Download Regulatory regions and regulatory elements - ENS-phys

Regulatory Sequence Analysis
Regulatory regions
and regulatory elements
Jacques van Helden
[email protected]
Tra
ns
cri
be
tiv
e
rep
eti
no
n
-co
g
din
s/M
ne
co
Mb
ge
ne
Ge
Siz
e
Organism
Ye
ar
s
b
din
g
d
The non-coding genome
%
%
%
%
Mycoplasma genitalium
Haemophilus influenzae
Escherichia coli
1995
1995
1997
0.6
1.8
4.6
481
1 717
4 289
802 90
954 86
932 87
10
14
13
Saccharomyces cerevisiae
1996
12
6 286
524 72
28
Arabidiopsis thaliana
Caenorhabditis elegans
Drosophila melanogaster
Homo sapiens
2001 120
1998
97
2000 165
2001 3 200
27 000
19 000
16 000
31 000
225 30
196 27
97 15
10 3
70
73
85
97
46 28
Transcriptional activation
Transcriptional
activator
activation
domain
RNA
polymerase
DNA-binding
domain
enhancer
initiation
Transcriptional repression
Prevent RNA polymerase from
accessing DNA
Competition for factor binding site
Factor titration
Prevent transcription factor from
interacting with RNA-polymerase
(bind with activation domain)
Transcription factor-DNA interfaces
A
C
B
D
Phosphate utilization in yeast
PHO2
PHO3
acid phosphatase
expression
codes for
acid phosphatase
up-regulates
Up-regulates
issecretion
secreted
expression
codes for
expression
up-regulates
PHO5, 11,12
catalyzes
up-regulates
Pho2p
Pi transporter
up-regulates
expression
codes for
orthophosphoric monoester
PHO84, 86,87,88,89
alkaline phosphatase
up-regulates
Pho4p
expression
codes for
H2O
orthophosphoric monoester
PHO8
3.1.3.1
H2O
PHO4
up-regulates
(nucleus)
catalyzes
3.1.3.2
PHO81
up-regulates
transports
facilitates
alcohol
expression
codes for
is
translocates
tranferred
Pi
expression
codes for
is tranferred
transport
Pho4p-Phosphate
Pho4p
alcohol
Pi
2.7.1.-
catalyzes
inhibits
Pho81p
Pho80p Pho85p
Pho85-Pho80 complex
inhibits
(cytoplasm)
extracellular space
Pho4p binding sites
gene
PHO5
PHO5
PHO5
PHO8
PHO8
PHO81
PHO84
PHO84
PHO84
PHO84
PHO84
PHO5
PHO5
PHO5
start end sequence
-260 -242 ..GCACTCACACGTGGGACTA
-260 -245 ..GCACTCACACGTGGGA
-262 -239 TGGCACTCACACGTGGGACTAGCA
-540 -522 ...TCGGGCCACGTGCAGCGAT
-736 -718 ..ttacccgCACGCTTaatat
-350 -332 ...TTATGGCACGTGCGAATAA
-421 -403 ..TTTCCAGCACGTGGGGCGG
-442 -425 ...TAGTTCCACGTGGACGTG
-879 -874 .aaaagtgtCACGTGataaaaat
-267 -250 ..taatacgCACGTTTTTaa
-592 -575 ....TTACGCACGTTGGTGCTG
-368 -349 ...AATTAGCACGTTTTCGCATA
(?)
(?)
..AAATTAGCACGTTTCGC
-370 -347 .TAAATTAGCACGTTTTCGCATAGA
IUPAC ambiguous nucleotide code
A
C
G
T
R
Y
W
S
M
K
H
B
V
D
N
A
C
G
T
A or G
C or T
A or T
G or C
A or C
G or T
A, C or T
G, C or T
G, A, C
G, A or T
G, A, C or T
Adenine
Cytosine
Guanine
Thymine
puRine
pYrimidine
Weak hydrogen bonding
Strong hydrogen bonding
aMino group at common position
Keto group at common position
not G
not A
not T
not C
aNy
Pho4p binding specificity - matrix descriptions
C
A
C
G
T
Pho4p
14 0
5
7
6
0 26 0
0
0
0
3
2
8
5 16 6 26 0 26 0
1
0
4
4
2
1
1 12 0
0
0 26 0 16 12
6 16 15 2
2
0
0
0
0 25 10 7
A
C
G
T
Pho4p.cacgtg
2 17 0
0
0
0
2
1
16 0 18 0
0
0
6
3
0
1
0 18 0 18 9 12
0
0
0
0 18 0
1
2
D
E
A
C
G
T
7
0
0
1
0
1
0
7
2
1
0
5
5
3
0
0
Pho4p.cacgtt
1
0
8
0
3
8
0
8
4
0
0
0
0
0
0
0
8
4
2
4
5
5
5
3
5
0
2
11
13
1
1
3
0
0
8
0
0
0
0
8
0
0
0
8
1
0
2
5
Sequence logo
Rap1
Rpn4
Gcn4
HSE
Mig1
Cbf1
Shannon uncertainty

