Characteristics of the CArG-SRF binding context in mammaliangenomes

Wenwu Wu • Xia Shen • Shiheng Tao

Received: 15 September 2009 / Accepted: 11 November 2009 / Published online: 3 December 2009

� Springer Science+Business Media, LLC 2009

Abstract Serum response factor (SRF), a member of the

MADS family, binds a 10-bp cis element known as the

CArG box. However, despite our extensive knowledge of

SRF and the CArG box, limited information is available on

the CArG-SRF binding context or how CArG flanking

sequences are defined. We used statistical tests and com-

puter simulation to find characteristics of the CArG-SRF

binding context. Based on the combination of published

literature and a search of DBTSS, 150 and 136 functional

CArG boxes together with 10 bp flanking on each side

were found in mouse and human genomes, respectively. By

statistical analysis of the 30 positions we found some new

conserved positions of interest (P \ 0.005) such as -15,

-8, and ?8, in addition to the ten highly conserved posi-

tions of the CArG box. Intriguingly, studies comparing the

flanking positions between consensus CArG boxes and

CArG-like boxes showed that there are more conserved

positions in the latter. Moreover, CpG content within the

CArG-SRF binding context is much higher than that within

introns. Collectively, these results suggest that there are

some special pre-existing features in the flanking sequen-

ces of functional CArG boxes probably contributing to SRF

selectively recognizing and binding to the functional CArG

from millions of functionless CArG boxes in mammalian

genomes. This is a significant step toward understanding

the mechanism of transcriptional regulation of SRF-depen-

dent genes.

Introduction

Serum response factor (SRF) as a 62-67 kDa protein first

purified from the HeLa nucleus (Prywes and Roeder 1987;

Treisman 1987) is a highly conserved, widely expressed,

and versatile transcription factor in mammals (De Folter

and Angenent 2006; Miano 2003; Shore and Sharrocks

1995). By binding to the CArG box, SRF can orchestrate

disparate programs of gene expression linked to mesoderm

formation (Arsenian et al. 1998), cardiac development and

maintenance (Miano et al. 2004; Parlakian et al. 2005), and

cell motility (Alberti et al. 2005; Fleige et al. 2007). SRF-

null mouse embryos (Niu et al. 2005), for example, failed

to gastrulate and form mesoderm, indicating that SRF is an

obligatory factor for mesoderm development. Moreover,

SRF has been linked to an increasing number of diseases,

including cancer (Bell et al. 2009; Koegel et al. 2009; Petit

et al. 2008; Zhang et al. 2005). For example, in the mouse,

cardiac SRF transgenesis (Chai and Tarnawski 2002)

showed that overexpression of SRF resulted in hypertro-

phic cardiomyopathy, indicating a link between the level of

SRF expression and heart disease.

Given the essential roles of SRF throughout the body,

elucidating the SRF binding site provides key information

for understanding the transcriptional regulation mechanism

of SRF-dependent genes. Over the last 20 years, hundreds

of genes have been shown to be transcriptionally regulated

by SRF and the regulatory regions of these genes have a

common 10-bp cis element called the CArG box (Miano

2003; Sun et al. 2006; Treisman 1992), which is directly

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00335-009-9238-x) contains supplementarymaterial, which is available to authorized users.

W. Wu � X. Shen � S. Tao (&)

College of Life Science, Northwest A&F University,

712100 Yangling, Shaanxi, China

e-mail: shihengt@nwsuaf.edu.cn

W. Wu � X. Shen � S. Tao

Bioinformatics Center, Northwest A&F University,

Yangling, Shaanxi, China

123

Mamm Genome (2010) 21:104–113

DOI 10.1007/s00335-009-9238-x

bound by SRF. Subsequently, Miano’s group (Miano et al.

2007; Sun et al. 2006) proposed a theoretical definition of a

functional CArG box in which the 10-bp consensus can

deviate by no more than 1 bp, yielding 1216 potential

sequences that could be bound by SRF. According to the

statistical frequency of CArG boxes, there would be more

than 3 million CArG boxes in the mouse genome (Sun

et al. 2006). However, so far fewer than 200 CArG boxes

have been experimentally verified functional (Miano 2008;

Sun et al. 2006). Thus, we want to answer the basic

question: what kind of factors discriminate the functional

CArG boxes from millions of functionless boxes and urge

SRF binding to the functional boxes?

Although in past years the CArG box per se (De Folter

and Angenent 2006; Hendrix et al. 2005; Huet et al. 2005;

Leung and Miyamoto 1989; Stepanek et al. 2007; Sun et al.

2006) and the distribution of its position in relation to

transcriptional start sites (TSS) (Shen et al. 2009; Sun et al.

