Full Title of the paper Regulation of CAPRICE Transcription by MYB

Plant Cell Physiol. 46(6): 817–826 (2005)

doi:10.1093/pcp/pci096, available online at www.pcp.oupjournals.org

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Regulation of CAPRICE Transcription by MYB Proteins for Root Epidermis Differentiation in Arabidopsis

Yoshihiro Koshino-Kimura 1, 2, Takuji Wada 2, Tatsuhiko Tachibana 1, 4, Ryuji Tsugeki 1, Sumie Ishiguro 1, 5

and Kiyotaka Okada 1, 2, 3, 6

1 Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan 2 Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan 3 Core Research for Evolutional Science and Technology (CREST) Research Project, Japan Science and Technology Agency, Kawaguchi, Saitama,

322-0012, Japan

;

Epidermal cell differentiation in Arabidopsis root is

studied as a model system for understanding cell fate speci-

fication. Two types of MYB-related transcription factors

are involved in this cell differentiation. One of these,

CAPRICE (CPC), encoding an R3-type MYB protein, is a

positive regulator of hair cell differentiation and is prefer-

entially transcribed in hairless cells. We analyzed the regu-

latory mechanism of CPC transcription. Deletion analyses

of the CPC promoter revealed that hairless cell-specific tran-

scription of the CPC gene required a 69 bp sequence, and a

tandem repeat of this region was sufficient for its expres-

sion in epidermis. This region includes two MYB-binding

sites, and the epidermis-specific transcription of CPC was

abolished when base substitutions were introduced in these

sites. We showed by gel mobility shift experiments and by

yeast one-hybrid assay that WEREWOLF (WER), which is

an R2R3-type MYB protein, directly binds to this region.

We showed that WER also binds to the GL2 promoter

region, indicating that WER directly regulates CPC and

GL2 transcription by binding to their promoter regions.

Keywords: CAPRICE — Cell differentiation — DNA binding

— MYB protein — Transcription factor — WEREWOLF.

Abbreviations: bHLH, basic helix–loop–helix; CPC, CAPRICE;

DTT, dithiothreitol; GL, GLABRA; GST, glutathione S-transferase;

GUS, β-glucuronidase; EGL, ENHANCER OF GLABRA; TTG,

TRANSPARENT TESTA GLABRA; WER, WEREWOLF.

Introduction

Cell fate determination is a critical step in the develop-

mental process of multicellular organisms, in which transcrip-

tion factors are known to play important roles. Because of its

well-characterized genetics, short generation time and ease of

observation, Arabidopsis epidermal cells have been used as a

simple but significant model for understanding cell differentia-

tion. Epidermal cells are generated at the root apical meristem

and differentiate into two cell types, hair cells and hairless

cells, in a cell position-dependent manner (Dolan et al. 1994).

Epidermal cells contacting two cortical cells differentiate into

hair cells, whereas cells touching only one cortical cell develop

into hairless cells. Wild-type Arabidopsis ecotypes have eight

hair cell files aligned longitudinally along the root.

Several mutants defective in epidermal cell differentiation

have been isolated. The glabra2 (gl2) and werewolf (wer)

mutants show conversion of hairless cells to root hair cells

(Masucci et al. 1996, Lee and Schiefelbein 1999). GL2 encodes

an HD-Zip protein and WER encodes an R2R3-type MYB pro-

tein (Rerie et al. 1994, Di Cristina et al. 1996, Masucci et al.

1996, Lee and Schiefelbein 1999). Expression of GL2 induces

differentiation into hairless cells, and WER has been suggested

to induce GL2 expression (Lee and Schiefelbein 2002). Two

basic helix–loop–helix (bHLH) proteins, GLABRA3 (GL3)

and ENHANCER OF GLABRA3 (EGL3), work on hairless cell

differentiation in a redundant manner (Bernhardt et al. 2003,

Zhang et al. 2003). In the gl3 egl3 double mutant, most hair-

less cells are converted to root hair cells, though gl3 and egl3

single mutants show a slight increase of hair cells (Bernhardt et

al. 2003). Like WER, these bHLH proteins also regulate GL2

expression in hairless cells (Bernhardt et al. 2003, Zhang et al.

2003). Using the yeast two-hybrid system, GL3 and EGL3

were shown to interact with WER (Bernhardt et al. 2003) and

with a WD40 protein TRANSPARENT TESTA GLABRA1

(TTG1) (Payne et al. 2000, Esch et al. 2003, Zhang et al.

2003), suggesting that a transcriptional complex including

MYB, bHLH and WD40 protein regulates GL2 transcription.

The caprice (cpc) mutant has fewer root hairs apparently

because some hair cells are converted to hairless cells (Wada et

al. 1997). The CPC gene encodes an R3-type MYB protein.

4 Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka, 412-8513 Japan.5 Present address: Department of Cellular Mechanisms and Functions, Graduate School of Bio-Agricultural Sciences, Nagoya University,

Nagoya, 464-8601 Japan.6 Corresponding author: [email protected]; Fax, +81-75-753-4257.

