Download pdf - Autoregulatory binding sites in the zebrafish six3a promoter region define a new recognition sequence for Six3 proteins

Autoregulatory binding sites in the zebrafish six3apromoter region define a new recognition sequence forSix3 proteinsClotilde S. Suh, Staale Ellingsen*, Lars Austbø, Xiao-Feng Zhao, Hee-Chan Seo and Anders Fjose

Department of Molecular Biology, University of Bergen, Norway

Introduction

Vertebrate Six3 proteins have important roles during

the development of eyes and forebrain, and belong to

the Six ⁄Sine oculis family. This family represents a

divergent group of the homeodomain (HD) superfam-

ily of transcription factors [1,2]. The 60 amino acid

HD, which is a DNA-binding domain, has a conserved

global fold consisting of three a-helices and a flexible

N-terminal arm that becomes more ordered upon

DNA binding [3–6]. Binding to specific DNA

sequences is mediated by interactions between particu-

lar amino acids in the ‘recognition helix’ and bases in

the major groove, and specific contacts between the

N-terminal arm and the minor groove [4,7,8].

Specific base contacts in the minor groove involve

the first two nucleotides in the TAAT core, and are

achieved through interactions with residues at posi-

Keywords

chromatin; eye development; homeobox;

transcription factor; transgenic

Correspondence

A. Fjose, Department of Molecular Biology,

University of Bergen, PO Box 7803, N-5020

Bergen, Norway

Fax: +47 555 89683

Tel: +47 555 84331

E-mail: [email protected]

*Present address

National Institute of Nutrition and Seafood

Research, NIFES, PO Box 2029 Nordnes,

N-5817 Bergen, Norway

(Received 11 September 2009, revised

22 December 2009, accepted 29 January

2010)

doi:10.1111/j.1742-4658.2010.07599.x

The homeodomain (HD) transcription factor Six3, which is a member of

the Six ⁄Sine oculis family, is essential for development of the eyes and fore-

brain in vertebrates. It has recently been claimed that the HDs of Six3 and

other members of the Six family have a common recognition sequence,

TGATAC. However, a different recognition sequence including the typical

TAAT core motif, which has not yet been fully defined, has also been pro-

posed for the Six3 HD in mice. Our study of the zebrafish orthologue

six3a, which has an identical HD, shows that it binds in vitro to multiple

TAAT-containing sites within its promoter region. Comparison of the dif-

ferent binding affinities for these sequences identifies three high-affinity

sites with a common TAATGTC motif. Notably, this new recognition

sequence, which is supported by our analysis of the influence of single-

nucleotide substitutions on the DNA-binding affinity, is distinct from all

of the DNA-binding specificities previously described in surveys of HDs.

In addition, our comparison of Six3a HD binding to the novel TAATGTC

motif and the common recognition sequence of Six family HDs

(TGATAC) shows very similar affinities, suggesting two distinct DNA-

binding modes. Transient reporter assays of the six3a promoter in zebrafish

embryos also indicate that the three high-affinity sites are involved in auto-

regulation. In support of this, chromatin immunoprecipitation experiments

show enrichment of Six3a binding to a six3a promoter fragment containing

two clustered high-affinity sites. These findings provide strong evidence that

the TAATGTC motif is an important target sequence for vertebrate Six3

proteins in vivo.

Abbreviations

ChIP, chromatin immunoprecipitation; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; GFP, green

fluorescent protein; GST, glutathione-S-transferase; HD, homeodomain; hpf, hours postfertilization.

FEBS Journal 277 (2010) 1761–1775 ª 2010 The Authors Journal compilation ª 2010 FEBS 1761

tions 2, 3 and 5–8 in the N-terminal arm. Also, an

arginine at position 5 is important in most HDs

[4,5,9]. Similarly, the recognition helix makes specific

contacts with several nucleotides in the core motif, and

its residues at positions 47, 50 and 54 also specify two

adjacent nucleotides 3¢ of the TAAT core [5,10]. For

example, HDs containing Lys50 and Gln50 have

been shown to bind specifically to TAATCC and

TAATGG, respectively [11–13]. Recent studies indicate

that the sequence recognition also depends on a few

additional flanking nucleotides, and this variation in

specificity may include more than 60 distinct DNA-

binding activities [14].

The Six ⁄Sine oculis family proteins also have a con-

served Six domain of 115–119 amino acids involved in

protein–protein interactions [1,15], and can be subdi-

vided into three subfamilies, Six1 ⁄2, Six4 ⁄ 5, and

Six3 ⁄ 6, on the basis of their HD sequence divergence

and characteristic tetrapeptides in the N-terminal arm

[16]. The absence of Arg5 in their N-terminal arms

may explain, in part, why regulatory DNA sequences

that bind Six1 ⁄ 2 and Six4 ⁄ 5 do not contain the TAAT

core [17]. Although the Six3 ⁄ 6 proteins also lack Arg5,

their HDs are more distinct from those of the members

of the other two subfamilies, and various studies have

suggested that their DNA-binding specificity is differ-

ent [1,16,18]. In a previous investigation of murine

Six3, a common TAAT core motif was identified by

in vitro binding site selection from a randomized pool

of oligonucleotides, and autoregulatory binding sites

containing the TAAT core were also identified in the

promoter of the Six3 gene [18]. However, more recent

studies have provided evidence that Six3 ⁄ 6 proteins

have similar in vitro DNA-binding specificities to those

of the other Six family members [10,14]. Further analy-

sis of the binding affinities of functional Six3 target

sites and how they function in vivo may help to clarify

uncertainties regarding the recognition sequences of

these HD proteins.

Two orthologues of the murine Six3 gene, six3a and

six3b, are present in the zebrafish, Danio rerio, owing

to the extra genome duplication that occurred before

the teleost radiation [15,19]. An additional Six3-like

gene in zebrafish, six7, was probably generated by an

independent gene duplication event [20]. Several stud-

ies of Six3 homologues in mouse, fish and Xenopus

have demonstrated that these genes are essential for

forebrain and eye development, and their importance

is also reflected in human mutant phenotypes [21–25].

In these processes, Six3 proteins have been shown to

act both as transcriptional activators and repressors,

and as regulators of cell proliferation through interac-

tions with the cell cycle inhibitor Geminin [26,27].

Studies on Six3 proteins in zebrafish have contrib-

uted to our understanding of their functional roles in

forebrain and eye development [22,28–30], and how

they can act as transcriptional repressors through

interactions with members of the Groucho family of

corepressors [31]. Relatively little is known about the

regulation of zebrafish six3 genes during development

[32,33], but essential cis-regulatory elements have been

identified in one of the gene homologues in medaka

fish [34].