2006) and SRF and its cofactors (Cooper et al. 2007; Miano

et al. 2007; Sun et al. 2006) have been studied extensively,

much less information is available on the properties of

CArG flanking sequences. To our knowledge, the experi-

ment (Pellegrini et al. 1995) showed that the 5-methyl

group of T?8 downstream of the CArG box has hydro-

phobic contact with Thr159 and Ser162 of SRF, and certain

point mutations in CArG flanking sequences (Leung and

Miyamoto 1989; Mack et al. 2000) would reduce, enhance,

or not influence SRF binding to the box. Thus, for at least

some adjacent positions of functional CArG boxes, we

assume that the function of binding to SRF constrains the

flanking sequences surrounding the box. In particular, the

flanking 10 bp on each side of the functional CArG box

would provide a suitable biochemical environment to be

recognized by SRF, before the core CArG box eventually

binds to the factor uniquely.

In this study, using the v2 test and computer simulation

we identified some new conserved positions (-15, -8, ?7,

and ?8) and found high CpG content in the CArG-SRF

binding context. In addition, we confirmed the conserved

positions in the flanking positions of the CArG-like boxes

but also identified some new conserved positions (-12, -7,

and -6). Together, our studies revealed the characteristics

of the CArG-SRF binding context in mammalian genomes,

a new perspective to understanding the importance of the

flanking sequences surrounding the functional CArG boxes.

Materials and methods

Isolation of target fragments

The primary strategy for isolating target fragments, which

include functional CArG boxes and their flanking

sequences, was generalized as follows: First, SRF target

genes in mouse and human genomes were collected from

the published literature (Cooper et al. 2007; Sun et al.

2006). In most cases these genes were accompanied by

CArG sequences and their positions with respect to TSS.

Subsequently, the promoters of these genes were retrieved

using transcript IDs against DBTSS (Wakaguri et al. 2008),

and then a Perl script was developed to capture CArG-SRF

binding context through CArG sequences and their corre-

sponding positions to TSS. Finally, 150 and 136 target

fragments (30 nucleotides long), including functional

CArG boxes, were found in mouse and human genomes,

respectively (see Supplementary Material).

Statistical analysis

Tables 1 and 2 present the status of each position within

the 30-nucleotide-long fragments in mammalian genomes

obtained using the v2 goodness-of-fit test. v2 was calculated

using the following equation:

v2 ¼XA;T;G;C

i¼A

ðOi � EiÞ2

Ei

where Oi is the observed frequency of base i and Ei is the

expected frequency of base i, determined by the frequen-

cies of the four nucleotides (A, T, G, and C) in CArG

flanking sequences. By computer simulation, p values were

obtained as the proportion of the 10,000 replications in

which the simulated v2 values exceeded the v2 values

calculated from the real data.

Using the v2 test for stochastic independence, Table 3A

and B presents dinucleotide distributions in CArG flanking

sequences and in the intron fragments randomly chosen

from the Exon–Intron Database (EID) (Shepelev and Fe-

dorov 2006). v2 was calculated using the following

equation:

v2ij ¼ðOij � EijÞ2

Eij

where Oij is the observed dinucleotide (ij) counts, Eij is the

expected dinucleotide (ij) counts calculated from the

mononucleotide counts, and the ratio between them was

calculated as R = Oij/Eij. Table 4A presents the associations

of four consecutive positions in the middle of functional

CArG boxes with only A or T taken into account. In

Table 4A, v2 was calculated using the following equation:

v2ijkn ¼

ðOijkn � EijknÞ2

Eijkn

where Oijkn is the observed tetranucleotide (ijkn) counts,

Eijkn is the expected tetranucleotide (ijkn) counts calculated

from the four mononucleotide counts, and Yates’

W. Wu et al.: Implication of CArG-SRF binding context 105

123

correction (Yates 1934) was used when necessary

(Eijkn \ 5). The p value for each tetranucleotide (ijkn) was

obtained using the computer simulation strategy as above.

Results

Based on the published literature (Cooper et al. 2007; Sun

et al. 2006) and the Database of Transcription Start Sites

(DBTSS, version 6.0) (Wakaguri et al. 2008), we found 150

and 136 functional CArG boxes with 10-nucleotide-long

fragments flanking each side in the mouse and human

genomes, respectively (see Materials and methods). The 20

adjacent positions, which refer to 10 bp upstream and 10 bp

downstream of the CArG box, can be regarded as CArG-

SRF binding context (CSBC), and we focused our analysis

mainly on the CSBC of the 286 functional CArG boxes in

mammalian genomes (see Supplementary Material).