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Gene regulation in root epidermis differentiation818


The phenotype of cpc suggests that CPC promotes hair cell dif-

ferentiation. Though CPC is a positive regulator of hair cell

differentiation, CPC is preferentially transcribed in hairless

cells (Wada et al. 2002). CPC transcription was decreased in

root hairless cells in ttg and wer, whereas it was ectopically

observed in plants where the maize bHLH gene R was constitu-

tively overexpressed (Lee and Schiefelbein 2002, Wada et al.

2002). This genetic evidence has revealed that TTG1, WER

and some bHLHs seem to regulate the CPC transcription pat-

tern. Lee and Schiefelbein (2002) proposed that transcriptional

feedback loops between the WER, CPC and GL2 genes help to

establish the CPC transcription pattern.

Many of the same or similar components involved in root

epidermis differentiation also regulate shoot epidermis differ-

entiation. MYB protein, GL1 and bHLH proteins, GL3 and

EGL3, are predominantly expressed in trichome cells and posi-

tively regulate GL2 expression (Bernhardt et al. 2003, Zhang et

al. 2003). TTG1 is also involved in trichome differentiation.

TRIPTYCHON (TRY) encodes a CPC-homologous MYB-

related transcription factor and regulates leaf trichome distribu-

tion (Schnittger et al. 1998, Schnittger et al. 1999, Schellmann

et al. 2002). In the cpc mutant, the number of trichomes is

increased, suggesting that CPC has a role in long-range lateral

inhibition of trichome formation in leaves (Schellmann et al.

2002).

Genetic evidence suggests that CPC and GL2 transcrip-

tion are regulated positively by WER and negatively by CPC.

However, little is known about the details of these transcrip-

tion relationships; in particular, there is no evidence of whether

WER and CPC directly regulate transcription of the target

genes or not. To understand the transcriptional regulation of

CPC, we analyzed the CPC promoter region and identified a

cis-acting element. We also evaluated the binding affinity of

transcription factors for this element.

Results

Deletion analyses of the CPC promoter

In order to understand the cell file-specific expression of

CPC, we examined the structure of the CPC promoter

sequence by constructing a series of deletions (Fig. 1B). First,

the transcription start site was identified in position –156 rela-

tive to the translational start codon by screening of an

Arabidopsis cDNA library of the root and by 5′ rapid amplifi-

cation of cDNA ends (5′ RACE) (Fig. 1A, Wada et al. 1997).

Then, we used an approximately 1.2 kb fragment from posi-

tion –1,252 to –63 as a control to examine the CPC promoter

activity, [designated pCPC(-1252); Fig. 1A, B], because this

fragment is sufficient to complement the cpc mutation when it

was fused to the CPC coding sequence (Wada et al. 1997).

When the pCPC(-1252) fragment was fused to the β-glucuroni-

dase (GUS) gene and introduced into wild-type Arabidopsis,

GUS staining was observed in hairless cells at the root meristem

(Fig. 2A, F). Weak GUS staining was observed in root stele

cells (including the pericycle and vascular tissue) (Fig. 2F). In

leaves, GUS staining was observed in trichome cells exclu-

sively (Fig. 2K). On younger developing leaves, GUS staining

was observed more broadly (Fig. 2K arrow). These GUS stain-

ing patterns were consistent with the results of in situ RNA

hybridization (Schellmann et al. 2002, Wada et al. 2002).

When the promoter was truncated up to –681 [pCPC

(-681)], GUS staining was observed in hairless cells and stele

cells in the root, and in the trichome cells in leaves (Fig. 2B, G,

L). This is the same pattern observed for full-length promoter

up to –1,252. When using the –492 promoter, GUS staining

was observed in hairless cells and stele cells, as well as trich-

ome cells (Fig. 2C, H, M). In pCPC(-492)::GUS, however, the

staining was not observed in epidermal cells near the root tip

where GUS staining was observed in pCPC(-1252)::GUS and

pCPC(-681)::GUS plants (Fig. 2A–C). About 10 epidermal

cells close to their initial cells are not stained (Fig. 2C). This

result suggests that the region between –492 and –681 of the

CPC promoter is required for CPC transcription in the early

stage of root epidermis development. When the promoter was

deleted up to –423 [pCPC(-423)], GUS staining was detected

neither in hairless cells at the root (Fig. 2D, I) nor in leaf tri-

chome cells (Fig. 2N). However, the staining remained in the

stele (Fig. 2D, I). With the –394 promoter, the GUS staining

pattern was similar to that of the –423 promoter (Fig. 2E, J, O).

These results indicate that the 69 bp region between –492 and –

423 of the CPC promoter (termed the CWB region; Fig. 1B, C)

is required for epidermis-specific transcription, specifically in

root hairless cells and leaf trichome cells. The GUS staining in

root stele cells, which was observed with the full-length CPC

promoter, was retained in all of the truncated promoters. The

region that is responsible for CPC transcription in stele cells

should be located downstream of –394.

