Báo cáo khoa học: Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4 docx

Slc12a2 is a direct target of two closely related homeobox
proteins, Six1 and Six4
Zen-ichi Ando, Shigeru Sato, Keiko Ikeda and Kiyoshi Kawakami
Division of Biology, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan

Keywords
dorsal root ganglia; Six1; Six4; Slc12a2;
transcriptional targets
Correspondence
K. Kawakami, Division of Biology, Center for
Molecular Medicine, Jichi Medical School,
Yakushiji, Minamikawachi, Tochigi,
329-0498, Japan
Fax: +81 285 44 5476
Tel: +81 285 58 7312
E-mail:
(Received 13 December 2004, revised
15 March 2005, accepted 11 April 2005)
doi:10.1111/j.1742-4658.2005.04716.x

Six genes are homologs of Drosophila sine oculis and encode transcription
factors that are characterized by a conserved Six domain and homeodomain. Of the six family members (Six1–Six6) in mice, Six1 and Six4
show similar expression patterns during embryogenesis. Six1– ⁄ – mice show
defective formation of various organs such as inner ear, nose, skeletal muscle, kidney and thymus, whereas Six4– ⁄ – mice show little anomaly in organogenesis. To understand the molecular basis for the differential function of
Six1 and Six4 in vivo, we screened target genes of Six1 and Six4 and found
that Six1 and Six4 differentially regulated a set of target genes. Gel-retardation assays indicated that the promoter region of one of the targets, sodium–
potassium–chloride cotransporter 1 (Slc12a2), contains multiple Six1-binding
sites and one common binding site of Six1 and Six4, suggesting that the
DNA-binding specificity of Six1 is distinct from that of Six4. This underlies
the differential regulation of common target genes by Six1 and Six4. Furthermore, in situ hybridization demonstrated that the expression of Slc12a2
was reduced in the developing dorsal root ganglia of Six1– ⁄ – ⁄ Six4– ⁄ – mice,

suggesting that Six1 and Six4 regulate Slc12a2 in vivo.

The Six homeobox gene is characterized by the conserved Six domain (SD) and homeodomain (HD),
both of which are required for specific DNA binding
[1,2]. The prototype of this gene family is Drosophila
sine oculis, which is essential for compound eye formation [3,4]. Six members (Six1–Six6) of the Six
family gene have been identified in mouse and
human [1]. Six3 and Six6 are essential for forebrain
formation and eye development [5–11], whereas Six5
is involved in cataractogenesis and spermatogenesis
[12–14].
Among the Six family genes, Six1 and Six4 show
a remarkably similar expression pattern [1,15,16]. Both
Six1 and Six4 bind to the MEF3 site in the myogenin
promoter and positively regulate the activity of the
promoter in conjunction with their coactivator Eya
proteins [17,18]. Based on these observations, Six1 and

Six4 are thought to be functionally similar in vivo.
Six4– ⁄ – mice showed little anomaly in embryogenesis
including skeletal muscles [16]. This was explained by
the compensatory function of Six1 considering the
similar expression pattern of both genes and the functional similarity in activating their target gene myogenin. In contrast, Six1– ⁄ – mice showed anomalies in
the development of various organs such as the inner
ear, nose, thymus, kidney and skeletal muscle [19–23].
These results indicate that Six4 does not compensate
for the functional loss of Six1, whereas Six1 compensates for the function of Six4, suggesting a distinct
function of Six1 and Six4 in vivo. To understand the
basis for the difference between Six1 and Six4, we
screened possible target genes by using an approach

similar to that applied in our previous study for
searching the targets of Six5 protein [24]. We identified

Abbreviations
Atp1a1, sodium–potassium ATPase alpha 1 subunit; Clcn5, chloride channel 5; Coll2a1, procollagen type 2 alpha 1; Figf, c-fos induced
growth factor; Gas1, growth arrest-specific 1; GST, glutathione S-transferase; HD, homeodomain; MCK, muscle creatine kinase; SD, Six
domain; Slc12a2, sodium–potassium–chloride cotransporter 1; Trex, transcriptional regulatory element X.

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Z-i. Ando et al.

three putative categories of targets, Six1-specific
targets, Six4-specific targets and common targets. We
then focused our analysis on one of the typical common target genes, sodium-potassium-chloride cotransporter 1 (Slc12a2) to understand the molecular basis
for the distinct functions of Six1 and Six4.

Results
Identification of potential downstream target
genes for Six1 and Six4
Studies of Six1– ⁄ – mice revealed that Six1 is required
for inner ear, nose, thymus, skeletal muscle and kidney
formation [19–23]. Six4 is also reported to be
expressed in these regions [16]. To explore the target
genes of Six1 and Six4 in the physiological context of
development, we took advantage of using the cell line
mK4 isolated from the kidneys of transgenic mice that

express SV40 T-antigen under the control of Hoxa11
promoter. The mK4 cells are thought to represent
embryonic metanephric mesenchyme that undergoes
epithelial conversion and expresses genes typical of late
mesenchyme such as E-cadherin, Wnt4, Pax2, Lim1,
Pax8 and Bmp7 in addition to Six1, Six4, Eya2 and
Eya3 [25] (data not shown). By overexpressing transcriptionally constitutive active fusion proteins VP16–
Six1 and VP16–Six4, the direct target genes are expected to be activated through VP16–Six1 or VP16–Six4,
respectively, tethered to target gene promoters in the
mK4 cells. As a control, fusion proteins VP16–
Six1W171R and VP16–Six4W263R, which are defective in DNA binding (data not shown), were overexpressed. The mK4 cells were infected with recombinant
adenovirus expression vectors expressing VP16–Six
fusion proteins and cultured for 24 h, then used as the
source of poly(A)+ RNA to prepare hybridization
probes. Expression profiling was performed by hybridization of an oligo microarray containing 20 371 mouse
cDNAs. Scatter plot analysis of the microarray data
showed that most of the data points fell along the
diagonal, indicating that most of the genes were
equally expressed in the two samples (supplementary
Fig. S1). The genes that showed more than 1.5-fold
higher level of expression in cells infected with VP16–
Six1 adenovirus compared with cells infected with
VP16–Six1W171R were considered potential Six1 target genes, whereas 1.5-fold higher level of expression
in cells infected with VP16–Six4 compared with VP16–
Six4W263R were considered potential Six4 target
genes. A total of 363 Six1 target genes and 149 Six4
targets genes were identified. Of these, 63 were common target genes. The data were deposited in GEO
FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