Conservation status of CSBC in mammalian genomes

Using a v2 goodness-of-fit test and computer simulation

(see Materials and methods), we obtained the frequencies

of nucleotides A, T, G, and C; v2 and the P value at each

indicated position is given in Table 1A and B. The table

shows that there are similarities and differences in the

Table 1 Conservation of CArG flanking sequences in human (A) and

mouse (B) genomes

Position A T G C v2 P

(A) Human

-15 31 16 39 50 19.7 0.0001

-14 30 30 33 43 3.5 0.3369

-13 29 46 29 32 4.1 0.2551

-12 28 34 30 44 3.7 0.3067

-11 37 35 24 40 2.7 0.4495

-10 35 32 27 42 2.4 0.4905

-9 42 30 30 34 2.8 0.4350

-8 33 55 20 28 15.8 0.0015

-7 29 36 35 36 1.3 0.7317

-6 33 44 30 29 3.0 0.4058

-5 5 2 1 128 335.0 0.0000

-4 3 7 0 126 321.2 0.0000

-3 70 60 0 6 109.0 0.0000

-2 18 117 1 0 256.4 0.0000

-1 103 20 8 5 188.2 0.0000

?1 25 102 4 5 174.5 0.0000

?2 110 23 1 2 232.7 0.0000

?3 72 61 2 1 119.8 0.0000

?4 5 4 127 0 382.1 0.0000

?5 4 3 125 4 366.0 0.0000

?6 32 33 31 40 1.1 0.7730

?7 34 32 17 53 16.3 0.0012

?8 60 34 23 19 28.9 0.0000

?9 25 41 50 20 20.9 0.0001

?10 38 36 35 27 2.7 0.4534

?11 29 41 33 33 1.8 0.6258

?12 42 33 29 32 2.4 0.5006

?13 33 35 35 33 0.6 0.8947

?14 29 36 45 26 2.5 0.4731

?15 35 35 36 30 1.4 0.6963

(B) Mouse

-15 28 21 48 53 16.9 0.0008

-14 37 38 33 42 1.7 0.6406

-13 35 38 38 39 0.1 0.9903

-12 32 29 35 54 9.9 0.0195

-11 31 45 29 45 6.2 0.1009

-10 29 41 38 42 2.0 0.5781

-9 46 32 33 39 5.3 0.1518

-8 30 70 25 25 38.3 0.0000

-7 25 43 54 28 11.5 0.0090

-6 27 53 31 39 10.1 0.0172

-5 1 1 3 145 408.7 0.0000

-4 1 5 0 144 401.5 0.0000

-3 67 78 2 3 139.7 0.0000

-2 37 112 0 1 222.5 0.0000

-1 97 39 12 2 161.5 0.0000

Table 1 continued

Position A T G C v2 P

?1 29 107 5 9 181.2 0.0000

?2 102 45 3 0 199.7 0.0000

?3 64 80 4 2 137.1 0.0000

?4 6 0 144 0 375.3 0.0000

?5 2 1 145 2 381.9 0.0000

?6 39 37 35 39 1.0 0.8042

?7 37 23 29 61 23.1 0.0000

?8 73 37 24 16 59.2 0.0000

?9 30 47 45 28 6.3 0.0952

?10 46 24 54 26 17.1 0.0007

?11 29 39 53 29 7.7 0.0516

?12 41 37 42 30 2.7 0.4403

?13 32 24 42 52 10.8 0.0126

?14 35 33 49 33 3.4 0.3306

?15 22 41 54 33 11.1 0.0109

The positions relative to the CArG box are indicated numerically

from -15 to ?15. The frequencies of nucleotide occurrence are

investigated numerically in each cell and the numbers corresponding

to v2 and P represent the v2 and probability values relating to each

recording position (see Materials and methods). Bold numbers with

low P values exhibit conserved positions in the corresponding species

106 W. Wu et al.: Implication of CArG-SRF binding context

123

CArG boxes and their flanking sequences between human

and mouse genomes. First, the ten positions (from -5 to

?5) of the CArG box have significant p values almost

equal to 0, represented as highly conserved binding posi-

tions to SRF in mouse and human genomes. Also, there is a

preference for C at -5 and -4, G at ?4 and ?5, T at -2

and ?1, A at -1 and ?2, and W at -3 and ?3, which

seems to indicate that the CArG box has a reversible motif:

CCWTA/TAWGG. Second, some flanking positions (-15,

-8, ?7, and ?8) of interest, sharing a similar conservation

status in human and mouse genomes, are more conserved

(P \ 0.005) than other flanking positions. From these

conserved positions there is a bias for C or G at -15, T at

-8, C at ?7, and A at ?8. Third, there are still a few

different conserved positions between the human and

mouse genomes. These are -7 and ?10 conserved posi-

tions (P \ 0.01) in the mouse genome but not conserved in

the human genome, and ?9 conserved positions (P \ 0.01)

in the human genome but not conserved in the mouse

genome. Perhaps in line with the differences in CArG

flanking sequences between mouse and human genomes,

the differences in orthologous SRF proteins between mouse

and human are presented in Fig. 1. This figure shows that

orthologous SRF-core residues (amino acids 132-223) are

exactly the same in mouse and human. Nonetheless, a few

mismatches are still observed in the flanking sequences of

orthologous SRF-core regions (see Discussion).