The CWB region is sufficient for hairless cell- and trichome

cell-specific transcription

To clarify whether the CWB region between –492 and –

423 of the CPC promoter was sufficient for hairless cell- and

trichome cell-specific transcription, we constructed an 8-fold

tandem repeat of the CWB fragment fused to the 35S minimal

promoter and GUS gene, designated pCPC(CWBx8):mp::GUS

(Fig. 1D). GUS activity was preferentially observed in trich-

ome cells and hairless cells (Fig. 3A–C), and was not observed

in stele cells (Fig. 3B). These results indicate that the CWB

region is sufficient for specific expression in trichome cells and

hairless cells.

When the same promoter was connected to the CPC cod-

ing region, designated pCPC(CWBx8):mp::CPC, and the result-

ing construct was used to transform the cpc mutant, the

transformant complemented the cpc phenotype (Fig. 3E, Table

1). This result suggests that the CWB region has an important

role for the activity of CPC that leads to the development of nor-

mal root hairs. It is also suggested that CPC transcription in stele

cells is not related to the CPC function in root hair formation.


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Putative MYB-binding sites are required for epidermis-specific

transcription

In the sequence of the CWB region, we found two

putative MYB-binding sites (termed CPCMBS1 and 2; see

Materials and Methods). To determine whether these CPC-

MBSs were related to the epidermis-specific transcription of

CPC, we introduced base substitutions in both CPCMBSs (m1),

in CPCMBS2 (m2) or in CPCMBS1 (m3) to the pCPC(-492)::

GUS construct, and checked their promoter activity (Fig. 1C,

4A–F). When pCPC(m1) or pCPC(m2) was used, GUS stain-

ing was not observed in epidermal cells of roots (Fig. 4A, B, D,

E). In the case of pCPC(m3), only very weak GUS staining

could be detected in some root epidermal cells (Fig. 4F). In

leaves, we were not able to detect GUS staining in trichome

cells or in any other cells in any of the transgenic plants (Fig.

4G–I). These results indicate that the two CPCMBSs have

important roles for transcription in hairless cells and trich-

ome cells.

WER binds to the CWB region of the CPC promoter

Previous genetic studies using mutants suggested that

CPC transcription was regulated by two MYB proteins, WER

and CPC itself (Lee and Schiefelbein 2002, Wada et al. 2002).

In the wer mutant, GUS staining was reduced and rarely

observed in epidermal cells (Fig. 4J, N), whereas it was

ectopically observed in hair cells in the 35S::WER plant (Fig.

4K, O) (Lee and Schiefelbein 2002). On the contrary, the GUS

staining was observed in all of the epidermal cells in the cpc

mutant (Fig. 4L, P), whereas it disappeared from the epidermis

in the 35S::CPC transformant (Fig. 4M, Q) (Lee and Schiefel-

bein 2002, Wada et al. 2002). These results suggest that CPC

transcription is positively regulated by the WER protein and is

negatively regulated by the CPC protein itself.

To determine whether WER can directly bind to the CWB

region of the CPC promoter, we carried out a gel mobility shift

assay using WER recombinant protein. Because the full-length

WER protein could not be obtained as a glutathione S-trans-

Fig. 1 Sequence of the CPC promoter

and constructions for generating trans-

genic plants. (A) The sequence of the

CPC promoter region. The number indi-

cates the distance from initiation codon

ATG. The CWB region is underlined and

CPCMBS1 and CPCMBS2 are indi-

cated in bold. (B) A series of deletions in

the 5′ regulatory region of the CPC gene.

Each fragment is connected to the GUS

coding region and nopaline synthase ter-

minator. (C) The CWB region sequence

located from position –492 to –423 and

the modified promoters of the –492 frag-

ment which are mutated in both or either

CPCMBSs. Red stripes in pCPC(m1),

pCPC(m2) and pCPC(m3) indicate the

position where the base substitutions

were introduced. Wild-type CWB frag-

ment and m1–m4 fragments were used

for gel mobility shift assay. (D)

pCPC(CWBx8) has eight tandem repeats

of the region from –492 to –423 (arrows)

connected to the minimal CaMV 35S

promoter from –46 to –1 (box).


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ferase (GST)-fused recombinant protein (data not shown), we

used the N-terminal fragment of WER (residues 1–126; includ-

ing two MYB domains), which was expressed as a GST-fused

protein in Escherichia coli (GST–WERMYB). The DNA-bind-

ing activity of this fusion protein was tested by gel mobility

shift assays using fragments of the wild-type CWB region (Fig.

5A). A shifted band was observed (Fig. 5A, lane 2), indicating

that the MYB domain of the WER protein directly binds to the

wild-type CWB fragment. Any shifted bands were not

observed in the absence of dithiothreitol (DTT) (Fig. 5A, lane

4), indicating that the reduced state of the WER protein is

required for binding to the CWB region.