Target genes of Six1 and Six4

database GSE2043 (a complete list of these genes
appears in Table S1 and a partial list is shown in
Table 1). Because only a single microarray was used
for each condition, genes with a relatively low level of
expression were excluded. Examples of target genes
included the cyclin-dependent kinase inhibitor 1C
(Cdkn1c), a cell-cycle regulator expressed in the kidney
and cochlea, which controls the number of podocytes
and glomerular size in the kidney [26]. Another example is the sodium–potassium–chloride cotransporter 1
(Slc12a2) that plays an important role in dorsal root
ganglia function, spermatogenesis and inner ear formation, and mutation of this gene causes impairment of
hearing [27–30]. Other examples include the sodium–
potassium ATPase alpha 1 subunit (Atp1a1), which was
originally identified as the target gene of Six4 [2,31];
c-fos induced growth factor (Figf), which shows remarkably similar patterns of Six1 and Six4 expression [32];
growth arrest-specific 1 (Gas1), which encodes a sonic
hedgehog inhibitor [33,34] and whose mutation causes
cerebellar and eye defects [33]; chloride channel 5
(Clcn5), which is mainly expressed in the kidney, and
mutation of this gene causes kidney stone disease
[35,36]; and procollagen type 2 alpha 1 (Col2a1), whose
missense mutation causes spondyloepiphyseal dysplasia, hearing loss and retinoschisis [37].
To confirm the above microarray results, we performed semiquantitative RT-PCR using RNA prepared from recombinant virus-infected mK4 cells. As
summarized in Table 2, the results of RT-PCR confirmed > 1.5-fold difference in the expression levels of
13 of 14 genes examined. The result seems to suggest
that our strategy using microarray as an initial screening to search for potential Six1- and Six4-target genes
was largely successful.
Differential regulation of potential target genes
by Six1 and Six4

To verify that the above identified putative target
genes are regulated by Six1 or Six4, we performed
transient transfection assays and examined the effects
of Six1 and Six4 on the promoter activity. We used
luciferase reporter constructs harboring the Gas1 promoter region of )3408 to +19 (Gas1)3408Luc), the
Clcn5 promoter region of )1325 to +2529 (Clcn5)
1325Luc) and the Slc12a2 promoter region of )1938
to +149 (Slc12a2)1938Luc). Gas1 promoter showed
similar extent of activation by both Six1 and Six4 in
a dose-dependent manner (Fig. 1A), although it was
listed as a potential Six1-specific target gene. Clcn5 promoter showed a moderate repression by Six1 and a
strong repression by Six4 in a dose-dependent manner
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Target genes of Six1 and Six4

Z-i. Ando et al.

Table 1. A partial list of potential target genes regulated by Six1 and ⁄ or Six4 in mk4 cells. Expression profiling was performed using the
Mouse Development Oligo Microarray containing 20 371 mouse cDNAs (Agilent Technologies, Palo Alto, CA). Total RNA was prepared as
described previously [24] from mK4 cells infected with recombinant viruses. To prepare fluorescence-labeled cDNA probes, total RNA
(20 lg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP and single-stranded cDNAs were coupled with
Cy3 (VP16–Six1wt and VP16–Six4wt infected samples) or Cy5 (VP16–Six1W171R and VP16–Six4W263R samples) dyes. Hybridization to the
microarray was carried out at 65 °C for 17 h according to the instructions provided by the manufacturer. The arrays were washed, dried and
scanned using ScanArray 5000 (GSI Lumonics Inc., ON, Canada). Cy3 and Cy5 intensities for each spot on the array were determined by
QUANTARRAY software (GSI Lumonics Inc.). The raw data were processed and the Cy3 to Cy5 ratios were calculated as follows: (a) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, (b) normalization of the entire data set using the global normalization method, (c) elimination of spots with high background intensity for either dye, (d)
determination of the Cy3 to Cy5 ratios. The microarray data were deposited in the GEO database under accession number GSE2043. Among
the genes with a Cy3 ⁄ Cy5 ratio of > 1.5, those with a relatively high level of expression (normalized signal value > 500 in both dyes) were
considered to be potential Six1- and Six4-target genes. Data are from a single microarray experiment and no dye-swap experiment was carried out.

Symbol

VP16–Six1a
wt ⁄ W171R

Six1–Six4 common target genes
Ogn
5.10
Amot
3.73
Hs6st2
3.07
Rhoe
2.98
Nid2
2.57
Acsl3
2.47
Acadm
2.41
Sdc2
2.30
Add3
2.15
Cav1
2.12
Kif5b
2.11
Stag2

2.05
Sh3bgrl
2.00
Cdkn1c
1.90
Slc12a2
1.80
Six1-specific target genes
Atp1a1
2.89
Figf
2.87
Cpd
2.65
Gas1
2.17
Laptm4a
2.12
Fmr1
2.05
Bmi1
1.96
Nrp2
1.86
Irs1
1.85
Mbnl1
1.84
Clcn5
1.83

Cav2
1.73
Aqp1
1.66
Bmpr2
1.62
Six4-specific target genes
Cyb561d2
Ifitm3
Gpd2
Thbs1
Col2a1
Matn2
Tmem2

VP16–Six4b
wt ⁄ W263R

Kidneyc function
development

3.71
2.09
1.82
2.60
1.84
2.58
2.09
1.69
1.74

1.55
1.58
1.59
1.64
2.41
1.94

Yes

Yes

Yes

Yes
Yes
Yes

Yes
Yes
Yes
2.29
2.27
2.24
2.21
2.20
2.05
2.01

Description

Ref.

Osteoglycin
Angiomotin
Heparan sulfate 6-O-sulfotransferase 2
ras homolog gene family, member E
Nidogen 2
Acyl-CoA synthetase long-chain family member 3
Acetyl-Coenzyme A dehydrogenase, medium chain
Syndecan 2
Adducin 3 (gamma)
Caveolin, caveolae protein 1
Kinesin family member 5B
Stromal antigen 2
SH3-binding domain glutamic acid-rich protein like
Cyclin-dependent kinase inhibitor 1C
Solute carrier family 12, member 2
ATPase, Na+ ⁄ K+ transporting, alpha 1 polypeptide
c-fos induced growth factor
Carboxypeptidase D
Growth arrest specific 1
Lysosomal-associated protein transmembrane 4 A
Fragile–mental retardation syndrome 1 homolog
B lymphoma Mo-MLV insertion region 1
Neuropilin 2
Insulin receptor substrate 1
Muscleblind-like 1 (Drosophila)
Chloride channel 5
Caveolin 2
Aquaporin 1

Bone morphogenic protein receptor, type II

[50]

[51]

[50]

[52]
[53]
[54]

[55]
[50,56]
[57]

Cytochrome b-561 domain containing 2
Interferon induced transmembrane protein 3
Glycerol phosphate dehydrogenase 2, mitochondrial
Thrombospondin 1
Procollagen, type II, alpha 1
Matrilin 2
Transmembrane protein 2

a

Ratio of expression level in VP16–Six1wt-expressing mK4 cells to expression level in VP16–Six1W171R-expressing cells. b Ratio of expression level in VP16–Six4wt-expressing mK4 cells to expression level in VP16–Six4W263R-expressing cells. c Genes involved in kidney development or function.

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Z-i. Ando et al.