In addition, we specifically performed analysis on -8

and ?8 positions that we have identified as conserved

Table 2 Comparison of the flanking positions between consensus

CArG boxes (A) and CArG-like boxes (B)

Position A T G C v2 P

(A) CArG boxes

-15 34 19 44 52 15.9 0.0009

-14 37 30 34 48 4.4 0.2284

-13 37 39 39 34 0.6 0.8980

-12 33 33 38 45 2.4 0.5020

-11 36 36 31 46 2.9 0.4217

-10 39 35 35 40 0.4 0.9392

-9 46 32 27 44 6.4 0.0925

-8 35 70 21 23 42.2 0.0000

-7 30 40 43 36 2.8 0.4308

-6 31 45 38 35 3.1 0.3894

-5 0 0 0 149 439.9 0.0000

-4 0 0 0 149 439.9 0.0000

-3 67 82 0 0 152.8 0.0000

-2 35 114 0 0 235.0 0.0000

-1 116 33 0 0 240.2 0.0000

?1 34 115 0 0 239.3 0.0000

?2 108 41 0 0 208.3 0.0000

?3 71 78 0 0 150.2 0.0000

?4 0 0 149 0 452.7 0.0000

?5 0 0 149 0 452.7 0.0000

?6 41 29 39 40 2.3 0.5139

?7 40 25 25 59 19.9 0.0003

?8 72 32 23 22 44.5 0.0000

?9 26 51 45 27 13.6 0.0033

?10 49 33 41 26 8.1 0.0429

?11 28 43 46 32 6.4 0.0898

?12 45 38 36 30 3.2 0.3735

?13 30 33 40 46 4.0 0.2658

?14 30 33 54 32 5.8 0.1212

?15 29 43 44 33 4.8 0.1852

(B) CArG-like boxes

-15 25 18 43 51 20.8 0.0001

-14 30 38 32 37 0.4 0.9284

-13 27 45 28 37 4.0 0.2646

-12 27 30 27 53 12.8 0.0044

-11 32 44 22 39 6.3 0.0979

-10 25 38 30 44 4.5 0.2150

-9 42 30 36 29 5.3 0.1454

-8 28 55 24 30 13.6 0.0032

-7 24 39 46 28 7.9 0.0484

-6 29 52 23 33 10.6 0.0127

-5 6 3 4 124 306.7 0.0000

-4 4 12 0 121 288.2 0.0000

-3 70 56 2 9 104.8 0.0000

-2 20 115 1 1 239.4 0.0000

-1 84 26 20 7 115.3 0.0000

Table 2 continued

Position A T G C v2 P

?1 20 94 9 14 126.6 0.0000

?2 104 27 4 2 221.6 0.0000

?3 65 63 6 3 105.5 0.0000

?4 11 4 122 0 306.9 0.0000

?5 6 4 121 6 298.1 0.0000

?6 30 41 27 39 2.6 0.4512

?7 31 30 21 55 17.7 0.0003

?8 61 39 24 13 43.0 0.0000

?9 29 37 50 21 13.5 0.0033

?10 35 27 48 27 10.3 0.0137

?11 30 37 40 30 1.9 0.5858

?12 38 32 35 32 1.9 0.5868

?13 35 26 37 39 4.0 0.2592

?14 34 36 40 27 1.6 0.6585

?15 28 33 46 30 5.8 0.1180

Italic numbers represent the same conservation level with significant

P values at corresponding positions in A and B, and bold numbers

represent different conservation levels at corresponding positions in A

and B

W. Wu et al.: Implication of CArG-SRF binding context 107

123

positions in CArG flanking sequences. Of the 150 CArG

boxes in the mouse genome, nearly half (73) have nucle-

otides T or A at the two positions, which is approximately

twice as many as the expected value of 37.5.

Conservation status of CSBC between consensus

and CArG-like boxes

Based on Miano’s study on CArG boxes (Miano et al.