Binding of WER to the CWB region was also assayed

using the yeast one-hybrid system. Eight tandem repeats of the

CWB region were fused to the LacZ gene, and then served as a

reporter construct, designated CWBx8::LacZ. Three types of

WER corresponding to full-length WER, the N-terminal frag-

ment (including MYB domains) and the C-terminal fragment

of WER were fused at the C-terminus of the yeast GAL4 acti-

vation domain, and then served as effector constructs. When

the full-length WER or the N-terminal fragment of WER was

expressed, the LacZ expression level was increased approxi-

mately 9-fold or 5-fold compared with the vector control,

respectively (Fig. 6). These results indicate that the MYB

domain of the WER protein recognizes and binds to the CWB

region of the CPC promoter.

Fig. 2 Promoter activity of the 5′-

deleted series of the CPC promoter. GUS

activity was observed by whole mount of

the root (A–E), and transverse sections of

the root (F–J) and young leaves (K–O).

Each plant carried one of a series of CPC

promoter::GUS fusions; pCPC(-1252)::

GUS (A, F and K), pCPC(-681)::GUS

(B, G and L), pCPC(-492)::GUS (C, H

and M), pCPC(-423)::GUS (D, I and N)

and pCPC(-394)::GUS (E, J and O).

GUS staining in hairless cells in the root

was observed in (A), (B), (C), (F), (G)

and (H). The young leaves were stained

broadly in (K) (arrow). Arrowheads in (I)

and (J) indicate the lack of GUS staining

in hairless cells. Bars = 100 µm.

Fig. 3 Tandem repeats of the CWB region induce epidermis-specific

transcription. (A–C) GUS staining pattern in pCPC(CWBx8):mp::GUS

transgenic plants. GUS staining was observed in hairless cells in the

developing region of the root (A, whole mount; B, transverse section)

and developing trichome cells in leaves (C). (D–F) Complementation

test by the CPC gene driven by the CWB tandem promoter. Pheno-

types of root hairs in cpc (D), transgenic plant (E) and wild-type (F).


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We also estimated the effect of base substitutions in CPC-

MBSs of the CWB region on binding of WER by competition

gel mobility shift assay. The m1 fragment did not compete with

the labeled CWB fragment as effectively as the wild-type com-

petitor (Fig. 5B). As well as the m1 fragment, the competition

was less effective when we used the m4 fragment, in which

base substitutions were introduced more broadly than in m1

(data not shown). These results indicate that WER recognizes

the CPCMBSs of the CWB region. The m3 fragment did not

compete effectively but the m2 fragment did, indicating that

CPCMBS1 is more important for the recognition of WER than

is CPCMBS2 (Fig. 5B).

CPC does not bind to the CPC promoter but suppresses WER

binding

As described previously, the CPC protein has been sug-

gested to be a negative regulator of CPC transcription (Fig. 4L,

M, P, Q). We examined whether the CPC protein could bind to

the CWB region of the CPC promoter. When GST-fused CPC

protein was added to the CWB fragment in gel mobility shift

assays, no shifted bands were observed (Fig. 5C, lane 4). Yeast

one-hybrid assays using CWBx8::LacZ and GAL4 activation

domain-fused CPC showed no significant increase of β-galac-

tosidase activity (Fig. 6). These results suggest that CPC could

not bind to the CWB region of the CPC promoter.

However, when both full-length WER protein and CPC

protein were co-expressed in yeast, LacZ activity was lower

than in yeast with WER only (Fig. 6), suggesting that the exist-

ence of CPC interferes with the binding of WER to the CWB

region. We also tested this interference using gel mobility shift

assay. The efficiency of the shift was not affected when the

CPC protein was added in the binding solution of WER and the

CWB fragment, suggesting that CPC does not directly inter-

fere with the binding of WER to the CPC promoter. We dis-

cuss later the mechanism of interference by the CPC protein.

WER also binds to GL2 promoter

Another key player in epidermis differentiation, GL2, is

transcribed in hairless cells and trichome cells (Hung et al.

Table 1 The ratio of root hair formation per cell (%) in H

position cells (that make contact with two cortical cells) and in

N position cells (that make contact with only one cortical cells)

in wild-type, cpc mutant and transformant [pCPC(CWBx8):mp::

CPC in cpc].

H position N position

Wild-type(WS) 90.0 (n = 10) 0.0 (n = 14)

cpc 11.9 (n = 42) 0.0 (n = 39)

Transformant 89.9 (n = 148) 0.6 (n = 168)

Fig. 4 The effects of base substitutions in CPCMBSs in epidermis

and the involvement of MYB-related mutants and transgenic plants in

CPC transcription. (A–I) The GUS staining pattern of transgenic

plants that carried a mutated promoter and GUS coding sequences was

observed. In pCPC(m1)::GUS (A, D and G) and pCPC(m2)::GUS (B,

E ad H), we could not detect GUS staining either in hairless cells in

root or in leaves. Faint staining was observed in the pCPC(m3)::GUS

plant, though not in its leaves (C, F and I). (J–Q) GUS staining of

pCPC(-1252)::GUS in wer (J and N), 35S::WER (K and O), cpc (L

and P) and 35S::CPC (M and Q). Ectopic expression in hair cells is

indicated by the arrowhead in (O) and (P). Bars = 100 µm.