Target genes of Six1 and Six4

Table 2. RT-PCR analysis of potential Six1- and Six4-target genes
identified by microarray. Total RNA (10 or 100 ng) prepared from
mk4 cells infected with adenoviruses overexpressing VP16–Six1 or
VP16–Six4 fusion proteins was subjected to RT-PCR. Aliquots of
PCR products were removed from the thermal cycler at multiple
cycle numbers, separated on a 5% acrylamide gel, stained with the
fluorescent dye, scanned and quantitated. Linearity of PCR amplification was maintained over several cycles, and the amount of PCR
products at 16–28 cycles, depending on the gene, was selected for
comparison. Three independent RT-PCR reactions were set up
from the same RNA samples. Data are shown as mean ± SEM.
We could not find 1.5-fold difference of expression in Lampt4A.

Six1–Six4 common targets
Ogn
1.91 (0.07)
Amot
1.99 (0.20)
Rhoe
2.71 (0.24)
Nid2
1.62 (0.06)
Acadm
1.67 (0.01)

Cdkn1c
2.09 (0.10)
Slc12a2
1.60 (0.04)
Six1-specific target genes
Figf
2.11 (0.08)
Gas1
2.15 (0.10)
Clcn5
1.86 (0.03)
Six4-specific target genes
Ifitm3
1.90 (0.02)
Thbs1
0.81 (0.05)
Col2a1
1.33 (0.06)

Six4wt ⁄ Six4W263Rb
Mean (± SEM)

Cyclesc

1.88
2.02
2.59
1.63
2.04
3.10

2.28

(0.02)
(0.01)
(0.02)
(0.04)
(0.13)
(0.03)
(0.11)

20
20
20
24
24
24
22

1.51 (0.15)
1.28 (0.09)
2.03 (0.09)

24
20
20

5.52 (0.27)
2.77 (0.12)
1.63 (0.04)

20
16
28

A

a
Ratio of expression level in VP16–Six1wt-expressing mk4 cells to
expression level in VP16–Six1W171R-expressing cells. b Ratio of
expression level in VP16–Six4wt-expressing mk4 cells to expression level in VP16–Six4W263R-expressing cells. c The number of
PCR cycles at which the relative amount of PCR products were
determined.

B

10

1.2
1.0

8

0.8
Fold activation

Six1wt ⁄ Six1W171Ra
Mean (± SEM)

Fold activation

Symbol

(Fig. 1B). In our screening, Clcn5 was listed as a potential Six1-specific target and it is reasonable that even
the genes naturally repressed by Six1 were activated by
VP16–Six1 in our screening condition. Slc12a2 promoter was strongly activated by Six1, but weakly activated
by Six4 in a dose-dependent manner (Fig. 1C). The
apparent discrepancy in the activation ⁄ repression profiles of Six1 and Six4 based on the results of transient
transfection assays and microarray analyses may be
explained by the involvement of other transcription factors in cells used in transfection assays. VP16-fusion

6

4

2

0.6
0.4
0.2
0

0
Gas1-3408Luc
C

Clcn5-1325Luc
D

35

25

30
20

FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

Fold activation

Fig. 1. Differential regulations of the potential target genes by Six1
and Six4. Transient transfection assays with 175 ng of the indicated
promoter-luciferase reporter construct were carried out as described in Experimental procedures. Luciferase activity was normalized to the protein content and expressed relative to the value in
the presence of 75 ng pFLAG-CMV2 (white bars), which was set at
1. As effectors, increasing amounts (25 and 75 ng) of pfSix1 (black
bars) and pfSix4 (gray bars) were cotransfected into COS7 cells.
Data are mean ± SEM of three independent experiments (each performed in duplicate). (A) Trans-activation of Gas1 promoter by Six1
and Six4. (B) Regulation of Clcn5 promoter by Six1 and Six4. (C)
Trans-activation of Slc12a2 promoter by Six1 and Six4. (D) Transactivation of myogenin promoter by Six1 and Six4. (E) Gel-retardation assays of Flag–Six1 and Flag–Six4 in nuclear extracts (800 ng
protein for Six1 and 860 ng protein for Six4) from transiently transfected cells using 5 fmol of C3 oligonucleotide probe. Arrows indicate the positions of specific retarded complexes and arrowhead
indicates the position of nonspecific complex.

Fold activation

25
20
15
10

10

5

5
0

0
Slc12a2-1938Luc

E

15

pGL3MG-185

Six1
Six4
Gas1

Clcn5

Slc12a2

pGL3MG

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Target genes of Six1 and Six4

protein may bypass the effects of other transcription

factors that may affect the promoter activity in cells.
The control myogenin promoter was moderately activated by Six1 and strongly by Six4 (Fig. 1D). To ensure
that the differential regulation by Six1 and Six4 is not
due to the altered expression levels of cotransfected
Six1 and Six4, we performed gel-retardation analysis
of nuclear extracts from cotransfected cells. Similar
amounts of gel-retarded complexes of Flag–Six1 and
Flag–Six4 (Fig. 1E) were detected among each set of
nuclear extracts from the transfected cells except in the
case of Flag–Six1 in the presence of the myogenin
reporter. These findings indicate that Six1 and Six4
differentially regulate their target genes.

Z-i. Ando et al.

A
0

1

2

Relative luciferase activity
3
4
5
6
7
8

Slc12a2-820Luc

Slc12a2-97Luc

pGL3-basic

B
-1938

-97

+1

+77

Cfr10I

+103

+149

HinfI MspI

SacI

probe A
probe B
probe C

Identification of Six1- and Six4-responsive

elements in the Slc12a2 promoter

3030

10

Slc12a2-1938Luc

NarI

We focused our analysis on Slc12a2 promoter to determine the molecular basis of the differential regulation
by Six1 and Six4. To localize Six1 and Six4 responsive
elements in the Slc12a2 promoter, we prepared a set
of deletion constructs harboring the Slc12a2 promoter
regions )1938 to +149 (Slc12a2)1938Luc), )820 to
+149 (Slc12a2–820Luc) and )97 to +149 (Slc12a2–
97Luc). Activation by Six1 and Six4 was observed in
the series of deletion constructs and similar activation
level was still observed in the shortest construct,
Slc12a2–97Luc (Fig. 2A).
To explore the possibility that Six1- and Six4-binding sites lie within )97 to +149, we next performed
gel retardation assays using probes of NarI–HinfI fragment ()97 to +77, Fig. 2B, probe A), Cfr10I–MspI
fragment (+2 to +103, Fig. 2B, probe B) and HinfI–
SacI fragment (+75 to +149, Fig. 2B, probe C). The
expressed glutathione S-transferase (GST) fusion protein of Six1 (GST–Six1) bound to probes A, B and C,
whereas GST fusion protein of Six4 (GST–Six4) bound
to probe C, but not to probes A and B (Fig. 2C).
These results suggest the existence of at least two Six1specific binding sites, and only one binding site common to Six1 and Six4.
To analyse the location of each binding site, we synthesized the double-stranded competitor oligonucleotides (oligo 1 to oligo 9) that covered various portions
of the promoter region (Fig. 3A). The formation of a

complex by Six1 was strongly competed by oligos 2, 3
and 6 for probe A, by oligos 6 and 7 for probe B and
by oligos 6, 7 and 9 for probe C (Fig. 3B). In contrast,
the formation of a complex by Six4 was competed only
by oligo 9 for probe C (Fig. 3C).
To precisely localize the binding element for Six1
and Six4, we generated 4-bp substitution mutations