2007), we classified the 286 functional CArG boxes into

two categories: 149 consensus CArG boxes that are CC(A/

T)6GG with a high SRF-binding affinity, and 137 CArG-

like boxes that are a 1-bp deviation from consensus with a

much weaker SRF-binding affinity. We then compared the

characteristics of the flanking positions between the two

Table 3 Observed and expected counts, v2, and their corresponding

ratios for each dinucleotide (see Materials and methods)

Base A C G T Sum

(A)

A

Observed 200 117 213 125 655

Expected 154.5 160.4 171.8 168.4

Ratio 1.294 0.729 1.240 0.742

v2 13.4 11.7 9.9 11.2

C

Observed 190 189 93 209 681

Expected 160.7 166.7 178.6 175

Ratio 1.182 1.134 0.521 1.194

v2 5.4 3.0 41.0 6.6

G

Observed 157 196 197 156 706

Expected 166.6 172.8 185.1 181.5

Ratio 0.942 1.134 1.064 0.860

v2 0.5 3.1 0.8 3.6

T

Observed 90 159 205 204 658

Expected 155.2 161.1 172.5 169.1

Ratio 0.580 0.987 1.188 1.206

v2 27.4 0.0 6.1 7.2

Sum 637 661 708 694 2700

(B)

A

Observed 194 133 191 182 703

Expected 180.5 155.4 164.5 202.6

Ratio 1.091 0.856 1.161 0.898

v2 1.0 3.2 4.3 2.1

C

Observed 196 162 27 226 611

Expected 156.8 135.1 142.9 176.1

Ratio 1.250 1.199 0.189 1.283

v2 9.8 5.4 94.0 14.1

G

Observed 151 135 170 166 622

Expected 159.7 137.5 145.5 179.3

Ratio 0.946 0.982 1.168 0.926

v2 0.5 0.0 4.1 1.0

T

Observed 156 173 250 212 791

Expected 203 174.9 185.1 228

Ratio 0.768 0.989 1.351 0.930

v2 10.9 0.0 22.8 1.1

Sum 700 603 638 786 2727

Values represent the distributions of dinucleotides in CArG flanking

sequences (A) and in intron sequences (B), which serve as the control.

The greatest differences between A and B are in boldface

Table 4 Association analysis of (A) tetranucleotide in the middle of

10-nt CArG boxes and (B) any two positions from -15 to ?15

A

Association Obs_N Exp_N Obs_F Exp_F v2 P

TATA 80 80.78 0.3493 0.3527 0.0075 0.8523

TTTA 28 25.53 0.1223 0.1115 0.2384 0.4518

TATT 27 25.53 0.1179 0.1115 0.0843 0.6580

TAAA 24 23.14 0.1048 0.1011 0.0317 0.7944

AATA 20 21.99 0.0873 0.0960 0.1799 0.5305

AAAA 13 6.30 0.0568 0.0275 7.1246 0.0031

TTTT 13 8.07 0.0568 0.0352 3.0106 0.0342

AATT 8 6.95 0.0349 0.0304 0.1584 0.6413

TTAA 6 7.32 0.0262 0.0319 0.2366 0.5732

ATAT 3 0.63 0.0131 0.0027 5.5582 0.0108

ATAA 2 1.99 0.0087 0.0087 0.1213 0.4626

ATTA 1 6.95 0.0044 0.0304 5.0945 0.0078

ATTT 1 2.20 0.0044 0.0096 0.2211 0.3938

TAAT 1 7.32 0.0044 0.0319 5.4523 0.0060

AAAT 1 1.99 0.0044 0.0087 0.1213 0.4673

TTAT 1 2.31 0.0044 0.0101 0.2854 0.3545

B

PLT0.001 PLT0.0001

Positions v2 Positions v2

[-15-14] 32.8641 [-8-7] 44.8884

[-12-11] 31.2061 [?7?8] 61.8528

[-11?13] 28.7352 [?8?9] 34.5924

[?6?7] 31.6810 [?9?10] 39.1402

[?11?12] 50.1089

Obs_N and Exp_N represent observed and expected numbers and

Obs_F and Exp_F represent observed and expected frequency. Prepresents the probability of the corresponding tetranucleotide

occurrence. PLT0.001 and PLT 0.0001 represent P \ 0.001 (rather

than say [0.0001) and P \ 0.0001

108 W. Wu et al.: Implication of CArG-SRF binding context

123

categories in Table 2A and B. The table shows some

positions with different conservation status (-12, -7, -6,

and ?10) in the flanking sequences of the two categories,

in addition to the same conserved positions (-15, -8, ?7,

?8, and ?9). Upstream of the CArG-like boxes, -12, -7,

and -6 positions show statistically significant (P * 0.05)

results, whereas these positions of the consensus CArG

boxes show insignificant results. Similarly, downstream of

the CArG-like boxes, ?10 shows a little more significance

than the consensus boxes (see Discussion).

