 50
MUC6(c) / GGTATAGGTGGGATAGAGGTGGTA / CCCTCCCAAAAAAAATTTTAATC / pos.-1235/-1014 (222bp) / 50
MUC6(d) / TGGGGTAATTTTGGTGATTATTTAG / TATACTCCTAACCCCTTCAAACAA / pos.-990/-818 (173bp) / 50
MUC6(e) / AGTAGGGTTTTTTTTAAGTTGGGTTAG / AATCCTACAAACACCCCCTACATA / pos.-421/-231 (191bp) / 50
MUC6(f) / TGTTTGTAGGATTTTTTAAAGAAAGT / ACAAACCTACTACTACCATCCATAC / pos.-175/+28 (203bp) / 50

Supplemental table 2. Sequences of the pairs of primers used for bisulfite sequencing studies.Sizes of the PCR products (bp) and primer position (pos.) referring to the proximal transcription start site (+1) are indicated.

Supplemental data

Legend to supplemental data 1:Sequence of the human MUC6promoter. The transcription start site +1 (bold and underlined) is located 24 nucleotides downstream of the TATA box (-28/-24) and 61 nucleotides upstream of the first ATG. The CpG island (-157/+6) is coloured in grey.

Legend to supplemental data 2:Influence of epigenetics on the expression of Sp1 and c-fos transcription factors in LS174T cells. RT-PCR was performed as described in Materials and Methods section. The expected size for PCR products of Sp1 and c-fos are 821 and 333bp, respectively. Untreated (-) or treated (+)cellswith 5-aza or TSA. Sp1/28S and c-fos/28S ratio are indicated.

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