9

C

GST-Six1
probe

A

B

C

GST-Six4
A

B

C

complex

free probe

free probe
free probe
1

2

3

4

5

6

Fig. 2. Identification of Six1 and Six4 responsive regions in the
Slc12a2 promoter. (A) Effects of Six1 or Six4 on the Slc12a2 promoter in COS7 cells. In these studies, 175 ng of luciferase reporter
constructs containing a series of deletions of Slc12a2 promoter
fragments were cotransfected with increasing amounts (25 and
75 ng) of pfSix1(black bars) and pfSix4 (gray bars). Luciferase activity was normalized to the protein content and expressed relative to
the value in the presence of 75 ng pFLAG-CMV2 (white bar), which
was set at 1. (B) Schematic representation of the Slc12a2 promoter
with the position of a major transcription start site (+1). Solid bars
at the bottom show positions of probes used in gel retardation
assays. (C) Binding of Six1 and Six4 to the promoter DNA fragments of Slc12a2. Gel-retardation assays were performed using
bacterially expressed 10 ng of GST–Six1 and 30 ng of GST–Six4
proteins with each 5 fmol probe indicated in (B). Lanes 1–3 and
lanes 4–6 were from separated lanes in the same gel. Free probes
are indicated by the arrows and shifted complexes are indicated by

the bracket.

in various regions of oligo 3, oligo 6 and oligo 9
(Table 3) and each mutated oligo was added to the gel
retardation assay mixture to examine the competition
to the binding of Six1 and probes A, B and C. Oligo
FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS


Z-i. Ando et al.

probe

A

probe A

Target genes of Six1 and Six4

-97

+77

probe B

+2

oligo 1
oligo 2

+75
-99

+149

-62
-31

-68

oligo 3
competitor

+103

probe C

+3

-37

oligo 4

+34

-4

oligo 5

+65

+28

oligo 6

+93

+59

oligo 7
oligo 8
oligo 9

+115
+86
+109
+138
+115
+152

B

1 2 3 4 5 6 7
probe
competitor
binding

8 9 101112

1314151617

A

B

C

1 2 3 4 5 6

4 5 6 7

6 7 8 9

1.0 0.5 0.3 0.2 0.5 0.4 0.1

1.0 0.5 0.5 0.1 0.2

1.0 0.3 0.3 0.6 0.2

C

1 2 3 4 5 6 7 8 9 10
probe
competitor
binding

C
1 2 3 4 5 6 7 8 9

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.1 1.2 0.6

Fig. 3. Mapping of Six1 and Six4 binding elements in the Slc12a2
promoter. (A) Positions of probes A, B and C are indicated in the
upper panel. Positions of competitors (oligos 1–9) are indicated in
the lower panel. (B) Gel retardation assays with GST–Six1 in the
presence (lanes 2–7, 9–12 and 14–17) or absence (lanes 1, 8 and
13) of competitors; 500-fold molar excess competitor oligonucleotides were added to the reaction. The protein amount of GST–Six1
was 16 ng for probe A (lanes 1–7), 15 ng for probe B (lanes 8–12)
and 13 ng for probe C (lanes 13–17). The intensities of the retarded
bands relative to that in the absence of competitors, which was
set at 1 (lanes 1, 8 and 13), are shown at the bottom. (C) Gel-retardation assays with 30 ng of GST–Six4 in the presence (lanes 2–10)
or absence (lane 1) of competitors; 500-fold molar excess competitor oligonucleotides were added. The intensities of the retarded
bands relative to that in the absence of competitors, which was
set at 1(lane 1), are shown at the bottom. Free probes are indicated
by the arrow and shifted complexes by the bracket in (B) and (C).

FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

3mut()13 ⁄ )10) showed the most reduced competition
among the mutated oligos examined (Fig. 4A, lane 7).
The reduction of Six1 binding was confirmed by comparing the binding of Six1 to the oligo 3wt probe and
the oligo 3mut()13 ⁄ )10) probe. The amount of the
retarded complex composed of Six1 and oligo
3mut()13 ⁄ )10) was approximately threefold less compared with oligo 3wt (Fig. 4B, lanes 2 and 4). As for
probe B, all substitution mutations of oligo 6 showed
similar competition compared with oligo 6wt (data not
shown), suggesting the presence of multiple binding
sites for Six1 in oligo 6. We also tested deletion oligos
and found that oligo 6 with 10-bp deletion in +65 to
+74 [oligo 6del(+65 ⁄ +74)] slightly reduced the competition compared with oligo 6wt (Fig. 4A, lanes 11

and 12). The double mutation oligo 6 containing
del(+65 ⁄ +74) and substitution mutation in +89 to
+92 [del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92)] showed further
reduced competition against the probe B–Six1 protein
complex formation (Fig. 4A, lane 13). The reduction
in the binding was confirmed by comparing the binding of Six1 to the oligo 6wt probe and oligo
6del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92) probe and the binding was approximately threefold weaker in the latter
compared with the former (Fig. 4B, lanes 6 and 10).
The oligo 9mut(+135 ⁄ +138) showed the most
reduced competition among the oligos examined
against the probe C–Six1 protein complex formation
(Fig. 4A, lane 19). The reduction in the binding was
confirmed by comparing the binding of Six1 to the oligo 9wt probe and the oligo 9mut(+135 ⁄ +138) probe
and the Six1 binding to the latter was approximately
threefold less than the former (Fig. 4B, lanes 12 and
14). Likewise, the oligo 9mut(+135 ⁄ +138) showed
the most reduced competition to the probe C–Six4
complex formation (Fig. 4C, lane 6). The binding of
Six4 to the oligo 9wt probe was compared with the
oligo 9mut(+135 ⁄ +138) probe and in this case, the
reduction of binding was  10-fold (Fig. 4D, lanes 2
and 4).
In summary, the three Six1-specific binding sites
reside around )13 to )10, +65 to +74 and +89 to
+92 and the single common binding site resides
around position +135 to +138 in the promoter region
of Slc12a2 (Fig. 4E).
Activation of the Slc12a2 promoter
by Six1 and Six4
To confirm that Six1 activates through the aboveidentified binding sites, we introduced substitution

mutations used in the gel-retardation assays into the
luciferase reporter constructs. We prepared the following
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Z-i. Ando et al.