We have investigated the significance of the high

density of TAAT sequences present in the zebrafish

six3a promoter region. Our comparison of the relative

binding affinities of these potential target sites for the

Six3a HD identified several strong binding sites that

defined the sequence TAATGTC as a recognition

motif. Results from chromatin immunoprecipitation

(ChIP) experiments and transient reporter assays of

the six3a promoter in zebrafish embryos supported the

functional role of these high-affinity sites in mediating

autoregulation. Hence, it is also likely that many of

the target genes of vertebrate Six3 proteins are recog-

nized on the basis of high-affinity binding to sequence

elements containing this motif.

Results

A 3.6 kb promoter region of six3a recapitulates

early embryonic expression

The genomic region upstream of the translational start

site in zebrafish six3a is syntenic with a 4.5 kb pro-

moter region of the orthologous medaka (Oryzia latipes)

gene olSix3.2, which contains cis-regulatory elements

responsible for its spatiotemporal regulation in embryos

[34]. Additional evidence that the corresponding

promoter region of zebrafish six3a contains cis-acting

elements required for early expression in the eyes and

forebrain was obtained from transient expression assays

with injected reporter constructs [33].

To analyse the significance of the six3a promoter

region, we fused a 3.6 kb genomic fragment to the

ORF of an enhanced green fluorescent protein (EGFP)

reporter gene in a Tol2 vector (Fig. 1A), and used this

construct to establish transgenic lines of zebrafish (see

Experimental procedures). From 53 founders crossed

to wild-type fish, we identified three transgenic lines

with EGFP expression comparable to that of the

endogenous six3a gene (data not shown). The trans-

genic line Tg(3.6S3a:EGFP) was chosen for direct

comparison of EGFP expression with the spatial distri-

bution of endogenous six3a transcripts by in situ

hybridization. At 12 h postfertilization (hpf), EGFP

New recognition sequence for Six3 C. S. Suh et al.

1762 FEBS Journal 277 (2010) 1761–1775 ª 2010 The Authors Journal compilation ª 2010 FEBS

expression was detected in the optic vesicles and ros-

tral brain, where six3a transcripts were also shown to

accumulate (Fig. 1B). These results confirm that the

3.6 kb promoter region included in the six3a:EGFP

transgene contains regulatory sequences sufficient to

drive expression mimicking early six3a endogenous

expression.

Differences in Six3a HD binding to TAAT core

motifs within its promoter region

The promoters of murine Six3 and human SIX3

contain autoregulatory binding sites [18,35]. In the case

of the murine Six3 promoter, it has been shown that

negative autoregulation involves clustered TAAT core

motifs and interaction with Groucho-related corepres-

sors [18]. Sequence analysis of the 3.6 kb promoter

region of six3a revealed enrichment and clustering of

the same sequence motif (Fig. 2). Within this promoter

region, the common core motif is present at 43 posi-

tions in both orientations (TAAT or ATTA). The ratio

between TAAT and ATTA on the coding strand is

25 : 18 (Fig. S1). In initial studies of several of the 18

ATTA sites by electrophoretic mobility shift assays

(EMSAs), we observed the strongest shift for the a1

site (data not shown). Therefore, a1 was selected as a

reference for comparisons of the binding affinities of

the 18 different ATTA-containing sites. In this study,

we used a biotin-labelled 27 bp DNA fragment con-

taining a1 as a probe in EMSAs, and tested the influ-

ence of this on HD complex formation in the presence

of excess amounts of unlabelled fragments representing

the individual a1–a18 sites (Fig. 2B). Whereas a 200-

fold excess of unlabelled a1 competitor almost

completely prevented the formation of probe–HD

complexes in EMSAs, only a few of the other sites

were able to compete significantly under the same

conditions. Notably, the competitor representing a2,

which is located a short distance (� 10 bp) upstream

of a1 (Fig. S1), also caused a strong reduction in

formation of the probe–HD complex.

In addition to their clustering and high binding

affinities for the Six3a HD, the flanking nucleotides of

the core ATTA motifs in a1 (G1T2C3A4T5T6A7G8G9)

and a2 (G1A2T3A4T5T6A7T8G9) have common Gs at

positions upstream (G1) and downstream (G9). Taking

into consideration that the binding specificities of HDs

have been shown to depend mainly on the two nucleo-

tides 5¢ to the ATTA core motif [10–14], the common

G1 was likely to be important. However, three addi-

tional sites (a6, a9, and a11), which have a G in the

same 5¢-position relative to the ATTA core, showed

much weaker binding, indicating that other nucleotide

positions also influence the binding affinity (Fig. 2B).

To address the functional importance of the G1 nucle-

otide, an inspection of the 25 TAAT sites was per-

formed, and this identified four sites (t2, t13, t15, and

t17) containing a G in this 5¢-position. Among these

sites, only t15 showed similar binding affinity to the

Six3a HD as a1 and a2 (Fig. 2C). This further indi-

cated that flanking nucleotides other than G1 have sig-

nificant influence on the binding affinity. Notably,

three ATTA sites (a10, a14, and a18) without the G1

flanking nucleotides also bound quite strongly to the

Six3a HD (Fig. 2B). However, among the 21 TAAT

sites lacking the G1 nucleotide, none showed significant

competition with the a1 probe in EMSAs (Fig. S2).

Hence, these comparative analyses showed that the

frequency of high-affinity sites was significantly higher

among the G1-containing sites. An additional compari-

son of the relative strengths of the various high-affinity

binding sites conducted with lower amounts of

competitor still showed strongest binding for the

three G1-containing sites, a1, a2, and t15 (Fig. S3).

six3a promoter region

ATG

EGFP

730 bp3635 bp

A

B C DTg (3.6S3a:EGFP) six3aEGFP

Fig. 1. A 3.6 kb promoter region of the

six3a gene is sufficient to recapitulate its

early expression. (A) Schematic representa-

tion of the six3a promoter region including

the 5¢-UTR, fused to the coding region of

EGFP. Arrows indicate the position of the

transcription start site and the initiation

codon. (B) Lateral view of EGFP expression

in a Tg(six3a:EGFP) embryo at 12 hpf.

(C) Detection of EGFP transcripts in a

Tg(six3a:GFP) embryo by in situ

hybridization (lateral view of 12 hpf stage).

(D) Detection of endogenous six3a

transcripts at 12 hpf (lateral view).

C. S. Suh et al. New recognition sequence for Six3


Therefore, we aimed to investigate the functionality of

these sites in vivo and the relative importance of the

different nucleotide positions flanking their ATTA core

motifs.

Deletion analysis of the six3a promoter indicates

autoregulatory binding sites

To determine whether any of the strong Six3a

HD-binding sites identified by EMSA might have a

function in vivo, we made several promoter–reporter

constructs with small deletions of regions containing

particular sites. These constructs, which were made

from the construct pS3aPG used to make the trans-

genic line (Fig. 1A; see Experimental procedures), were

tested in transient reporter assays based on microinjec-

tion into fertilized eggs and measuring the number of

EGFP-expressing cells at 12 hpf (Fig. 3). Notably,

when six3a mRNA and pS3aPG were coinjected, we

observed a more than two-fold increase in the number

of EGFP-expressing cells as compared with injection of

pS3aPG alone. This indicated that overexpression

of Six3a caused an increase in EGFP expression

through binding to one or more sites within the

promoter region in the pS3aPG reporter construct.