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1998, Szymanski et al. 1998). Genetic analyses showed that

CPC and GL2 expression is controlled by same or similar

components (Wada et al. 2002). As well as the CPC pro-

moter, the GL2 promoter has two putative MYB-binding-sites

[GL2MBS1 (GACTAACGGTAAG) and GL2MBS2 (TACTA-

ACAGTATA)], which are required for expression in trichome

cells and root hairless cells (Hung et al. 1998, Szymanski et al.

1998). To estimating whether the WER protein can also bind to

these sites, we carried out the gel mobility shift assay. When

the GST-fused MYB domain of WER protein was added, a

shifted band was observed (Fig. 5D). This indicates that WER

directly binds to the GL2MBSs in the GL2 promoter.

Discussion

CPC promoter can be divided into three regions

As shown in Fig. 2, expression of CPC is regulated in a

spatial and temporal manner. CPC transcription is restricted to

hairless cells of root epidermis and to trichome cells of leaves

at relatively early stages, and expression was not observed in

fully developed cells. A series of promoter truncations clearly

showed that the CPC promoter includes at least three function-

ally distinctive regions. The first is the CWB region located

between –492 and –424, which is responsible for hairless cell-

and trichome cell-specific expression. When we deleted this

region, epidermis-specific expression was completely absent.

The second is a region between –681 and –492 which is

involved in determining at which stage of hairless cell CPC

transcription starts. When this region was absent, expression

was not observed in hairless cells of very early stages, but was

detected in cells at later stages (Fig. 2A, C). With the full-

length promoter [pCPC(-1252)], the expression level gradually

decreased in fully developed epidermal cells. The timing of the

decrease was, however, not affected by the presence or absence

of this region (data not shown). The third region between –423

and –1 is apparently not responsible for expression in epider-

mal cells. It is suggested that this region is responsible for

expression in stele cells because the GUS staining was

observed in pCPC(-423)::GUS.

An artificial promoter combining repeats of the CWB

region and a minimal promoter [pCPC(CWBx8):mp] showed

exactly the same expression pattern as the control promoter

Fig. 5 The DNA binding properties of WER

and CPC. (A) DIG-labeled DNA of the CWB

region was used as the probe. Either GST-

fused WER MYB domain (W) or GST pro-

tein (G) was added. A 200-fold excess of

competitor was added in lane 3. (B) Sequence

specificity of the binding was checked by add-

ing base-substituted CPCMBSs as competi-

tors. See Fig. 1C for m1–m3 fragments. Each

competitor DNA was added at 20-fold or 200-

fold excess (lanes 3–10). (C) GST–CPC was

also examined for binding to the CWB region

or for inhibiting the binding of WER to the

CWB region. The CPC protein could not bind

to the CWB region (lane 4) and could not

inhibit the WER-binding activity to the CWB

region (lanes 6 and 7). (D) DIG-labeled DNA

of GL2MBS1 (lanes 1–3) or GL2MBS2 (lanes

4–6) was used as the probe. WER MYB pro-

tein was added with or without a 200-fold

excess of competitor. Arrows indicate shifted

bands and arrowheads indicate free probes.


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carrying –1,252 to –1, except for expression in stele cells. In

addition, the CPC coding sequence driven by pCPC(CWBx8):

mp complemented the cpc mutation (Fig. 3). These results con-

firmed that the CWB region is necessary and sufficient for

hairless cell- and trichome cell-specific expression, and that the

CWB region also controls the amount of CPC transcripts

required for normal differentiation of epidermal cells. Addi-

tionally, there is little possibility that other regions of the CPC

gene are responsible for the precise control of its expression.

Expression of CPC in the stele was not observed when GUS

was driven by a tandem repeat of the CWB region, suggesting

that expression in stele makes no contribution in epidermis dif-

ferentiation. It remains to be determined whether the interven-

ing space is required between the CWB region and the

transcription start site.

Two MYB protein-binding sites in the CWB region are responsi-

ble for epidermal cell-specific expression

We showed that the CWB region includes two MYB-bind-

ing sites, CPCMBS1 and 2. These sites are responsible for epi-

dermal cell-specific expression, because the specific expression

pattern was absent when the binding consensus sequence was

disrupted at either site (Fig. 4). The disruption of one site

resulting in almost complete loss of transcription in vivo sug-

gests that binding of MYB protein to one of the two sites is

insufficient and binding to both sites is required for hairless

cell- and trichome cell-specific expression, although the base

sequences of the two sites are not identical. In addition, these

results strongly suggest that it is less likely that sequences out-

side the MYB-binding sites have some role in epidermal cell-

specific expression.

We showed that WER could bind to two CPCMBSs and

two GL2MBSs. A comparison of their base sequences has

identified the octameric sequence (CC/TAACNG) as the WER

protein-binding consensus sequence. This sequence is similar

to the previously reported MYB-binding sites of maize P1 pro-

tein (CCT/AACC) (Grotewold et al. 1994, Williams and Grote-

wold 1997) and vertebrate MYB protein v-MYB (T/AAACGG)

(Howe and Watson 1991, Weston 1992). Recently, Heine et al.