Table 3. Double-stranded oligonucleotides used in Fig. 4. Nucleotide substitutions in the mutation oligonucleotides are represented in bold.
Name

Sequence

mutation
reporters,
Slc12a2–97mut()13 ⁄ )10)Luc,
Slc12a2–97del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92)Luc, and the
combination of the two, Slc12a2–97mut()13 ⁄ )10) ⁄
del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92)Luc as Six1-binding mutations. We also prepared Slc12a2–97mut(+135 ⁄ +138)Luc,
which abolished Six4 binding. We performed reporter
gene assays and analysed the effects of Six1 (Fig. 5A)
and Six4 (Fig. 5B). Cotransfection of pfSix1 showed
activation of Slc12a2–97Luc in a dose-dependent manner to an  11-fold increase in the level of luciferase
activity. The activation level was decreased to approximately sixfold in Slc12a2mut()13 ⁄ )10)Luc, whereas
Slc12a2del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92)Luc showed comparable luciferase activity to the wild-type reporter
construct, Slc12a2–97Luc. In contrast, the combina3032

tion of these two mutations, Slc12a2mut()13 ⁄ )10) ⁄
del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92)Luc, showed only 1.3fold activation by Six1 (Fig. 5A). These results

clearly indicate that the three Six1-binding sites
located at around )13 ⁄ )10, +65 ⁄ +74 and
+89 ⁄ +92 are the responsible element as a whole for
the activation by Six1. As for the effects of Six4,
cotransfection of pfSix4 showed 2.6- to 4.6-fold activation in the wild-type reporter construct Slc12a2–
97Luc in a dose-dependent manner. By contrast, the
luciferase activity of Slc12a2mut(+135 ⁄ +138) was
enhanced to 1.6- to 2.3-fold by Six4 (Fig. 5B).
These results indicate that Six4 activates the Slc12a2
promoter
through
its
binding
site
around
+135 ⁄ +138.
FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS


Z-i. Ando et al.

Target genes of Six1 and Six4

A

B

C

D

oligo 9wt

1.0

oligo 9mut(+135/+138)

1 2 3 4

probe C
9wt
9mut(+117/+120)
9mut(+123/+126)
9mut(+129/+132)
9mut(+135/+138)
9mut(+141/+144)
9mut(+147/+150)

0.6

oligo 9mut(+135/+138)

1.0

0.3

1.0

0.3

+1

-13/-10

1.0 0.3 0.3 0.3 0.6 0.8 0.3 0.3

0.3

E
-97

1 2 3 4 5 6 7 8

1.0

1.0 0.4 0.3 0.4 0.5 0.8 0.3 0.4

oligo 9wt

1.0 0.1 0.2 0.3

9wt
9mut(+117/+120)
9mut(+123/+126)
9mut(+129/+132)
9mut(+135/+138)
9mut(+141/+144)
9mut(+147/+150)

3wt

3mut(-37/-34)
3mut(-31/-28)
3mut(-25/-22)
3mut(-19/-16)
3mut(-13/-10)
3mut(-7/-4)
3mut(-1/+3)

1.0 0.2 0.2 0.1 0.2 0.1 0.3 0.2 0.2

oligo 6del(+65/+74)
/mut(+89/+92)

probe C
oligo 6del(+65/+74)

probe B

5 6 7 8 9 10 11121314

oligo 6wt

probe A

1 2 3 4
oligo 3mut(-13/-10)

1415161718192021

oligo 3wt

10111213

6wt
6del(+65/+74)
6del(+65/+74)
/mut(+89/+92)

1 2 3 4 5 6 7 8 9

+65/+74 +89/+92 +135/+138

: Six1-specific binding site
: Six1/Six4 common binding site

0.1

Slc12a2 is regulated by Six1 and Six4 in vivo
To confirm that Six1 and Six4 regulate the expression of
Slc12a2 in vivo, we examined the expression of Slc12a2
in the developing embryos of wild-type, Six1– ⁄ – and
Six1– ⁄ – ⁄ Six4– ⁄ – mice. The expression of Slc12a2 was
observed in various adult organs such as the central nervous system, dorsal root ganglia and renal cortex [38].
Because Six1– ⁄ – and Six1– ⁄ – ⁄ Six4– ⁄ – mice die soon after
birth and they show developmental defects in various
organs [23] (unpublished observation), we compared the
FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

+149

expression pattern of Slc12a2 in these embryos. The
expression level of Slc12a2 was too low to allow precise
comparison of its expression level in the nephrogenic
cord. Therefore, we analysed the expression of the gene
by in situ hybridization in the dorsal root ganglia, where
Six1 and Six4 were abundantly expressed and some
developmental abnormalities were observed in Six1– ⁄ – ⁄
Six4– ⁄ – mice (K. Ikeda & K. Kawakami, unpublished
observation). The antisense probe detected expressions
of Slc12a2 in the dorsal root ganglia in E18.5 fetus
where significant expression was reported [38] (Fig. 6C),
3033


Target genes of Six1 and Six4

Z-i. Ando et al.

Fig. 4. Identification of Six1- and Six4-binding sites in the Slc12a2 promoter by gel retardation assays. The competitor oligonucleotides harboring mutations are shown in Table 3. (A) Competition assays were performed with GST–Six1 protein in the absence (lanes 1, 10 and 14)
or presence of 500-fold molar excess of various competitor oligonucleotides (lanes 2–9, 11–13 and 15–21). Probes A, B and C indicated in
Fig. 2 were used. The protein amount of GST–Six1 was 20 ng for probe A (lanes 1–9), 15 ng for probe B (lanes 10–13), and 16 ng for probe
C (lanes 14–21). The intensities of the retarded bands relative to that in the absence of competitors for each probe, which was set at 1
(lanes 1, 10 and 14), are shown at the bottom. Shifted complexes were least competed by oligo 3mut()13 ⁄ )10) (lane 7), oligo 6del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92) (lane 13), and oligo 9mut(+135 ⁄ +138) (lane 19). (B) Comparison of binding of GST–Six1 to the wild-type (oligo 3wt, lane 2; oligo 6wt, lane 6; oligo 9wt, lane 12) and to the mutated probes [oligo 3mut()13 ⁄ )10), lane 4; oligo 6del(+65 ⁄ +74) and
oligo 6del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92), lanes 8 and 10, respectively; oligo 9mut(+135 ⁄ +138), lane 14]. Sixty-seven nanograms of GST–Six1
was used in the reactions. Binding of GST–Six1 to oligo 3mut()13 ⁄ )10) was  30% compared with that of oligo 3wt (lanes 2 and 4). Binding
of GST–Six1 to oligo 6del(+65 ⁄ +74) and oligo 6del(+65 ⁄ +74) ⁄ mut(+89 ⁄ +92) was  60 and 30%, respectively, compared with that of oligo 6wt (lanes 6, 8 and 10). Binding of GST–Six1 to oligo 9mut(+135 ⁄ +138) was  30% compared with that of oligo 9wt (lanes 12 and 14).
Lanes 5–6 and lanes 7–10, lane 11 and lanes 12–14 are from separate lanes of one gel. (C) Competition assays were performed with GST–
Six4 protein in the absence (lane 1) or presence of 500-fold molar excess of various competitor oligonucleotides (lanes 2–8). Probe C indicated in Fig. 2 was used. Thirty nanograms of GST–Six4 was used in the reaction. The intensities of the retarded bands relative to that in the
absence of competitors for the probe, which was set at 1(lane 1), are shown at the bottom. Shifted complexes were most weakly competed by the oligo 9mut(+135 ⁄ +138) (lane 6). (D) Comparison of binding of GST–Six4 to the wild-type (oligo 9wt, lane 2) and mutated probes
[oligo 9mut(+135 ⁄ +138), lane 4]. Thirty nanograms of GST–Six4 was used in the reactions. Binding of GST–Six4 to oligo 9mut(+135 ⁄ +138)