Consistent with the expression pattern of the endoge-

a1 a11

a12

a13

a14

a15

a16

a17

a18

x 200

a1 a2 t6 a9 a11

t2 t13

t15

t17

x 200

a1 a2 a3 a4 a5 a6 a7 a8 a9 a10

x 200

+++++

++++

+/–

+/–

+/–

–

+/–

+/–

+/–

++++

+/–

–

+/–

+++

+

–

+/–

++

–

+/–

+++++

+/–

a1

a2

a3

a4

a5

a6

a7

a8

a9

a10

a11

a12

a13

a14

a15

a16

a17

a18

t2

t13

t15

t17

Relative

competition

Motif

18A

B

C

D

17 16 15 14 13 12 1110 9 8 7 6 5 4 3 2

t2

1**

t13t15t17*

six3a promoter region (pS3aP)

Fig. 2. Distribution and relative binding affinities of potential Six3a HD target sites within the six3a promoter region. (A) Distribution of ATTA

motifs within the 3.6 kb promoter region of zebrafish six3a (pS3aP). Vertical bars (numbered 1–18) indicate ATTA motifs in the forward

strand. Vertical bars indicate 25 ATTA motifs in the reverse strand, and the four GNNATTA sites in the reverse strand are labelled (t2, t13,

t15, and t17). Stars indicate the three high-affinity GNNATTA sites (a1, a2, and t15). An arrow indicates the transcription start site. (B) EMSAs

with the Six3a HD and biotin-labelled a1 probe. Competition was performed using a · 200 molar excess of unlabelled fragments containing

the ATTA motifs a1–a18 (forward strand). The left lane shows the control (labelled probe together with Six3a HD). (C) Competitive EMSA

with biotin-labelled a1 probe and a · 200 excess of unlabelled fragments representing all GNNATTA sites (a1, a2, a6, a9, a11, t2, t13, t15,

and t17). (D) Table showing the relative competition of the different sites as compared to a1, from highest competition (+++++) to lack of

competition ()).



nous six3a gene [15], we observed EGFP-positive cells

mainly in the rostral region of the head at 12 hpf

(Fig. S5).

Among the seven deletion constructs coinjected

with six3a mRNA, we observed significant reductions

in the numbers of EGFP-expressing cells for three

constructs in which the deletions included specific

high-affinity sites (Fig. 3). The deletion construct

lacking the two strongest sites, a1 and a2

(pS3aPGDa1), showed a reduction of about 50%

relative to coinjections of pS3aPG and six3a mRNA.

The three additional binding sites (t1, t2, and t3) that

were deleted in this construct showed only weak

affinity for the Six3a HD (Fig. 2A), suggesting that

reduction of EGFP expression could be due to the

loss of the two G1-containing sites (a1 and a2). For

another reporter construct (pS3aPGDt15) in which

the deletion included two clustered high-affinity sites

(a10 and t15) and five weak binding sites (a8, a9,

a11, t14, and t16), the reduction in the number of

EGFP-expressing cells was somewhat lower (� 35%).

Similarly, when a fragment containing one high-affin-

ity site (a18) and eight additional ATTA core motifs

(a16, a17, and t20–t25) was deleted (pS3aPGDa18),the reduction in EGFP expression was � 45%. How-

ever, reporter gene expression was not significantly

reduced for one of the constructs (pS3aPGDa14) in

which a relatively strong binding site (a14) was

deleted together with two low-affinity sites (a13 and

a15). Notably, the effects on EGFP expression were

also weak or insignificant for the three constructs

(pS3aPGDa3, pS3aPGDa6, and pS3aPGDa12) in

which none of the high-affinity sites had been deleted.

Hence, we observed a correlation between the

presence of strong Six3a HD-binding sites, particu-

larly the three ATTA motifs with a flanking G1

nucleotide (a1, a2, and t15), and the ability to respond

to six3a overexpression in vivo.

Relative influence of ATTA core flanking

nucleotides on Six3a HD binding

To analyse the contribution of individual flanking

nucleotides and their importance relative to specific

positions within the ATTA core, we investigated how

single-nucleotide substitutions influenced the binding

affinity of a1. Modified a1 sites with single-nucleotide

mutations in nine positions (G1T2C3A4T5T6A7G8G9)

were compared for their ability to compete with

unmodified a1 in EMSAs (Fig. 4A). This in vitro anal-

ysis showed that substitution of G1 by T had moderate

effects on the binding affinity for the Six3a HD (G1T

in Fig. 4A). However, changes introduced at posi-

tions 2 (T2C) and 3 (C3G) affected the binding more

severely (Fig. 4A). Hence, these two 5¢-nucleotidesappeared to be essential for Six3a HD-binding affinity.

In fact, these substitutions resulted in less competition

than observed for individual mutations in three of the

positions within the ATTA core (A4C, T5G, and T6G).

Only a substitution at the fourth position (A7C) of this

core motif showed a similar deleterious effect on the

binding. In addition, we observed that mutation of the

3¢-flanking G8 nucleotide (G8A) had a moderate effect,

similar to the G1T substitution. By contrast, a change

of G9 (G9T) did not cause any reduction of the bind-

ing affinity, suggesting an insignificant role of this

3¢-flanking position. Overall, these results showed that

positions 2 and 3 immediately 5¢ of the ATTA core are

EGFP

pS3aPGΔa1

pS3aPGΔa3

pS3aPGΔa6

pS3aPGΔt15

pS3aPGΔa12

pS3aPGΔa14

pS3aPGΔa18

pS3aPG

0.5

–+

++

++

++

+

1.0 1.5 2.0 2.5

EGFP-expressing cellsSix3a

mR

NA

*

*

*Δ386 bp

Δ406 bp

Δ368 bp

Δ470 bp

Δ424 bp

Δ444 bp

Δ497 bp

Fig. 3. Deletion of particular fragments within the six3a promoter region affects reporter gene expression in embryos. Schematic represen-

tation of promoter–reporter deletion constructs and their corresponding activity in vivo. The pS3aPG vector (shown in Fig. 1A) and deletion

constructs derived from it were coinjected with six3a mRNA into one-cell embryos. EGFP-expressing cells from trypsinated embryos

(12 hpf) were detected by flow cytometry. The ratio between the EGFP expression from the reporter constructs coinjected with six3a

mRNA and from those without six3a mRNA was calculated. Standard deviations were calculated from three repeated experiments. Asterisks

indicate significant difference from the unmodified construct (pS3aPG) at P < 0.05. PS3aPGDa1, pS3aPGDt15 and pS3aPGDa18 showed

significantly less increase in EGFP expression when coinjected with six3a mRNA.



particularly important for binding affinity for the

Six3a HD. It also seemed that the Gs at positions 1

and 8 contributed moderately.