In addition, there is an incidental finding in the core

CArG boxes (Table 2B) that G is not observed at -4 and C

is not observed at ?4.

Distribution of dinucleotides in CSBC

High content of the dinucleotide CpG is generally associ-

ated with promoters in mammalian genomes (Antequera

2003; Rozenberg et al. 2008). Thus, we investigated the

contents of dinucleotides 10 bp upstream and 10 bp

downstream of the 150 CArG boxes in the mouse genome

(see Materials and methods) and compared them with the

contents of dinucleotides in intron sequences, which served

as the control (see Table 3A and B). In CArG flanking

sequences, the lowest ratios of observed to expected

dinucleotide counts are 0.521 for CpG and 0.580 for TpA,

followed by the ratios of ApC and ApT dinucleotides.

When compared to the control, CSBC has a far greater

ratio (0.521) for CpG and an apparently lower ratio (0.580)

for TpA. Besides, higher ratios for ApA, TpT, and GpC and

lower ratios for ApT and TpG in the CSBC compared with

those in the control also exhibit some pre-existing features

in CArG flanking sequences (see Discussion).

Association analysis of CArG boxes and CSBC

Statistical frequencies of nucleotides (see Tables 1 and 2)

showed that the CArG box is represented as the consensus

sequence C-5C-4W-3T-2A-1/T?1A?2W?3G?4G?5, which

probably demonstrates two points. First and obviously, the

CArG box is strongly biased and represented as a palin-

dromic sequence. Second, the C-5C-4W-3T-2A-1

sequence seems to be strongly linked to its complement,

T?1A?2W?3G?4G?5. To investigate whether this linkage

exists, we performed an association analysis on -2, -1,

?1, and ?2 positions in the middle of the CArG boxes in

Table 4A (see Materials and methods). The table surely

demonstrates that the observed number of T-2A-1/T?1A?2

is the most; however, surprisingly, T-2A-1 is not statisti-

cally significantly linked to its complement, T?1A?2, which

is roughly consistent with the small amount of mutual

information (see Fig. 2) between any two positions in the

functional CArG boxes produced from enoLOGOS online

(Workman et al. 2005). However, the tetranucleotides

A-2A-1A?1A?2, A-2T-1T?1A?2, and T-2A-1A?1T?2 are

Fig. 1 Alignment of orthologous SRF proteins in human and mouse.

Dots represent amino acid identity to human SRF. Underlinedresidues (132-223 amino acids) represent SRF-core region involved in

DNA binding, dimerization, and transcriptional activation. Amino

acid mismatches or indel residues are italicized

Fig. 2 Logo of CArG boxes with mutual information generated from

286 functional CArG boxes in mammalian genomes using enoLO-

GOS (Workman et al. 2005). Mutual information of any two positions

in the CArG box is lower than 0.07 bite (see Supplementary Material)

W. Wu et al.: Implication of CArG-SRF binding context 109

123

statistically significantly linked at P = 0.0031, 0.0078, and

0.0060, respectively (see Discussion).

Furthermore, we tested the associations of any two

positions in Table 4B. The table shows that there are no

associations in the functional CArG boxes at P \ 0.001,

which is consistent with the small amount of mutual

information shown in Fig. 2. By contrast, there are some

significant associations in both upstream and downstream

regions of the CArG boxes. Intriguingly, the downstream

region has more associations (P \ 0.0001) than the

upstream region (see Discussion).

Discussion

The regulation of SRF-dependent genes requires the spe-

cific recognition and binding of the CArG box by SRF,

however often the same CArG box is bound specifically by

SRF in one context but not in another. Thus, the small

consensus CArG box is not sufficient to account for the

binding specificity. However, other features such as the

sequences flanking the box (Hendrix et al. 2005; Leung and

Miyamoto 1989), the distance between multiple CArG

boxes (De Folter and Angenent 2006; Miano 2003), and the

position of the box in relation to TSS (Shen et al. 2009; Sun

et al. 2006) should play more important roles in CArG-SRF

recognition. Besides, the presence of SRF cofactors

(Cooper et al. 2007; Long et al. 2007; Miano 2003; Miano

et al. 2007; Pellegrini et al. 1995; Rensen et al. 2006; Sun

et al. 2006; Verger and Duterque-Coquillaud 2002), which

can bind to the SRF’s transcriptional activation domain,

regulates the expression of cell-specific SRF-target genes.

Moreover, epigenetics (Cooper et al. 2007; McDonald et al.

2006; Shaw et al. 1989; Verger and Duterque-Coquillaud

2002) and SRF with itself alternatively spliced (Kemp and

Metcalfe 2000; Patten et al. 2004) and phosphorylation

(Iyer et al. 2006) contribute to SRF and CArG binding

affinity and expression of downstream genes.