was  10% compared with that of oligo 9wt (lanes 2 and 4). (E) Schematic representation of binding elements of Six1 and Six4 in the
Slc12a2 promoter. Six1-specific binding elements reside in the region of )13 ⁄ )10, +65 ⁄ +74 and +89 ⁄ +92 (black ovals) and a bipartite Six1and Six4-binding element in the region of +135 ⁄ +138 (gray box).

whereas the control sense probe gave only background
signals (Fig. 6D). The expression level of Slc12a2 was
apparently lower in Six1– ⁄ – ⁄ Six4– ⁄ – embryo at E16.5
compared with the wild-type, whereas that in Six1– ⁄ –
was similar to the wild-type (Fig. 6H–J). We observed
similar reductions of Slc12a2 expression in the dorsal
root ganglia of Six1– ⁄ – ⁄ Six4– ⁄ – embryos at E17.5 and
E18.5 (data not shown). In the choroid plexus, where no
Six1 and Six4 were expressed, the expression level of
Slc12a2 was similar in each genotype (Fig. 6N–P).
These results suggest that the low expression level of
Slc12a2 in Six1– ⁄ – ⁄ Six4– ⁄ – embryos is due to the
absence of Six1 and Six4 in the dorsal root ganglia
and indicate that Slc12a2 is upregulated by Six1 and
Six4 in the developing dorsal root ganglia of wild-type
embryo.
A
0

2

Slc12a2-97Luc

Fold activation
4 6 8 10 12 14
-97

To confirm that Six1 and Six4 regulate the other
putative target genes in vivo, we analysed the expression of Figf and Col2a1 in E10.5 embryos of wild-type
and Six1– ⁄ – ⁄ Six4– ⁄ – by RT-PCR. The expression levels
in Six1– ⁄ – ⁄ Six4– ⁄ – embryo were significantly reduced
(to 36.4% for Figf and to 58.1% for Col2a1 compared
with the wild-type), suggesting that these genes are also
regulated by Six1 and Six4 in vivo (Fig. 7).

Discussion
Screening of putative target genes of Six1 and
Six4 and effectiveness of the screening method
To understand the function of transcription factors
in organ development, it is essential to identify direct

+1

+149

Slc12a2-97mut(-13/-10)Luc
Slc12a2-97del(+65/+74)
/mut(+89/+92)Luc
Slc12a2-97mut(-13/-10)
/del(+65/+74)/mut(+89/+92)Luc

B
0
Slc12a2-97Luc
Slc12a2-97mut(+135/+138)Luc

3034

2

Fold activation
4 6 8 10 12 14
-97

+1

+149

Fig. 5. Transactivation of the Slc12a2
promoter by Six1 and Six4. (A, B) Slc12a2–
97Luc and four types of mutation reporter
constructs (shown in the right-hand panel,
70 ng each) were cotransfected with increasing amounts (25 and 75 ng) of pfSix1 or
pfSix4 in COS7 cells. Luciferase activity was
normalized to protein content and expressed
relative to the value in the presence of
75 ng pFLAG-CMV2 (white bar), which was
set at 1. Data are mean ± SEM of a typical
result of three independent experiments
(each performed in triplicate).

FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS


Z-i. Ando et al.

Target genes of Six1 and Six4

Sense

Antisense
A

Dorsal root
ganglion

B

C

D

Six1-/-

Wild type
E

Six1-/-/Six4-/-

F

drg

G

sp

drg

drg

sp

Dorsal root
ganglion

sp
v

v

v
I

J

K

Choroid
plexsus

H

L

M

N

O

P

Fig. 6. In situ hybridization of Slc12a2 in the dorsal root ganglia of mouse embryos. (A–D) Transverse sections (16 lm) were stained with
hematoxylin (A, B) and the adjacent sections hybridized with Slc12a2 antisense (C) or sense (D) probe. Specific hybridization signals were
detected in the dorsal root ganglia (encircled by the dotted line) of E18.5 embryo in (C) but not in (D). (E–J) Transverse sections (16 lm) from
E16.5 wild-type (E, H), Six1– ⁄ – (F, I) and Six1– ⁄ – ⁄ Six4– ⁄ – (G, J) embryos were stained with hematoxylin (E–G) and the adjacent sections
hybridized with Slc12a2 antisense probes (H–J). The expression levels of Slc12a2 in the developing dorsal root ganglia of the Six1– ⁄ –
embryos were similar to those in the wild-type (H, I), but markedly reduced in the Six1– ⁄ – ⁄ Six4– ⁄ – embryos (J). (K–P) As controls, the signals
on the choroid plexus from E16.5 wild-type (K, N), Six1– ⁄ – (L. O) and Six1– ⁄ – ⁄ Six4– ⁄ – (M, P) embryos stained with hematoxylin (K–M) and
with antisense probe (N–P) are shown. Similar expression levels of Slc12a2 were observed in all genotypes (N–P). Scale bar: 100 lm.

target genes and recognize the gene cascade as well as
regulatory mechanisms involved in proper cell growth,
differentiation, cell movement and functional maturaFEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

tion of the organ. In this analysis, we took advantage
of a model cell line that reflects a certain developmental stage of the kidney in order to identify the genes
3035


Target genes of Six1 and Six4

Z-i. Ando et al.

normally involved in the regulation, in conjunction
with endogenous Six proteins, thus our screening may

be biased in the identification of target genes. Still, the
identified genes were regulated either by Six1 or Six4
(Fig. 1) and some of the genes were downregulated
in Six1– ⁄ – ⁄ Six4– ⁄ – (Figs 6 and 7), indicating that this
approach is a powerful method for the identification of
direct target genes irrespective of whether they are activated or repressed by the proteins.