Comparisons of the three high-affinity sites (a1, a2,

and t15) showed considerable sequence identity at 11

positions, including the ATTA core, and a consensus

sequence could be defined (Fig. 4B). By contrast, all

of the low-affinity sites containing a G1 nucleotide

showed less identity with this consensus sequence

(Fig. 4B). In particular, differences were detected for

the critical nucleotides at positions 2 and 3. To inves-

tigate the significance of the consensus sequence, we

also determined its binding affinity for the Six3a HD.

This analysis clearly showed that the consensus

sequence bound more strongly than a1 (Fig. S3B).

Therefore, we used the high-affinity consensus

sequence site, which differed from a1 in only a single

position (A2) within the 27 bp probe fragment, as a

reference in further studies of the relative importance

of the flanking nucleotides. Our analysis tested the

effects of other substitutions for the flanking nucleo-

tides, and also included assays for the two additional

positions (10 and 11) in the consensus sequence

(Fig. 4C).

The assays for G1 substitutions in the consensus

sequence site showed that a change from G to T had a

similar effect (Fig. 4C) to the same substitution in a1

(Fig. 4A). Stronger reduction of the binding affinity

was observed when G1 was changed to C, but a

change to A had no detectable effect (Fig. 4C). Consis-

tent with the results obtained with the a1 probe

(Fig. 4A), A2 substitutions in the consensus sequence

caused a strong reduction in the binding affinity

(Fig. 4C). However, it seemed that the presence of A2

in the consensus sequence caused a reduction in the

relative importance of the C3 nucleotide (Fig. 4C).

Whereas two of the substitutions tested for G8 and

G9 caused some reduction in the binding affinity,

a1 G1T

T2C

C3G

A4C

T5G

T6G

A7C

G8A

G9T

x 200A

B

C

Relativecompetition

Motifsequence

Motifname

CO

N

x 200

CO

N

x 200

Relativecompetition

Motifsequence

MotifnameG

1A

G1T

G1C

C3G

C3A

A2G

A2C

G8A

G8C

G9C

G9T

C10

G

C10

T

G11

C

G11

T

G11

A

Fig. 4. Identification of ATTA core flanking

nucleotides critical for Six3a HD binding. (A)

EMSAs with the Six3a HD and biotin-

labelled a1 probe. Unlabelled fragments with

a single point mutation in a1 (table) were

used as competitors (· 200 molar excess)

and compared with a1 for their ability to

compete for Six3a HD binding. The two left

lanes show the controls (labelled probe and

labelled probe together with Six3a HD). The

table shows the mutated sites and their rel-

ative competition as compared with a1,

from highest competition (+++++) to lack of

competition ()). (B) Nucleotide similarities in

the GNNATTA sites. The three high-affinity

sites (a1, a2, and t15) are aligned at the top.

Identical nucleotides flanking the ATTA core

in the high-affinity sites are shown in red,

and are represented in the consensus

sequence (CON). GNNATTA sites with low

affinity are aligned below the consensus

sequence. (C) EMSAs with the Six3a HD

and biotin-labelled consensus sequence

probe. Unlabelled fragments with a single

point mutation in the consensus sequence

site (table) were used as competitors (· 200

molar excess) and compared with consen-

sus sequence for their ability to compete for

Six3a HD binding. The two left lanes show

the controls (labelled probe and labelled

probe together with Six3a HD). The table

shows the mutated sites and their relative

competition as compared with consensus

sequence, from highest competition

(+++++) to lack of competition ()).



single mutations of the other 3¢-nucleotides in the

consensus sequence (C10 and G11) did not have detect-

able effects (Fig. 4C). These results suggested marginal

roles for each of the four 3¢-flanking positions within

the consensus sequence. Information obtained from

investigations of the effects of swapping flanking

regions with sequences from a low-affinity binding site

also supported this conclusion (Fig. 5A; see below).

Our identification of similar high-affinity binding

sites within the promoter regions of the zebrafish six3b

and six7 genes is also consistent with the recognition

sequence defined by these analyses (Fig. S4). Hence,

the three Six3-like genes in zebrafish, which have par-

tially overlapping expression domains [15,20], may be

able to cross-regulate each other.

Evaluation of the integrity and relative binding

affinity of the recognition motif

Regulatory transcription factors generally bind to

short target sequences independently of the properties

of the adjacent flanking regions. To investigate

whether the Six3 HD recognition motif displays such

integrity, we compared the relative binding affinities of

hybrid sites, in which the a1 regions located 5¢ of G1

and ⁄or 3¢ of G8 in the optimized consensus sequence

site were replaced by completely different sequences

from the corresponding parts of the low-affinity bind-

ing site a9. This analysis showed that the strength of

binding was only moderately influenced by changes in

the sequences surrounding the recognition motif

(Fig. 5A). Hence, when flanked by a9 sequences, the

recognition motif displayed the same binding affinity

as in the context of a1. This suggests that the identified

recognition sequence may occur within different geno-

mic contexts, where it can function as a target site for

Six3a in vivo.

Conversion of the most essential part of the consen-

sus sequence, which includes the first seven positions

(GACATTA), to its reverse complementary sequence

(TAATGTC) facilitates direct comparisons with the

recently reported recognition motifs of Six3 and other

Six family proteins [10,14]. The two recognition

sequences determined by studies of Six family proteins

in Drosophila (TGATAC) and mice (TGATACC)

[10,14] show a difference of only one nucleotide, and

do not contain the TAAT core previously reported for

murine Six3 by Zhu et al. [18]. We made direct com-

parisons of the binding affinities of these recognition

motifs by replacing the seven nucleotide core

(TAATGTC) of the consensus sequence probe with

the four alternatives of TGATACN. The competitive

assays conducted for these fragments showed that the

Six3a HD binding affinity for these motifs is compara-

ble to the binding affinity for a1 (Fig. 5B). However,

consistent with the proposed importance of the consen-

sus, which was derived from the three high-affinity

sites a1, a2 and t15, we observed strongest binding to

the consensus sequence fragment.

