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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 (http://www.ensembl.org/Homo_sapiens/) 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
http://www.fruitfly.org/seq_tools/promoter.html 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
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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 CleanUp 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
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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).
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Primer
Pair
Methylated set (5’-3’)
forward/reverse
Revised ONC-2006-01229(EBM)R
Unmethylated set (5’-3’)
forward/reverse
Primer position
(product size)
MUC2
GGAGTTATAAAGAGATGATTTTCGA
ACGATATAAATTACGCCCGA
GGAGTTATAAAGAGATGATTTTTGA
AAAAACAATATAAATTACACCCAAA
M, pos.-182/+13 (195bp)
U, pos.-182/+17 (199bp)
MUC6 (I)
TGTTTGAAGGGGTTAGGAGTATATC
ACCCTAAAAACCTAAAAAACTACCG
GTTTGAAGGGGTTAGGAGTATATTG
CCTAAAAACCTAAAAAACTACCATA
M, pos. -830/-627 (204bp)
U, pos. -829/-629 (201bp)
Position of
the mapped
cytosines
Annealing
temperature
(°C)
-160/-6
55
-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.
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Vincent et al.
Primer Pair
Revised ONC-2006-01229(EBM)R
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.
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Supplemental data
Legend to supplemental data 1: Sequence of the human MUC6 promoter. 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 cfos 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 (+) cells with 5-aza or TSA. Sp1/28S and cfos/28S ratio are indicated.
-
-
+
2.6
0.8
0
c-fos/28S ratio 2.2 2.6
3.1
1.9
5-aza
TSA
-
+
-
Sp1
Sp1/28S ratio 2.0
c-fos
28S
6