Shannon uncertainty



Hs(j): uncertainty of a column of a PSSM
Hg: uncertainty of the background (e.g. a genome)
Properties of the uncertainty (for a 4 letter alphabet)

min(H)=0
•

H=1
•


H s ( j ) = −∑ f ij log 2 ( f ij )
i=1
A
H g = −∑ pi log 2 ( pi )
No uncertainty at all: the nucleotide is completely specified (e.g.
p={1,0,0,0})
R
seq
Uncertainty between two letters (e.g. p={0.5,0,0,0.5})
max(H) = 2
•
A
Complete uncertainty: one bit of information is required to
specify the choice between each alternative (e.g.
p={0.25,0.25,0.25,0.25})
i=1
( j) = Hg − Hs( j)
w
Rseq = ∑ Rseq ( j )
j=1
Schneider (1986) defines an information content Rseq
based on Shannon’s uncertainty.
€
Source: Schneider (1986)
Schneider logos

Schneider (1990) proposes a graphical
representation based on his previous entropy (H)
for representing the importance of each residue at
each position of an alignment. He provides a new
formula for Rseq




Hs(j)
Rseq(j)
e(n)
uncertainty of column j
information content of column j
correction for small samples
(pseudo-weight)
Remarks



A
H s ( j ) = −∑ f ij log 2 ( f ij )
i=1
Rseq ( j ) = 2 − H s ( j ) + e( n )
hij = f ij Rseq ( j)
€
This information content does not include any
correction for the prior residue probabilities (pi)
This information content is expressed in bits.
Boundaries
•
•
min(Rseq)=0
max(Rseq)=2
equiprobable residues
perfect conservation of 1 residue,
all the others are forbidden
http://www.lecb.ncifcrf.gov/~toms/icons/tata.gif
References - Sequence logoo



Schneider, T.D., G.D. Stormo, L. Gold, and A. Ehrenfeucht. 1986.
Information content of binding sites on nucleotide sequences. J Mol
Biol 188: 415-431.
Schneider, T.D. and R.M. Stephens. 1990. Sequence logos: a new way
to display consensus sequences. Nucleic Acids Res 18: 6097-6100.
Tom Schneider’s publications online
• http://www.lecb.ncifcrf.gov/~toms/paper/index.html
Methionine Biosynthesis in S.cerevisiae
Aspartate
biosynthesis
L-Aspartate
ATP
ADP
2.7.2.4
Aspartate kinase
HOM3
Aspartate semialdehyde
deshydrogenase
HOM2
Homoserine
deshydrogenase
HOM6
L-aspartyl-4-P
NADPH
NADP+; Pi
1.2.1.11
L-aspartic semialdehyde
Threonine
biosynthesis
NADPH
NADP+
1.1.1.3
L-Homoserine
AcetlyCoA
CoA
2.3.1.31
Met31p
met32p
Homoserine
O-acetyltransferase
MET2
O-acetylhomoserine
(thiol)-lyase
MET17
MET31
MET32
O-acetyl-homoserine
Sulfur
assimilation
Sulfide
4.2.99.10
MET28
Homocysteine
Cysteine biosynthesis
5-methyltetrahydropteroyltri-L-glutamate
5-tetrahydropteroyltri-L-glutamate
2.1.1.14
Methionine synthase
(vit B12-independent)
MET6
Cbf1p/Met4p/Met28p
complex
CBF1
MET4
Gcn4p
GCN4
L-Methionine
S-adenosyl-methionine
synthetase I
H20; ATP
2.5.1.6
S-adenosyl-methionine
Pi, PPi
synthetase II
S-Adenosyl-L-Methionine
SAM1
SAM2
Met30p
MET30
Met4p binding sites
gene
MET3
MET3
MET14
MET16
ECM17
ECM17
MET10
MET10
MET2
MET2
MET17
MET17
MET6
MET6
SAM2
SAM2
A
C
G
T
13
1
1
1
11
0
1
4
start
-367
-384
-235
-185
-311
-339
-255
-237
-360
-554
-306
-332
-540
-502
-329
-381
end
-349
-366
-217
-167
-293
-321
-237
-219
-342
-536
-288
-314
-522
-484
-311
-363
3
0
4
9
3
3
4
6
sequence
GAAAAGTCACGTGTAATTT
AAAAGGTCACGTGACCAGA
CTAATTTCACGTGATCAAT
ATCATTTCACGTGGCTAGT
ATTTCATCACGTGCGTATT
.TTTGTCCACGTGATATTTC
.CCACACCACGTGAGCTTAT
.TAGAAGCACGTGACCACAA
GTATTTTCACGTGATGCGC
TAATAATCACGTGATATTT
.AAATGGCACGTGAAGCTGT
TTGAGGTCACATGATCGCA
GCCACATCACGTGCACATT
AATATTTCACGTGACTTAC
.TCTACCCACGTGACTATAA
.TCTTCACATGTGATTCATC
2
0 16 0
1
0
0 12
0 16 0 15 0
0
0
0
4
0
0
0 15 0 16 4
10 0
0
1
0 16 0
0
Met31p binding sites
gene
MET14
MET2
MET17
MET6
SAM2
SAM1
MET19
MUP3
MET8
MET1
MET3
MET28
MET8
MET30
MET6
A
C
G
T
5
2
5
2
start
-202
-313
-227
-313
-306
-283
-173
-188
-184
-232
-259
-159
-434
-168
-405
end
-182
-293
-207
-293
-286
-263
-153
-168
-164
-212
-239
-139
-414
-148
-385
sequence
CCTCAAAAAATGTGGCAATGG
TGCAAAAAATTGTGGATGCAC
TCATGAAAACTGTGTAACATA
GTCGCAAAACTGTGGTAGTCA
GCTTGAAAACTGTGGCGTTTT
ACAGGAAAACTGTGGTGGCGC
ATAAGCAAACTGTGGGTTCAT
CGGAAAAAACTGTGGCGTCGC
GGAAAAAAAATGTGAAAATCG
CATAATAAACTGTGAACGGAC
ACAAAGCCACAGTTTTACAAC
CTAACACCACAGTTTTGGGCG
TCTTGTCCGCAGTTTTATCTG
GGGAAGCCACAGTTTGCGCGG
CTATCGAACTCGTTTAGTCGC
11 14 14 14 2
0
0
0
0
2
2
0
0
0 11 0
0
1
0
0
0
0
0
0
0
0 14 0 14 11
1
0
0
0
1 14 0 13 0
1
5
5
1
3
Characteristics of yeast regulatory sites