(2004) showed that two cysteines in the R2 domain of the P1

MYB protein seem to form a disulfide bond in the non-reduced

state and the reduction of these two cysteines is required before

the P1 protein binds to DNA. These same two cysteines are

conserved in the R2 domain of WER and, furthermore, DTT is

required for the binding of WER to the CPC CWB region.

Therefore, it is reasonable to assume that these cysteines in

WER protein have a critical role for the ability to bind to the

CPC CWB region. In addition, it is highly likely that the GL1

protein recognizes and binds to CPCMBS sites, because a

chimeric gene combining GL1 coding sequence with the

WER promoter could complement the wer mutation (Lee and

Schiefelbein 2001). The WER RNA could not be detected in

leaves by Northern blot analysis (Lee and Schiefelbein 1999).

Therefore, the GL1 protein could replace the WER protein as a

transcriptional regulator of the CPC gene in leaves though the

binding sequence of the GL1 protein is not known.

Mechanism of the self-regulation of the CPC transcription

Previous studies reported that the epidermal cell-specific

expression of CPC is controlled by a number of proteins

including two MYB proteins, WER and CPC itself (Lee and

Schiefelbein 2002, Wada et al. 2002), two bHLH proteins, GL3

and EGL3 (Bernhardt et al. 2003), and a WD40 protein, TTG1

(Wada et al. 2002). Lee and Schiefelbein (2001) reported that

the C-terminal region 24 amino acid sequence of WER pro-

motes transcription as an activation domain in yeast. Here we

have shown that WER protein binds to the CWB region in the

CPC promoter, indicating that the WER protein has functions

of both DNA binding and transcriptional activation.

bHLH proteins are also involved in activation of CPC

transcription. Wada et al. (2002) showed that CPC transcrip-

tion was observed in both hairless and hair cell files when

maize bHLH protein R was ectopically expressed in epidermal

cells, indicating that bHLH promotes CPC expression.

Recently, Arabidopsis bHLH proteins GL3 and EGL3 were

shown to promote CPC transcription (Bernhardt et al. 2003),

and both bHLH proteins interacted with WER in yeast

(Bernhardt et al. 2003). Therefore, it could be proposed that the

bHLH proteins act as co-activators of WER through protein–

Fig. 6 WER can bind to the CWB region of the CPC promoter in

yeast. One-hybrid assays were carried out using the CWB tandem

fragment. WER and CPC were expressed with the GAL4 activation

domain in yeast. The binding activities were estimated by ONPG

assay.


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protein association. There are several reports that bHLH pro-

teins directly bind to DNA, but it is not known whether GL3

and EGL3 could directly bind to DNA and help to form the

protein–DNA complex.

WD40 domains are known to aid in protein–protein inter-

actions. A WD40 protein, TTG1, has a role in promoting CPC

transcription, because CPC expression was decreased in the ttg

mutant (Walker et al. 1999, Wada et al. 2002). Recent reports

showed that TTG1 interacts with bHLH proteins GL3 and

EGL3 (Payne et al. 2000, Esch et al. 2003, Zhang et al. 2003).

These results strongly suggest that hairless cell-specific tran-

scription of CPC is promoted by a protein complex including

WER, GL3, EGL3 and TTG1. Similar transcription-promoting

complexes of MYB, bHLH and WD40 proteins were reported

in the pigment synthesis of Arabidopsis (Baudry et al. 2004). In

this system, a MYB (TT2), a bHLH (TT8) and a WD40

(TTG1) form a protein complex, and synergistically specify the

expression of BANYULS, a key gene of proanthocyanidin bio-

synthesis.

As described above, expression of CPC is enhanced in the

cpc mutant, and is decreased in CPC overexpression lines, sug-

gesting that CPC expression is negatively self-regulated. It is

of considerable interest how CPC controls its own expression.

The binding activity of the WER protein to the CWB region

was decreased when the CPC protein was co-expressed in

yeast, suggesting that CPC has some roles in interfering with

WER function on promoting CPC transcription. From its

amino acid sequence, the CPC protein is not predicted to have

DNA binding activity. The 37th Asp, 41st Lys and 42nd Asn in

the MYB R3 domain are not conserved in the CPC R3 domain

though these amino acids in MYB proteins are essential for

sequence-specific DNA recognition (Ogata et al. 1994, Sainz et

al. 1997, Wada et al. 2002). Indeed, we showed that CPC could

not bind to the CWB region of the CPC promoter, therefore, it

is less likely that CPC competes with WER for the binding to

the CWB region. On the other hand, CPC was shown to inter-

act with the N-terminal region of GL3 and EGL3, with which

the WER homolog GL1 protein also interacts (Payne et al.