Using the same probe fragments, we also tested the

binding properties of the two related zebrafish proteins

Six3b and Six7. The HDs of these two proteins [15,20],

which differ from Six3a in one and four residues,

a1 C

on a

9

a1 C

on a

1

a9 C

on a

1 a9

Con

a9

a9

LP-Con

A

B

Con -

TAATG

TCa1

- TA

ATGAC

brG -

TGATA

CG

nsA -

TGATA

CA

nsT

- TG

ATACT

brC -

TGATA

CC

LP-a1

Fig. 5. Influence of surrounding DNA sequences and comparison

with other recognition motifs. (A) EMSAs with the Six3a HD and

consensus sequence (CON) as a labelled probe (LP-CON) to ana-

lyse how changes in the surrounding sequences (from a1) influence

binding to the Six3a HD. Unlabelled fragments, in which the a1

sequences 5¢ to G1 and 3¢ to G8 in the consensus sequence were

replaced with the corresponding parts from the low-affinity site a9,

were used as competitors (· 100 excess). The two left lanes show

the controls (labelled probe and labelled probe together with Six3a

HD). Competition with unlabelled a9 fragment was also included as

a reference (right lane). Replacement of the surrounding sequences

had very limited effects on competition relative to the complete

consensus sequence site (a1CONa1). (B) Comparison with the

binding affinities of previously defined recognition sequences of

Six3 and Six family proteins [10,14]. Using a1 as a labelled probe

(LP-a1), competition was conducted with a · 100 excess of unla-

belled fragments containing the a1 site, the consensus sequence,

and the previously reported recognition motifs. The reverse com-

plementary sequences are shown, and all fragments included the

same additional flanking sequences (from a1).



respectively, showed very similar binding affinities to

that of Six3a (Fig. 6). These findings indicate a high

degree of overlap in binding sites for Six3a, Six3b,

and Six7, and that they might compete for the same

recognition motifs in regulatory networks.

In vivo effects of eliminating particular

high-affinity sites in the six3a promoter

Transient reporter assays suggested that deletion con-

structs lacking strong Six3a HD-binding sites

responded less to the increased amount of Six3a pro-

vided by coinjection of six3a mRNA (Fig. 3; see

above). However, because of the relatively large dele-

tions (� 450 bp) in these constructs, which included

several putative Six3a-binding sites and ⁄or other

potential control sequences, it could not be excluded

that the reductions in reporter expression reflected

other effects. To determine more directly whether par-

ticular high-affinity sites mediated responses to

increased levels of Six3a, we investigated reporter con-

structs with smaller deletions. One of these constructs,

pS3aPGDa1.2, in which a deletion of 29 bp removed

the closely spaced high-affinity sites a1 and a2, showed

� 40% reduction in the number of EGFP-expressing

cells as compared with the control (Fig. 7A). Similarly,

a construct with a 25 bp deletion, pS3aPGDt15.2,which eliminated a10 and t15, reduced expression of

the reporter gene by � 30%. Notably, these reductions

were almost as strong as for the corresponding con-

structs in which the deletions were larger (Fig. 3; see

above). Hence, the clear effects of deleting the two

pairs of high-affinity sites strongly indicated that they

were important in mediating responses to six3a overex-

pression. However, the possible existence of other con-

trol sequences within these small deletions could not

be completely ruled out.

To affect the individual binding sites more directly,

we made single-nucleotide substitutions in the most

critical position within each of the two clustered high-

affinity sites a1 (T2C) and a2 (A2C). We also mutated

this position in t15 (A2C), and made a small deletion

to eliminate its adjacent high-affinity site (a10). The

oligonucleotides designed to make these site-directed

mutations in the pS3aPG reporter construct were first

assayed for binding to the Six3a HD. When the short

double-stranded fragments generated by base pairing

of these oligonucleotides were used as unlabelled com-

petitors in EMSA, we observed very poor competition

(Fig. 7B). This demonstrated that the mutations to be

introduced in the reporter construct would severely

affect the binding affinities of the targeted sites.

The new reporter construct, which contained the

mutated a1 and a2 sites, showed the same reduced

ability to respond to six3a overexpression as the con-

struct with the a1 ⁄ a2 deletion (Fig. 7A). We also

observed similar effects for the construct in which the

other two high-affinity sites, t15 and a10, had been

mutated and deleted, respectively (Fig. 7A). These

results provided strong evidence that the clustered

high-affinity sites can mediate positive feedback from

Six3a in vivo. However, the transient reporter assay in

CO

N

G1C

G1A

G1T

A2G

A2C

C3G

C3ACO

N

Six3aA B C

a1 brC

brG

nsA

nsT

Six3b Six3b

a1 brC

brG

nsA

nsT

x 200x 200

x 150x 150

Six7

Fig. 6. Analysis of Six3b and Six7 HD binding to different recognition motifs. (A) EMSAs with the Six3b HD and biotin-labelled consensus

sequence (CON) probe. Unlabelled fragments with a single point mutation in the consensus sequence site (see table in Fig. 4B) were used

as competitors (lanes 4–11, · 200 molar excess) and compared with the consensus sequence for their ability to compete for Six3b HD bind-

ing. Six3a HD was used as an internal control (lanes 1 and 2). Lane 3 shows the labelled probe together with Six3b HD. (B) Six3b HD bind-

ing to a1 and previously defined recognition sequences of Six family proteins [10,14]. (C) Six7 HD binding to a1 and the previously defined

recognition sequences of Six family proteins [10,14]. Using the consensus sequence as a labelled probe, competition was conducted with a

· 150 excess of unlabelled fragments containing the a1 site and the previously reported recognition motifs.



injected embryos may not reflect the regulation of

six3a gene expression under normal conditions. To

gain further information about possible autoregulation

of the six3a gene during early embryonic development,

we investigated whether Six3a actually binds to some

of the high-affinity sites in the endogenous six3a

promoter (see below).

In vivo validation of Six3a-binding sites in the

six3a promoter region

The transient assays of reporter constructs, which were

conducted at the 12 hpf stage, did not provide any

information regarding the possible binding of Six3a to

the endogenous six3a promoter region in its natural

context. To facilitate such an investigation, which

required a specific antibody, we tagged Six3a with

EGFP and used an antibody against green fluorescent

protein (GFP) for ChIP assays on extracts from this

embryonic stage. Following injection of six3a–EGFP

mRNA into fertilized eggs (see Experimental proce-

dures), we detected high EGFP levels during embryo-

genesis (data not shown), and chromatin fragments

isolated from these embryos were immunoprecipitated

with the antibody against GFP. Using PCR primers

designed to amplify specific regions within the six3a

promoter (Fig. 8A), we investigated whether fragments

containing high-affinity sites were selectively amplified

(Fig. 8B). Consistent with the results obtained from

the transient reporter assays (Figs 3 and 7), a short

fragment (181 bp) containing the clustered a1 and a2

sites was amplified. In addition, we detected selective

amplification of a distal fragment, which included a

relatively strong binding site (a18) and five ATTA ⁄TAAT core motifs. This finding also correlates well

with results from transient reporter assays, where elim-

ination of a18 and eight adjacent core motifs by a

deletion caused a significant reduction in the response

to six3a overexpression (Fig. 3). Furthermore, cloning

and sequencing of the two amplified fragments con-

firmed their origins from the six3a promoter region

(data not shown).