In this study we identified some conserved positions like

-15, -8, and ?8 in CArG flanking sequences, with CpG

and TpA contents quite different from those in introns in

mammalian genomes. Besides, we found more conserved

positions in the flanking sequences of CArG-like boxes

than in those of consensus CArG boxes. These results

suggest that CArG flanking sequences are truly affected by

SRF’s constraints, probably because the flanking sequences

should help the CArG box make a special configuration

with a bend of 72� (Pellegrini et al. 1995) in order to be

recognized and bound by SRF.

It is no surprise that -5 to ?5 positions are highly

conserved since these positions are direct contacts for the

SRF-core a1 helix at the major groove (-4/-5 or ?4/?5)

and for the SRF-core N extension at the minor groove

(from -3 to ?3) (Pellegrini et al. 1995). The CArG box

shows a reversible motif: C-5C-4W-3T-2A-1/T?1A?2-

W?3G?4G?5 (Table 1), and there is a strong overrepre-

sentation of the association T-2A-1T?1A?2. However,

T-2A-1 is not statistically significantly linked to T?1A?2

(Table 4A), which implies two things: First, the presence

of T-2A-1 is independent of its complement T?1A?2 and

the behavior of the CArG box is determined mostly by

SRF. Second, these functional CArG boxes are not para-

logs but rather originate from other sources (Wu et al.,

unpublished). However, the observed number of associa-

tion A-2A-1A?1A?2 is significantly higher than expected,

and the observed numbers of associations A-2T-1T?1A?2

and T-2A-1A?1T?2 are both significantly lower than

expected. The reason for the appearance of these higher or

lower associations is most likely that the pattern of spon-

taneous mutation yields a higher observed number of AA

and lower observed numbers of TA and AT than expected,

a situation that is consistent with the distributions of

dinucleotides (Table 3A and B) probably stemming from

structural constraints at the DNA level (Antequera 2003;

Jabbari and Bernardi 2004). Taken together, the results

indicate that the CArG box is a highly conserved element

represented as CCWTATAWGG, but there is no significant

linkage disequilibrium between T-2A-1 and T?1A?2.

However, there is still significant linkage disequilibrium in

some tetranucleotides, indicating that although SRF has a

strong affect on the CArG box, the box is also affected by

spontaneous mutation, so long as the mutation does not

demolish the special configuration to be recognized by

SRF.

In the CSBC there are some highly conserved positions

with a strong bias for T at -8, A at ?8, and S at -15 in

mammalian genomes, implying that these positions have

direct contact with SRF or contribute to making the DNA

bend 72� and distribute around the dyad axis of the CArG

box bound by SRF (Pellegrini et al. 1995). Pellegrini et al.

1995 showed that the 5-methyl group of T?8 has hydro-

phobic contact with Thr159 and Ser162 in the SRF a1 helix,

and phosphate of A?9 has hydrogen contact with Thr191

and His193 in the SRF b loop. However, there are also some

positions with different conservation status between human

and mouse genomes. What factors result in these loci-

conserved differences? SRF proteins binding to a CArG

box have significant constraints on the structure of their

special target CArG box (Huet et al. 2005; Pellegrini et al.

1995; Stepanek et al. 2007). They probably also constrain

the adjacent positions surrounding the CArG box. Thus, the

mismatches in the orthologous SRF between human and

mouse may partly explain the differences of CArG flanking

sequences between human and mouse genomes.

In contrast to the flanking positions of consensus CArG

boxes, the flanking positions of CArG-like boxes are more

110 W. Wu et al.: Implication of CArG-SRF binding context

123

conserved, suggesting that given a low affinity of CArG-

like box binding with SRF, the flanking positions of the

CArG-like box would contribute more to SRF recognizing

and binding to the CArG-like box than those of the con-

sensus CArG box. In other words, the requirements are

more stringent on the flanking positions of the CArG-like

box than on those of the consensus CArG box, and there

should be some reasonable compensation in the flanking

positions for the CArG-like box binding with SRF, if the

consensus mutates 1 bp across the CArG box. Thus, we can

infer that the effect of natural selection is stronger on the

flanking sequences of the CArG-like box than on those of

the consensus CArG box. Besides, the functional CArG-

like box at regulatory regions in SRF target genes (Cooper

et al. 2007; Sun et al. 2006) often appears associated with

the occurrence of another CArG box or SRF cofactor

binding site, leading to an increase in the stability of the

CArG-like box binding with SRF when a third or even

fourth factor interacts to form a higher-order complex (De

Folter and Angenent 2006). This suggests that given a low

binding affinity of the CArG-like box with SRF, other SRF

or its cofactor could contribute to increasing the stability

and the flanking sequences. In addition, in these CArG-like

boxes, G is not observed at -4 and C is not observed at ?4.

