Supplementary data 4: Detailed Multiple-Algorithm Scans for ATF and
CREB Binding Sites on IL6 and IL12b promoters.
Methods:
• Sequences:
– IL6: 5K upstream plus ~2K past a UTR region into the coding
region. [chr5: 29,042,073-29,050,069 Mouse Genome Assembly
Build35]
–
•
IL12b: 5K upstream plus UTR.
[chr11: 44,352,816-44,360,815 Mouse Genome Assembly Build35]
Position Weight Matrix (PWM) Scanning algorithms:
To examine the validity of the MotifLocator predictions given in Supplement 3,
we performed a detailed analysis of the IL6 and IL12b promoter regions using
multiple prediction algorithms. Predictions found using multiple algorithms are
more likely to be correct than those identified by a single analysis.
Algorithm
Parameters
MotifScanner prior = 0.2 (Default)
Clover
threshold motif score = 6.0 (Default)
Version
3.0
Mar 3 2004
URLS: MotifScanner may be downloaded from:
http://homes.esat.kuleuven.be/~thijs/download.html
Clover may be downloaded from: http://zlab.bu.edu/clover/
Background models:
MotifScanner and MotifLocator used the background model as described
Supplement 3.
To generate its own internal background model, Clover used the same
496 5Kbasepair upstream sequences from the first 496 genes on mouse
chromosome 17. Due to a specific requirement of the algorithm, every 2
sequences were concatenated together to create ~250 10K basepair
sequences.
•
Matrices
– Motifs used are from TRANSFAC Professional Version 8.3. The
M***** identifiers used in the table below are those used within the
TRANSFAC database.
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–
ATF binding sites have strong overlap with CREB binding sites.
Therefore, CREB sites were included in the scan analyses.
The complete set of matrices used is listed below:
ATF1_Q6 (M00691)
ATF3_Q6 (M00513)
ATF4_Q2 (M00514)
CREB_01 (M00039)
CREB_02 (M00113)
CREB_Q2 (M00177)
CREB_Q2_01 (M00916)
CREB_Q3 (M00801)
CREB_Q4 (M00178)
CREB_Q4_01 (M00917)
CREBP1_Q2 (M00179)
CREBP1CJUN_01 (M0041)
NFKBAPPB65_01 (M00052)
NFKAPPAB_01 (M00054)
NFKB_C (M00208)
NFKB_Q6 (M00194)
NFKB_Q6_01 (M00774)
•
Binding site predictions made by MotifLocator were thresholded using the
per-matrix score cut-offs determined by the quantile method described in
Supplement 3.
•
Computational platform:
– All analyses were performed on an Intel desktop PC running Linux
(Fedora Core release 1 (Yarrow)).
•
Notes on the Figures
– Analyses were run on both the forward and reverse strands of the
sequences. Reverse strand predictions are projected onto the
forward strand in Figures S4a and S4b for ease of viewing.
– Both Figures S4a and S4b report more MotifLocator predicted sites
than listed in Table S3.3. The subset of results reported in that
table are representative examples of overlapping hits shown in both
figures. This is done for the sake of clarity.
– The rightmost NFKB prediction in Figure S4b is shown for
completeness. This site is however not included in Figure 2B in the
main text as it (a) only marginally above the computed cut-off for
the matrix, (b) is a prediction on the reverse strand of the sequence
and (c) none of the other two prediction algorithms report a
matching site.
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IL6 promoter filtered scan; zoomed (-181 to -68)
Figure S4a. Alignment of ATF3/CRE TF binding sites predicted in the IL6 promoter.
Promoter sequences were scanned using Motif Mogul and only statistically significant
sites, as determined versus a randomly derived DNA sequence, are filtered and
examined. (Clover – blue. MotifLocator – red. MotifScanner - green.)
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IL12b promoter filtered scan; zoomed (-290 to -112)
Figure S4b. Alignment of ATF3/CRE TF binding sites predicted in the IL12b promoter.
Promoter sequences were scanned using Motif Mogul and only statistically significant
sites, as determined versus a randomly derived DNA sequence, are filtered and
examined. (Clover – blue. MotifLocator – red. MotifScanner made no predictions.)
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