Notably, amplification was not detected with the

primer pairs for regions containing only low-affinity

TAAT ⁄ATTA core motifs or lacking these motifs

completely. However, we also observed no amplification

0.5 1.0 1.5 2.0 2.5

–+

++

++

EGFP-expressing cells

Six3a

mR

NA

EGFP

Δ29 bppS3aPGΔΔΔΔa1.2

pS3aPG a1T2C, a2A2C Δ25 bppS3aPGΔΔΔΔt15.2

pS3aPGΔΔΔΔa10, t15A2C

a1/a

2

a10/

t15

Mu

t1

Mu

t2

x 200

pS3aPG

A

B

Fig. 7. Mutation of high-affinity sites within the six3a promoter region affects reporter gene expression in embryos. (A) Schematic represen-

tation of the in vivo activity of promoter–reporter constructs containing deletions (Da1 ⁄ a2 and Da10 ⁄ t15) or mutations of clustered high-affin-

ity sites. The plasmid constructs were coinjected with six3a mRNA into one-cell embryos. EGFP-expressing cells from trypsinated embryos

(12 hpf) were detected by flow cytometry. The ratio between the EGFP expression from the reporter construct coinjected with six3a mRNA

and from that without six3a mRNA was calculated. Standard deviations were calculated from three repeated experiments. Significant differ-

ence from the complete promoter–reporter construct (pS3aPG) was calculated at P < 0.05. (B) Double-stranded oligonucleotides with point

mutations at position 2 of the Six3a HD-binding sites were used as cold probes in competitive EMSA to confirm loss of binding affinity.

Mut1 represents a1T2C,a2A2C, and Mut2 represents Da10,t15A2C.



of the fragment containing the high-affinity sites a10

and t15, despite strong evidence from the reporter

assays (Figs 3 and 7). Possibly, an unfavourable state

of the chromatin within this particular region of the

endogenous six3a promoter may prevent Six3a from

binding at this developmental stage (12 hpf). Hence, it

remains a possibility that the two sites may have

autoregulatory functions at later stages of development.

Discussion

In this study, we show that binding of zebrafish Six3a

to high-affinity sites in the promoter region of its own

gene may contribute to autoregulation. The Six3a HD,

which is identical to the corresponding DNA-binding

domain of orthologous Six3 proteins in other verte-

brates, displayed differential binding affinities for vari-

ous sites containing a TAAT core motif, and this

defined a specific recognition sequence (TAATGTC).

When we compared this with a previously reported

Six3-binding sequence (TGATAC), we observed simi-

lar binding affinity, suggesting that the Six3 HD has

several binding modes with distinct DNA-binding

specificities.

Using several experimental approaches, previous

investigations of the DNA-binding preferences of the

three Six protein subfamilies (Six1 ⁄ 2, Six3 ⁄ 6, and

Six4 ⁄ 5) have found a number of different target

sequences. Whereas analyses of native binding sites in

putative target genes identified a variety of sequences

[17,18,30,36–38], two independent studies based on dif-

ferent in vitro selection procedures established a com-

mon recognition sequence of the three subfamilies

[10,14,39]. These partially conflicting results may reflect

the fact that the HDs belonging to different subfami-

lies have almost identical recognition helices but diver-

gent N-terminal arms [16].

The HDs in the Six3 ⁄ 6 subfamily proteins show a

higher degree of divergence relative to the other two

subfamilies [16]. Consistent with this sequence diver-

gence, early studies of the Six3 ⁄6 proteins indicated

clear differences in DNA-binding specificity relative to

the Six1 ⁄ 2 and Six4 ⁄ 5 family members [1]. Hence,

murine Six3 did not bind to the ARE regulatory

element of the Na+ ⁄K+-ATPase a1-subunit gene,

GGTGTCAGGTTGC, which showed specific binding

to Six4 and other members of the Six1 ⁄ 2 and Six4 ⁄5subfamilies [17]. In further investigations of the DNA-

binding properties of murine Six3, in vitro binding site

selection demonstrated high-affinity binding to

sequences containing the TAAT core motif, and clus-

ters of these tetranucleotide sites were found in regula-

tory elements within the promoter regions of both

Six3 and Wnt1 [18,37]. Although these analyses dem-

onstrated an involvement of the clustered TAAT

motifs in mediating negative regulation by Six3,

–357

9 –3

290

–289

3 –2

704

–209

3 –1

872

–118

3 –9

80

–656

–47

7

–25

3 –7

2

six3a promoter region (pS3aP)

IP (+Ab, injected)

Input control

IP (+Ab, uninjected)

300 bp200 bp

300 bp200 bp

300 bp200 bp

18 17

A

B

16 15 14 13 12 1110 9 8 7 6 5 4 3 2

t2

1**

t13t15t17*

Fig. 8. Detection of Six3a binding to high-affinity sites in the six3a promoter in vivo. (A) Schematic representation of the promoter region of

six3a (see legend to Fig. 2A) and the fragments that were selected for PCR amplification. Filled boxes indicate the regions that were ampli-

fied, and empty boxes represent regions that showed no amplification. (B) ChIP PCR assay on 12 hpf embryos. The upper panel represents

PCR amplification of different regions (indicated by the boxes) within the six3a promoter following immunoprecipitation (IP) of DNA bound to

Six3a–EGFP fusion protein (Experimental procedures). The middle panel represents PCR amplification from uninjected zebrafish embryos

immunoprecitated with GFP antibody (Ab). The lower panel represents PCR amplification from total embryonic DNA.



additional features specifically favouring binding to

Six3 proteins were not identified.

Our examination of the interaction of zebrafish

Six3a with 43 TAAT-containing sequences, distributed

within the promoter region of its own gene, identified

three high-affinity sites that defined the recognition

sequence TAATGTC. Notably, this sequence prefer-

ence includes three adjacent nucleotides 3¢ to the

TAAT core, corresponding to the positions generally

involved in discriminating between different HDs.

The importance of this novel recognition motif was

also supported by comparisons of the relative binding

affinities of mutated sites and by in vivo assays of

reporter constructs. In addition, a direct comparison

with the recently reported generic recognition

sequence of Six family HDs, TGATAC [10,14],

showed the same strong binding affinity. Further-

more, major changes of the surrounding sequences

did not affect the binding affinity of the recognition

sequence, indicating that it is generally independent

of the sequence context. Importantly, the TAATGTC

motif is also distinct from all the different DNA-

binding specificities that were recently described in

surveys including the majority of the Drosophila and

mouse HDs [10,14]. This unique sequence preference

is likely to facilitate binding of Six3 monomers to

regulatory sites in their target genes without requiring

cooperating factors to achieve sufficient sequence

specificity.

It remains to understand how it is possible for the

Six3 HD to bind strongly to two distinct types of

recognition sequences. A complete elucidation of this

issue can only be achieved by analysing the crystal

and ⁄or solution structures of the different complexes

formed when the Six3 HD binds to each motif.

Although such structures have not been determined for

any of the Six family proteins, some clues may be

obtained by considering the divergent features of their

HDs. In contrast to the majority of HDs, which recog-

nize various sequences containing the TAAT core, the

N-terminal arms of Six family HDs lack several resi-

dues, such as Arg5, known to be involved in specifying

the first two positions in this tetranucleotide motif

[1,15]. Another characteristic of the Six family HDs,

which they share with several other atypical HDs, is

the presence of Arg55 in the recognition helix that

may specify a G in position 2 of the core motif [10].