Figf
Col2a1
b-actin
1

2

Fig. 7. RT-PCR analyses of RNA from E10.5 mouse embryos. One
hundred nanograms of total RNAs from each of Six1– ⁄ – ⁄ Six4– ⁄ –
(lane 1) and wild-type (lane 2) embryos at E10.5 were amplified by
RT-PCR using a specific set of primers for Figf, Col2a1 and control
b-actin. Typical results of three independent experiments (six
embryos).

that are driven by Six1 and ⁄ or Six4. A similar strategy
was previously applied to identify target genes of Six5
using P19 carcinoma cells, and direct target genes
Igfbp5 and Igf2 were successfully identified [24]. By
overexpressing constitutively transcriptionally active
forms of VP16–Six1 and VP16–Six4, we expected that
genes accessible by Six1 and Six4 will be activated in
the cells. In this screening, we used mK4 cells, which
possess the characteristics of embryonic metanephric

mesenchyme [25]. As expected, we identified the potential target genes that might be involved in cell growth,
differentiation and specific ion transport functions in
the kidney. Of these, numerous terminal differentiation
genes were observed such as Aqp1, Atp1a1, Clcn5,
Npr3 and Slc12a2. Although Six1 is involved in the
early phase of kidney development, such as aggregation of metanephric mesenchyme around the ureteric
bud [21], it is not surprising that Six1 and Six4 proteins also directly regulate the genes involved in the
terminal differentiation. Many terminal differentiation
genes have been identified as target genes of MyoD as
well as genes involved in muscle development [39].
Our initial screening analysis identified 300 Six1specific targets, 86 Six4-specific targets, and 63 common
targets. However, this classification may not necessarily
be definite considering the data from transient transfection assays. For example, one of the Six1-specific target
genes, Gas1, was also activated by Six4, and another
Six1-specific target, Clcn5, was more efficiently
repressed by Six4 than by Six1. In addition, we clearly
showed that Six1 can bind to Six4-binding sites, thus,
many of the Six4-regulated genes would be also identified as target genes of Six1. VP16 fusion Six proteins
are expected to activate their target genes independent
of other transcription factors or cofactors that are
3036

Response of potential target genes to Six1 and
Six4
We previously reported that Six2, Six4 and Six5 activated the myogenin promoter, and such activation was
enhanced by Eya proteins [18]. Furthermore, the extent
of activation was dependent on the combinations of
Six and Eya. In this study, we identified various types
of target genes that showed similar activation by Six1
and Six4 (Gas1), that were efficiently activated by Six1

compared with Six4 (Slc12a2) and that were repressed
by Six1 and even more strongly by Six4 (Clcn5). Such
differential regulation of each target gene by Six1 and
Six4 may reflect some aspects of the regulatory action
in vivo, which should be clarified in future analyses.
The presence of target genes activated and repressed
by Six1 is consistent with the recent finding that
Xenopus six1 affects ectodermal genes through both
transcriptional activation and repression [40].
To explore the molecular mechanism of the differential regulation by Six1 and Six4, we analysed the
responsive elements of Slc12a2 gene promoter. Our
results showed that the number of Six1-binding sites is
at least three, whereas there is one Six4-binding site,
indicating that the DNA-binding specificity of Six1 is
distinct from that of Six4. Another member of the
Six1 ⁄ 2 subfamily, Six2, showed similar features, i.e.
residual activation by Six2 even in a mutation myogenin promoter construct of the MEF3 site, which abolished Six4 and Six2 binding, due to the presence of
additional Six2-binding sites other than MEF3 that are
not identified in the myogenin promoter region [18].
Although we did not address the structural basis of
the differential regulation of the promoter by Six1 and
Six4 proteins, our results clearly demonstrated that
Six1 and Six4 could regulate their target genes differently. This predicts that Six1 and Six4 may each
express a unique function in some cases, whereas they
may play a common role in other cases. In fact, we
previously reported that Six1 could compensate for
the Six4 function but Six4 did not compensate for
Six1 function during the development of various
organs [16,23]. Furthermore, our recent studies of
FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

Z-i. Ando et al.

Six1– ⁄ – ⁄ Six4– ⁄ – mice revealed specific anomalies in earlier stages of otic and nasal development and in the
formation of branchial arch and some cranial ganglia,
which were not observed in Six4– ⁄ – mice and Six1– ⁄ –
mice (K. Ikeda & K. Kawakami, unpublished observation). These observations indicate that the Six1 and
Six4 mutually compensate for their functions in the
early stage of development, suggesting a common role.

Target genes of Six1 and Six4

cochlea would be important for understanding deafness
caused by the loss of Slc12a2.
RT-PCR analyses of E10.5 embryo showed that the
expression levels of Figf and Col2a1 were reduced
in Six1– ⁄ – ⁄ Six4– ⁄ – embryos compared with wild-type.
This result suggests that these genes are upregulated
by Six1 and Six4 in the developing embryo.

Experimental procedures
DNA-binding sequences of Six1 and Six4
As for the recognition sequence of Six1, which were
identified in this study, it is difficult to predict the consensus sequence of the binding sites. The binding site
of the most similar protein Six2 has recently been
reported in Gdnf gene promoter [41]. It contains two
Six2-binding sites that show similarity to the homeodomain binding core sequence TAAT [41]. However,
this core sequence was not found in binding sequences
of Six1 identified in this study. The binding of Six1

may not depend on the exact DNA sequence but
rather some structural features like nonsequence-specific HMG protein DNA recognition [42].
A new binding site of Six4, the transcriptional regulatory element X (Trex), which is the positive control
element within the muscle creatine kinase (MCK)
enhancer, has been recently reported [43]. We found
(G)ACCCGAG, a single mismatch sequence of the
MCK Trex, in the +129 ⁄ +138 region of the Slc12a2
promoter.
Direct target of Six1 and Six4 in vivo
Six1 enhanced the promoter activity of Slc12a2 more
efficiently than Six4. This was explained by the presence of multiple binding sites of Six1 that exceed in
number those of Six4 in the promoter region.
Although the expression profile of Slc12a2 has not
been precisely analysed in the context of development
of the kidney and dorsal root ganglion, differential
regulation by Six1 and Six4 might be relevant in certain developmental stages in a specific organ.
In our in situ hybridization analyses of the dorsal
root ganglia, the expression levels of Slc12a2 in Six1– ⁄ –
embryos were similar to those in the wild-type, whereas
the level of expression was markedly reduced in Six1– ⁄ – ⁄
Six4– ⁄ – embryos compared with wild-type. These
results indicate that Six4 can support the expression of
Slc12a2 in the absence of Six1 and that the common
binding site around +135 ⁄ +138 is important for the
expression of Slc12a2, at least in the developing dorsal
root ganglion. Similar analyses in the developing

FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

Plasmid construction

Construction of expression plasmids and luciferase reporter
plasmids is described briefly here and in full in the supplementary material.
Plasmids expressing FLAG-tagged Six1 and Six4 (pfSix1
and pfSix4) were constructed as described previously
[18,44]. To construct constitutively active Six1 and Six4, a
transcription activation domain of herpes simplex virus
virion protein 16 (VP16) [45] was fused to the N-terminal
end of full-length Six proteins [pCS2 + FLAG VP16–
Six1wt and pCS2 + FLAG VP16–Six4wt]. As a control,
mutated proteins harboring a tryptophan (W) to arginine
(R) substitution were used [pCS2 + FLAG VP16–
Six1W171R and pCS2 + FLAG VP16–Six4W263R]. The
corresponding mutation in the Six5 HD abolishes DNAbinding activity [24].
Plasmids harboring the 5¢ upstream region of Slc12a2
were constructed by inserting appropriate fragments prepared from pGL3-2065 [46] into pGL3-basic (Promega
Bioscience, San Luis Obispo, CA). To construct Clcn5)
1325Luc, a mouse Clcn5 promoter fragment ()1325 to
+2529) was amplified from C57BL ⁄ 6 genomic DNA and
ligated into pGL3-basic. To construct Gas1)3409Luc, a
Gas1 promoter fragment excised from pUBT–luc ⁄
Gas1 3.5 k [47] was ligated into pGL3-basic. pGL3MG185 which harbors )185 to +50 of the myogenin promoter was described previously [18].