2000, Zhang et al. 2003). These indicate that CPC would com-

pete with WER for the binding to bHLH proteins and would

dissociate the WER-associated protein complex that activates

CPC expression. In yeast, we observed the interference with

the binding between the WER protein and the CWB region by

the CPC protein, and it is possible that some endogenous pro-

teins, such as bHLH or WD40 proteins, mediate this interfer-

ing mechanism. CPC protein lacks the transcription activation

domain, thus the CPC–bHLH complex could not promote CPC

expression. For the function of the CPC protein, it is also possi-

ble that some modifications are required. In addition, a recent

report showed that a CPC-like R3-type MYB protein, TRY,

inhibited binding of MYB (GL1) and a bHLH (GL3) in yeast

(Esch et al. 2003). These results suggest that a small MYB pro-

tein such as CPC or TRY works as an inhibitor of the active

transcription complex.

Regulation of GL2 transcription resembles the CPC regulatory

system

Previous analyses using mutants and overexpression lines

showed that hairless cell-specific expression of GL2 is control-

led positively by WER, TTG1, GL3 and EGL3, and negatively

by CPC (Lee and Schiefelbein 2002, Wada et al. 2002, Bern-

hardt et al. 2003, Zhang et al. 2003), which is similar to the sit-

uation of CPC expression. In this study, we demonstrated that

WER protein binds to GL2MBSs in the GL2 promoter. These

results indicate that control of GL2 transcription is likely to be

similar to CPC, namely by a protein complex including the

same MYB, WD40 and bHLH proteins. CPC protein would

disrupt the protein complex by competitive binding with WER,

and repress GL2 expression.

The question of why both GL2 and CPC are predomi-

nantly expressed in hairless cells, though CPC controls the

development of hair cells, has frequently arisen. However, this

expression pattern would be reasonable if expression of both

genes is dependent on the specific expression of WER in hair-

less cells and on the inability of the WER protein to move to

neighboring cells. CPC protein moves from the hairless cells to

the neighboring hair cells and represses GL2 expression in root

hair cells (Wada et al. 2002).

Considering that the negative function of CPC on the

expression of CPC and GL2 is based on binding competition

with WER, the ratio of available protein between CPC and

WER would be important to determine the fate of epidermal

cells. Therefore, quantitative regulation of CPC expression

would be an important addition to cell type regulation. More-

over, it was shown that GL2 regulates cell differentiation in a

dose-dependent manner (Ohashi et al. 2002), suggesting that

GL2 expression is tightly regulated. Therefore, the existence

and function of the CPC protein not only in root hair cells but

also in hairless cells are required for precise control of GL2

transcription. Further analyses of components of the transcrip-

tion complex and their dynamics will clarify mechanisms of

the epidermis determination.

Materials and Methods

Plant materials and growth conditions

We used Arabidopsis Wassilewskija (Ws) ecotype as wild type.

The seeds of the wer mutant and 35S::WER transgenic plants were

provided by Dr. John Schiefelbein. Seeds were surface-sterilized and

planted in square Petri dishes containing 1.5% agar medium as

described previously (Okada and Shimura 1990). Seeds on plates were

incubated at 4°C for 3–4 d and then the plates were placed vertically in

an incubator at 22°C under continuous white light.

Construction of chimeric genes and transgenic plants

To make a series of CPC promoter::GUS constructs, we

amplified a series of the deleted CPC promoters by PCR using primer

sets CPC-RIF (5′-ATAAAGCTTGAATTCCTGAACTTTATATC-3′)

and CPCR1B2 (5′-ATATCTAGAAGAAGCCTTGGCTTTGCTC-3′)

for pCPC(-681), CPC-D1F (5′-ATAAAGCTTCCAGAGGAGAAGC-

TACT-3′) and CPCR1B2 for pCPC(-492), CPCD1-2F (5′-ACAAAG-


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CTTTAAAATAAGTAGTTATGG-3′) and CPCR1B2 for pCPC(-423),

and CPC-D2F (5′-ATAAAGCTTTCGTTCTTCTTTCTCTATG-3′) and

CPCR1B2 for pCPC(-394). Amplified fragments were digested with

HindIII and XbaI and ligated into pBluescript SK+ (Stratagene, CA,

USA) as pBS_681, pBS_492, pBS_423 and pBS_394, respectively.

Clones were digested with HindIII–XbaI and subcloned into binary

vector pBI101 (Clontech Laboratories, Inc., CA, USA). To create

pCPC(m1)::GUS, pCPC(m2)::GUS and pCPC(m3)::GUS, the pBS_492

plasmid described above was used as a template in PCR amplification

with the following primer sets; pCPCdm296FF (5′-AGGAATT-

TTTACGCATGCAAGTCAAAGGAT-3′) and pCPCdm296RR (5′-

CGTTTACAGACAGCGATAGAAATAGTAGCT-3′) for pCPC(m1),

pCPC296RR (5′-CGTTTACAGACAGTTGGA-3′) and pCPCdm296FF

for pCPC(m2), and pCPCdm296RR and pCPC296FF (5′-AGG-

AATTTTTACAACCGC-3′) for pCPC(m3). The fragments obtained

by the PCR were self-ligated and checked with these sequences.