Consistent with this, the HDs of all Six family proteins

in mouse and Drosophila were shown to recognize

sequences containing the core motif TGAT [10,15].

Although the sequence divergence in the specifying

residues of the Six family HDs is not consistent with

our identification of the TAATGTC motif, it is impor-

tant to consider that intramolecular interactions

involving residues outside of the protein–DNA binding

interface can also influence DNA recognition [10,40].

In particular, variations in the position of the N-termi-

nal arm have been shown to influence its recognition

properties [6,10]. Investigations of the binding proper-

ties of the Ultrabithorax protein have also demon-

strated that the N-terminal arm can strongly affect the

topological binding mode of the HD and influence the

nucleotide contacts made by the recognition helix [6].

Notably, these different binding modes seemed to

depend on the binding site sequence, and the binding

affinities were similar [6]. Currently, it is not known

whether such transitions between different binding

modes are common among HDs. However, multiple

binding modes have been proposed as an explanation

for the identification of several recognition sequences

for some of the mouse HDs [14].

Consistent with the previous report of autoregula-

tion of the murine Six3 gene [18], we found that the

high-affinity sites recognized by the Six3a HD in vitro

were clustered within the promoter region of its own

gene. Our investigation of the functional significance

of these clustered high-affinity sites included in vivo

assays of various promoter–reporter constructs in

zebrafish embryos and ChIP. In transient transgenic

assays, which provide more natural conditions than

cell culture transfections, the 3.6 kb promoter fragment

of the six3a gene showed a clear response to coinjec-

tion of six3a mRNA by increasing expression of the

EGFP reporter by more than 100%. The strong effect

of overexpressing Six3a was possibly due to direct

binding to high-affinity sites within the promoter

region. Consistent with this interpretation, we observed

reduced responses to Six3a overexpression when

regions containing the clustered high-affinity sites were

deleted from the reporter constructs. To rule out the

possible involvement of other control sequences, we

assayed the effects of removing the high-affinity sites

by introducing single-nucleotide substitutions that

severely affected their ability to bind the Six3a HD.

Remarkably, reporter constructs with these minimal

changes showed almost the same reduced ability to

respond to higher levels of Six3a in injected embryos

as the constructs with larger deletions.

Our results clearly demonstrated that the clustered

high-affinity sites mediate positive autoregulation of

the zebrafish six3a gene under experimental conditions

in vivo. In contrast, autorepression of the murine Six3

gene has been detected in cotransfection assays in

cultured cells [18]. The opposite effects observed for

the zebrafish six3a and murine Six3 genes may reflect

differences in the experimental conditions and ⁄or



involvement of different cofactors. The autorepression

of the murine Six3 gene was shown to depend on

interaction with members of the Groucho-related

family of corepressors [18]. Additional studies in

medaka fish (O. latipes) have provided evidence that

Six3 ⁄ 6 family proteins bind differentially to these core-

pressors and the related cofactor Aes, which may lead

to transcriptional activation [41]. Similar mechanisms

of derepression may have occurred when we

experimentally overexpressed Six3a in our transient

reporter assays of the six3a promoter in zebrafish

embryos. However, irrespective of the cofactors that

may have contributed, the activation was dependent

on the Six3a HD-binding sites shown to bind strongly

in vitro.

Other members of the Six3 ⁄ 6 family, which have simi-

lar or identical DNA-binding specificity to that of Six3a,

have partially overlapping expression at early stages of

eye and forebrain development [15,20]. Hence, tran-

scriptional control of these genes is likely to involve

both autoregulation and cross-regulation. Differences in

their interactions with Groucho-related cofactors may

be important for this regulation and may contribute to

the control of eye and forebrain development. This pos-

sibility is supported by previous studies in medaka fish

that showed different effects on eye size when the Six3

and Six6 activities were modulated by Aes and the

Groucho family corepressor Tle1 [41].

The chromatin structure of the injected reporter con-

struct may differ considerably from the native state of

the endogenous six3a gene, which could also influence

the responses to increased levels of Six3a. To obtain

more direct evidence that Six3a binds to the clustered

high-affinity sites in a native context, we investigated

the chromatin of the endogenous six3a promoter

region in embryos. Our ChIP analysis detected Six3a

binding specifically to a small fragment (� 180 bp)

containing the two strong binding sites that were

shown to mediate autoregulation of the reporter con-

struct. This finding, which is also consistent with the

importance of the Six3 HD recognition sequence sug-

gested by the in vitro studies, provides a basis for

future identification of Six3 target genes.

Experimental procedures

Maintenance of fish stock and in situ

hybridization

Zebrafish were maintained and bred at 28 �C. Whole

mount in situ hybridization on zebrafish embryos was

performed with digoxigenin-labelled probes as described

previously [15].

Generation of Tg(six3a:EGFP) line

One-cell-stage embryos were coinjected through the cho-

rion with 1 nL of a solution containing plasmid DNA

()3.6 kb six3a promoter in Tol2 vector) at a concentration

of 10 ngÆlL)1, along with Tol2 transposase mRNA at

15 ngÆlL)1, KCl at 200 mm, and phenol red at 0.05%, as

described previously [42]. Injected F0 embryos were raised

and crossed with the wild-type TAB strain [43]. F1 prog-

eny were screened for EGFP expression at 12 hpf.

Embryos and larvae exhibiting tissue-restricted EGFP fluo-

rescence were selected and raised to obtain stable trans-

genic lines.

Cloning, expression and purification of

glutathione-S-transferase (GST)–Six3aHD

fusion protein

The HD coding sequence of six3a was amplified by PCR,

using 5¢-TCAGGTCGGATCCATGGTTTTCAGA-3¢ as

forward primer and 5¢-CTGTGTGGAATTCATACGTCG

CATTC-3¢ as reverse primer, cloned in-frame into the

BamHI and EcoRI site of the pGEX-2T expression vector

(Pharmacia Biotech Inc., Uppsala, Sweden), and transformed

into Escherichia coli [BL21 (DE3) pLysS Competent Cells]

cells. E. coli cells transformed with the pGEX-2T–Six3-

aHD vector were grown in 250 mL of LB medium prior

to induction with 2 mm isopropyl thio-b-d-galactoside.The bacteria were pelleted after a 3 h induction period,

and whole cell extracts were prepared by using a French

press. The GST–Six3aHD fusion protein was purified by

batch adsorption onto glutathione–Sepharose 4B beads

(Pharmacia Biotech Inc.). After several washes of the

beads with NaCl ⁄Pi with Tween-20, the GST fusion pro-

teins were eluted with 50 mm reduced glutathione solution

and subsequently used in EMSAs.