Production of recombinant adenovirus
Recombinant adenoviruses were produced using the Adenovirus Expression Vector Kit (Takara Bio, Shiga, Japan) as
described previously [24]. Briefly, HindIII–XbaI fragments
encoding VP16–Six1wt and VP16–Six1W171R were excised
from pCS2 + FLAG VP16 constructs, blunted and ligated
into SwaI-cut pAxCAwt cosmid vector. Likewise, ClaI–
XbaI fragments encoding VP16–Six4wt and VP16–
Six4W263R were excised, blunted and ligated into pAxCAwt. The recombinant cosmids were cotransfected with

the EcoT221-digested AxCAwt DNA-terminal protein complex into 293 cells. The recombinant viruses were isolated,
propagated and checked for titer (plaque forming unit) and

3037


Target genes of Six1 and Six4

transgene expression. Virus titration was carried out using
293 cells and western blotting was used to assess the expression of recombinant proteins.
Cell culture mK4 cells were cultured as described previously [25]. COS7 and 293 cells were grown as described
previously [18,24].

Virus infection and transient transfection
Adenovirus-infection was carried out as described previously [24]. To adjust the expression levels of recombinant
proteins, we infected mK4 cells at the following multiplicity
of infection values: AxCAwt VP16–Six1wt (25), AxCAwt
VP16–Six1W171R (200), AxCAwt VP16–Six4wt (50) and
AxCAwt VP16–Six4W263R (200). Transfections were carried out using CellPhect (Amersham Biosciences, Piscataway, NJ) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
in 24-well plates. The cells were harvested after two days.
Luciferase activity was normalized for total protein content
in cell lysates. Data were shown as mean ± SEM.

Semiquantitative RT-PCR analysis
Total RNA used in the microarray analysis (10 or
100 ng) or total RNA prepared from mouse embryos
(100 ng) was subjected to RT-PCR using OneStep
RT-PCR kit (Qiagen, Hilden, Germany) and the PCR
primers listed in the supplemental Table S2. Each set of
PCR primers was derived from different exons except

that for the intronless Gas1. For RT-PCR analysis of
Gas1, RNA samples were treated with RNase-free DNaseI and checked for the absence of visible PCR products
even after 32 cycles of amplification as described previously [24]. Aliquots of PCR products were removed
from the thermal cycler at multiple cycle numbers, separated on a 5% acrylamide gel, stained with the fluorescent
dye Vistra Green (Amersham Biosciences) and scanned
using the STORM system (Amersham Biosciences). Quantitation of the amplified products was carried out using
the STORM system and imagequant software (Molecular Dynamics, Sunnyvale, CA). Linearity of PCR
amplification was maintained over several cycles, and the
amount of PCR products at 16–28 cycles, depending on
the gene, was selected for comparison. Three independent
RT-PCR reactions were set up from the same RNA
samples. For analysis of samples from mouse embryos,
b-actin mRNA was also amplified as an internal control
to monitor for embryo-to-embryo variations in the quality of input RNA.

Gel-retardation assay
Gel-retardation assays were carried out as described previously [48]. GST–Six1 and GST–Six4 (SMNTND1) were

3038

Z-i. Ando et al.

prepared as described previously [2,44]. Restriction fragments of Slc12a2 promoter and annealed double-stranded
oligonucleotides were end-labeled with [32P]dCTP[aP] by
Klenow fragment or with [32P]ATP[cP] by T4 polynucleotide kinase. Quantitation of the hybridization signals was
carried out using the STORM system (Amersham Biosciences) and imagequant software (Molecular Dynamics).
Nuclear extracts and C3 oligonucleotide probe were prepared as described previously [2].

In situ hybridization and ethical consideration
These experiments were conducted in wild-type, Six1– ⁄ –

and Six1– ⁄ – ⁄ Six4– ⁄ – mice of either sex at E16.5 and E18.5.
The in situ hybridization histochemistry was performed
essentially as described previously [49]. An antisense oligo
cDNA probe and a sense cDNA probe (complementary to
the antisense) for mouse Slc12a2 mRNA were designed as
follows; Slc12a2 antisense, 5¢-ATCTTCACAAGAAAAAT
CACCTGGTACCAAGGATGT; Slc12a2 sense, 5¢-ACAT
CCTTGGTACCAGGTGATTTTTCTTGTGAAGAT.
All experimental protocols described in this study were
approved by the Ethics Review Committee for Animal
Experimentation of Jichi Medical School.

Acknowledgements
We thank S. S. Potter for providing mK4 cells, R. de
Martin for pUBT-luc ⁄ Gas1 3.5 k and E. Delpire for
pGL3-2065 plasmids. We also thank M. Nakamura,
Y. Goto and Y. Takano for the expert technical assistance. This work was supported by grants from Ministry of Education, Culture, Sports, Science and
Technology of JAPAN (KK and SS), The Science
Research Promotion Fund from The Promotion and
Mutual Aid Corporation for Private Schools of Japan
(KK) and The Research Award to JMS Graduate
Student (ZA).

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oculis gene product, and implication in development.
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3 Serikaku MA & O’Tousa JE (1994) sine oculis is a
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Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB4716/EJB4716sm.htm

FEBS Journal 272 (2005) 3026–3041 ª 2005 FEBS

Target genes of Six1 and Six4

Table S1. A complete list of potential target genes
regulated by Six1 and ⁄ or Six4 in mk4 cells.
Fig. S1. Scatter plot analysis of microarray data. Logscale scatter plots of normalized fluorescent intensity
values (normalized data) obtained from Cy3 and Cy5
channels. The Agilent Mouse Developmental Oligo
Microarrays, which contains 20 371 genes, were

hybridized with cDNA probes prepared from mRNAs
derived from mk4 cells infected with recombinant
adenovirus overexpressing VP16–Six1 fusion proteins
(A) and VP16–Six4 fusion proteins (B). Normalized
data from Cy3 channel were plotted against Cy5
channel in log scale. (A) VP16–Six1wt (Cy3) vs. VP16–
Six1W171R (Cy5). (B) VP16–Six4wt (Cy3) vs. VP16–
Six4W263R (Cy5).

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