Located upstream the regulated gene
Short DNA sequences (5-30 bp)





Highly conserved core (5-8 bp), with partly conserved flanking
nucleotides
Pair of very shot oligonucleotides (3 nt) separated by a nonconserved segment (0-20 bp)
Strand-insensitive
Wihtin 800 bp from the start codon
Efficiency dos not depend on


strand
position
Pattern matching vs pattern discovery
Set of DNA sequences
Yes
Pattern matching
Matching
positions
Pattern
known ?
No
Pattern discovery
Putative regulatory
patterns
Questions and approaches
1.
If we know the consensus for a given transcription factor, can we predict its
binding sites in a DNA sequence ?

2.
Can we scan a sequence for matches with the consensus of all he
currently known transcription factor ?

3.
Pattern discovery within a sequence set
Can we detect regulatory signals by searching conserved elements in noncoding sequences of orthologous genes ?

5.
Matching a library of patterns against a sequence
Starting from a set of co-expressed genes, can we predict cis-acting
elements involved in their transcriptional regulation ?

4.
Pattern matching against a sequence
Phylogenetic footprinting
Can we predict gene regulation on the basis of the presence of regulatory
motifs in their regulatory regions ?

Gene classification on the basis of pattern scores
Typical situations : pattern discovery

Selected sequence set


e.g. family of 20 co-regulated genes, obtained from DNA chip
experiment
→ identify putative regulatory sites
Genome-scale pattern discovery
θ

e.g. all upstream sequences
→ identify transcription initiation signals
e.g. all downstream sequences
→ identify 3' maturation signals
Typical situations : pattern matching

Selected genes, selected patterns
θ
ν
Selected genes, library of patterns
θ

e.g. 10 genes known to be regulated by a factor
→ search matching positions
→ infer putative action of any previously known transcription
factor
All genes, selected patterns
θ
→ classify all the genes of a genome according to putative
regulatory properties
Differences between species
organism
location
yeast
upstream
coli
upstream
overlap. Initiation
distance range
-800 to -1 bp
-400 to +50 bp
higher organisms
upstream
downstream
within introns
over 100s of Kb
position effect
often irrelevant
often essential
often irrelevant
strand
insensitive
sensitive or symmetric
insensitive
spaced pair of 3nt
~5-8 conserved bp
rare
frequent
most common ~5-8 conserved bp
core
repeated sites
occasional
composite
elements
frequent