EMSAs

EMSAs were performed with 5¢-biotin-labelled oligonucle-

otides in 20 lL reactions containing 80 mm Tris ⁄HCl,

240 mm KCl, 2 mm dithiothreitol, 0.2% NP40, 10%

Ficoll, and 50 ng of poly(dI.dC), and incubated with

50 ng of purified GST–Six3aHD fusion protein. Each

27 bp DNA fragment contained the core ATTA motif

flanked by 13 nucleotides at the 5¢-end and 10 nucleotides

at the 3¢-end. For competition reactions, a · 200 molar

excess of unlabelled double-stranded oligonucleotide was

added to the binding reaction [40]. After incubation at

room temperature for 20 min, protein–DNA complexes

were separated on a 6% polyacrylamide gel in 0.5 · TBE

buffer. A chemiluminescent nucleic acid detection kit

(Pierce, Rockford, IL, USA) was used for the detection of

protein–DNA interactions, according to the manufacturer’s

instructions.



Generation of reporter constructs for transient

analysis

A 3.6 kb fragment spanning positions )3616 to +19 of the

cis-regulatory region of the six3a gene was amplified by

PCR, and cloned into the pT2AL200R150G vector in front

of EGFP [44], generating the construct pS3aPG. Deletions

in the 3.6 kb six3a promoter region were performed by

PCR [45]. Primers were designed to add a noncomplemen-

tary 5¢-sequence consisting of the restriction enzyme

sequence XmaI. The following deletion constructs were

made: pS3aPG, pS3aPGDa1,a2,t2 (or pS3aPGDa1),pS3aPGDa3–a5 (or pS3aPGDa3), pS3aPGDa6,a7,t13(or pS3aPGDa6), pS3aPGDa8–a11,t15 (or pS3aPGDt15),pS3aPGDa12,t17 (or pS3aPGDa12), pS3aPGDa13–a15(or pS3aPGDa14), pS3aPGDa16–a18 (or pS3aPGDa18),pS3aPGDa1.2, and pS3aPGDat15.2. Following PCR ampli-

fication of the whole plasmid except the region to be

deleted, the PCR product was digested with XmaI and

DpnI (to eliminate the template DNA). The plasmid was

self-ligated and transformed into E. coli cells. Specific

nucleotide mutations on pS3aPG were generated using the

quick-change site-directed mutagenesis kit (Stratagene, La

Jolla, CA, USA), according to the manufacturer’s instruc-

tions, to make pS3aPGa1T2C,a2A2C and pS3aPGDa10,t15A2C. All deletion and mutation constructs were verified

by DNA sequencing.

Generation of mRNA

The Six3a coding sequence was amplified from zebrafish

cDNA (10–12 hpf) and cloned into the pEGFP-N1 vector

(Clontech, Mountain View, CA, USA). PstI and XbaI were

used to cut out the six3a–EGFP fusion and subclone it

into the expression vector pCS2+ [46]. For six3a mRNA

transcription, the six3a coding sequence was cloned directly

into the pCS2+ vector. The six3a–GFP and six3a mRNAs

were generated with the mMessage mMachine kit (Ambion,

Austin, TX, USA). All RNA products were analysed by

agarose gel electrophoresis and stored at )80 �C until use.

Detection of promoter activity by flow cytometry

Zebrafish embryos at the one-cell stage were coinjected with

25–30 pg of reporter constructs (as described above) and

100 pg of six3a mRNA per embryo. Embryos were examined

for EGFP expression at 12 hpf. One hundred EGFP-positive

embryos at 10–12 hpf were selected and washed twice in

NaCl ⁄Pi. Preparation of embryos for cell cytometry was per-

formed as previously described [47]. Single-cell suspensions

from the embryos were then analysed using a FACSCalibur

flow cytometer equipped with cellquest from BD Bio-

sciences (San Jose, CA, USA). During flow cytometric analy-

sis, the cell suspensions were kept on ice. The time between

dissociation of cells and flow cytometry was minimized to

2 h. Using forward and side scatter, yolk cells and debris

were eliminated in the analysis. EGFP fluorescence was

detected using a 530 ⁄ 30 nm bandpass filter in the FL1 chan-

nel. For each sample, information on 25 000 events was

acquired. The calculation of significant differences between

mean values was determined using Student’s t-test. Each

value is the mean difference ± standard deviation from

three experiments. The level accepted for statistical signifi-

cance in all cases was P < 0.05.

ChIP PCR

Zebrafish embryos at the one-cell stage were injected with

six3a–EGFP mRNA in the yolk at 100 pg per embryo. The

embryos were dechorionated at 12 hpf. ChIP was performed

as described in [48], using mouse antibody against GFP from

Invitrogen (Carlsbad, CA, USA). Briefly, 12 hpf embryos

were crosslinked with formaldehyde, and chromatin was iso-

lated. The isolated chromatin was sonicated to an average

size of about 300 bp. Incubation with normal goat IgG

precleared the chromatin. Protein G magnetic beads were

incubated with antibody against GFP at 4 �C overnight.

Immunoprecipitation reactions were performed in duplicate

by incubating 10 lg of antibody against GFP with the chro-

matin overnight at 4 �C. Parallel controls with uninjected

zebrafish embryos were incubated with antibody against

GFP as a negative control. The immunoprecipitated chro-

matin complexes were washed several times and eluted with

50 mm Tris (pH 8). In each ChIP experiment, a portion of

the chromatin solution corresponding to 1% of that used in

the ChIP reaction was used as input DNA control. The

immunoprecipitated and uninjected control immunoprecipi-

tated samples were purified by phenol ⁄ chloroform extraction

and ethanol precipitation after the protein–DNA crosslinks

had been reversed by incubation at 65 �C. Immunoprecipi-

tated DNA and input DNA were used as templates for PCR

amplification with the five primer pairs (Table S1).

Acknowledgements

We thank H. Savolainen, R. Aanesen and G. Merkin

for their expert technical help in our zebrafish facility.

This study was funded by the Research Council of

Norway (Grant 174979 ⁄ I30), and the Faculty of Mathe-

matics and Natural Sciences at the University of Bergen.

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Supporting information

The following supplementary material is available

online:

Fig. S1. Overview of the DNA sequence of the six3a

promoter region showing the positions of sites contain-

ing ATTA (and TAAT) core sequences.

Fig. S2. Sites within the six3a promoter containing

oppositely oriented TAAT cores that showed weak

binding as compared with the a1 site in competition

EMSAs.

Fig. S3. Comparison of relative binding affinities of

the consensus sequence (CON) and the strongest bind-

ing sites located within the six3a promoter region.

Fig. S4. Comparison of high-affinity GNNATTA sites

located within the promoter regions of the zebrafish

genes six3a, six3b and six7 indicates a common recog-

nition motif.

Fig. S5. Autoregulatory activity of the six3a promoter

assayed in zebrafish embryos.

Table S1. Primer sequences used in ChIP assay.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

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should be addressed to the authors.