Although there has been no direct experimental evidence

indicating that C-4 cannot mutate to G-4 or G?4 to

C?4, indirect evidence (Huet et al. 2005; Stepanek et al.

2007) has shown that the mutations C-5 ? G-5 or

C-5C-4 ? G-5G-4 can significantly alter the binding

stoichiometry of core-SRF, which leads us to conclude that

the CpG doublet at -4-5 or ?4?5 is prone to demolish

the curvature of 72� bound by SRF.

Interestingly, there are more significant associations (see

Table 4B) and more conserved positions (Table 1) down-

stream than upstream of the CArG boxes. A classical

experiment (Pellegrini et al. 1995) showed that the CArG

box is bent around the SRF by 72�, with the curvature

nonuniformly distributed around the dyad axis of the box,

implying that there are some different requirements for the

downstream region than for the upstream region. Besides,

given that the downstream region is closer to the TATA

box or TSS than the upstream region, the downstream

region should contribute more to making SRF interact

directly with the TFIIF of the RNA polymerase II holo-

enzyme (Joliot et al. 1995; Zhu et al. 1994). Taken toge-

ther, we suggest that the downstream region plays a

stronger role in mediating the regulation and expression of

SRF target genes.

Under the hypothesis of neutral mutation, the distribu-

tion of universal dinucleotides in intron sequences is

approximately equal to that in the CSBC. Differences

between the two distributions probably have been due to

factors related to natural selection, linkage, or spontaneous

mutation locus (Nachman and Crowell 2000; Walser et al.

2008). By DNA methyltransferase, most CpGs are meth-

ylated on the C residue and prone to spontaneous deami-

nation to T, then CpG contents should decrease with

evolutionary time, whereas TpG (or CpA) contents should

increase in negative correction (Bird 1980; Coulondre et al.

1978; Ehrlich and Wang 1981; Walser et al. 2008). In this

regard, it is no surprise that Table 3 shows these sequences

to have low CpG, high TpG, and CpA contents in common.

However, the ratio for CpG in CArG flanking sequences is

far greater than that in introns. To our knowledge, in

mammals, approximately 60% of all promoters contain

CpG islands that have a higher CpG content than the

genome average (Antequera 2003). This could account for

the higher CpG content in CArG flanking sequences than

that in introns. Together, the result shows that natural

selection constrains spontaneous mutation in CArG flank-

ing sequences, demonstrating again that CArG-SRF bind-

ing has a relationship with the adjacent positions

surrounding the CArG box.

In addition, sometimes the flanking sequences of the

CArG box not only help SRF recognize and bind to the

CArG box, they also contribute to the SRF cofactor’s

recognizing and binding to their corresponding DNA

binding sites adjacent to the CArG box. For example, the

ETS binding site (GGAT) is located 2 bp proximal to a

CArG box that is bound by SRF in the c-fos promoter

(Treisman 1986). Another example is that TCF binding site

is adjacent to a CArG box in the SM22 promoter (Cen et al.

2004). Both examples indicate that the expression of

CArG-containing genes is affected not only by SRF bind-

ing sites, but some, at least, are affected by their flanking

sequences and SRF cofactor’s binding site.

In summary, we characterized functional CArG boxes

and, in particular, their flanking sequences. We suggest that

these pre-existing features of CSBC contribute to helping a

CArG box form a characteristic spatial structure to be

recognized and bound by SRF. Furthermore, the study

would be an important step toward developing a reliable

method to predict an SRF binding site and improve

experimental analysis of SRF-mediated CArG-containing

gene transcription. Finally, what is more intriguing and

significant is that this study gives us a hint that the flanking

sequences of other transcriptional factor binding sites, such

as the Ets-binding site, the GATA binding site (EBS), and

the MyoD binding sites, would also have some special

features to be recognized correctly by their cognate tran-

scriptional factors.

Acknowledgments We are grateful to Zhuoran Huang, Mingjing

Zhang, Xiaoqian Jiang, and Hongxia Pang at the Bioinformatics

Center of Northwest A&F University for many useful discussions. We

also thank Professor Zhao Xu at the College of Science of Northwest

A&F University for his suggestions on the v2 test. We are also

W. Wu et al.: Implication of CArG-SRF binding context 111

123

grateful to Donna Elizabeth at the Department of Foreign Languages

of Northwest A&F University and Associate Professor Mengjun Liu

at the College of Agronomy of Northwest A&F University for cor-

recting the English version of the manuscript. Finally, we thank the

anonymous reviewers for their insightful suggestions and criticisms.

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