Obtained plasmids were then digested with HindIII–XbaI and were

subcloned into binary vector pBI101. To create a tandem repeat of the

CWB region of the wild-type CPC promoter, CWB regions were am-

plified by PCR using the primers D1XbaF (5′-ACCTCTAGACC-

AGAGGAGAAGC-3′) and C1SpeR (5′-TCCACTAGTTCCTTT-

GACTTGC-3′). This PCR fragment was cloned into the XbaI–SpeI

sites of pBluescript SK+ as pBS_CWB. Multimers of the CWB region

were produced by recursively cloning the XbaI–SpeI fragment of the

CWB region into the XbaI site of a pBS_CWB derivative, creating

pBS_pCPC(CWBx8). Digestions verified the preservation of the XbaI

site, ensuring that all CWB region replicates were in the same orienta-

tion. pCPC(m1×8), pCPC(m2×8) and pCPC(m3×8) were created by

the same procedure described above, using pCPC(m1), pCPC(m2) and

pCPC(m3) as a template. The minimal 35S promoter of cauliflower

mosaic virus (CaMV) (from –46 to –1) (Benfey et al. 1989, Fang et al.

1989) was obtained by PCR using the primer set 35S-46sl (5′-AGAG-

TCGACGCAAGACCCTTCCTCTATATAAGG-3′) and glcSnaB1 (5′-

TCAGTTCGTTGTTCACACAAACGG-3′), with pBI221 (Clonteth)

as a template. The PCR product was digested with SalI and SnaBI,

cloned into pBI221, and designated pBI_minimalGUS. The tandem

repeats of the CWB region obtained above were subcloned into

pBI_minimalGUS and then into binary vector pBI101 (Clontech).

These binary plasmids were introduced into Agrobacterium tumefaciens

strain C58C1 (pGV2260) or C58C1 (pMP90) by electroporation using

a Gene Pulser (Bio-Rad Hercules, CA, USA). Transformation into

plants was performed by a vacuum transformation procedure (Bechtold

et al. 1993) or a floral dip method (Clough and Bent 1998) and select-

ed on a 0.5× MS agar plate containing 30 mg l–1 kanamycin.

Histological analysis

Primary roots of 5-day-old seedlings of transgenic plants were

excised and immersed in X-Gluc solution containing 1.0 mM X-

Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide), 1.0 mM K3Fe(CN)

6,

1.0 mM K4Fe(CN)

6, 100 mM NaPi (pH 7.0), 100 mM EDTA and 0.1%

Triton X-100. They were incubated under reduced pressure for 5 min

at room temperature, and then at 37°C for 5 h. For transverse sections,

stained samples were embedded in 5% low-melting point agarose

(Gibco-BRL) and sectioned to 50 mm thick at about the 150–250 mm

region from the root tip with a vibrating blade microtome VT1000 S

(Leica). Samples of seedlings and leaves were pre-fixed with ice-cold

90% acetone, stained as described above, and cleared with 70% ethanol.

Bacterial expression of proteins and purification of GST-fused proteins

The WER coding sequence was amplified by PCR using the

primers WERSmaF (5′-CGGCCCGGGAATGAGAAAGAAAGTAA-

GTAg-3′) and WERER1B (5′-ATAGAATTCTCAAAAACAGTGTC-

CATCTA-3′). Total root cDNA was used as a template. The PCR

fragment was digested with EcoRI and SmaI, and cloned into expres-

sion vector pGEX-2T (Amersham Biosciences Corp., NJ, USA),

designated pGEX_WER and used to transform E. coli strain Rosetta

(Novagen, WI, USA). To generate GST–MYBWER protein,

pGEX_WER was used as a template for PCR amplification with

primers WERR3R1 (5′-TTTCTGATCTTTGATTCCGA-3′) and

pGEX2TstopER1 (5′-TGAGAATTCATCGTGACTGA-3′) The PCR

product was self-ligated, its sequence was checked and it was trans-

formed into E. coli strain Rosetta. These transformed bacteria were

used for purifying the recombinant proteins based on a standard

method (Sambrook and Russell 2001). The GST–CPC fusion protein

used for this study is the same as reported previously (Wada et al.

2002).

Gel mobility shift assay

Oligonucleotides for gel mobility shift analysis were labeled with

a Roche DIG Gel Shift Kit, 2nd Generation (Roche, Germany). See

Fig. 1 for CPC CWB fragments. Sequences of GL2MBS1 and 2 are 5′-

TTCAACAACTCTTCTTTCCTGCCTTTACCGTTAGTCTAATTGTT-

TTCCTAATACTGCTAC-3′ and 5′-GGTTCCATGTAGCGCTGCA-

ATATACTGTTAGTAACTCTCTCTTACCCATATATTGTATAT-3′, re-

spectively. The DNA–protein binding reaction was basically per-

formed by incubating 32 fmol of digoxigenin (DIG)-labeled oligo-

nucleotide with 100 ng of each protein in 20 µl of binding buffer

[10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 10 mM DTT,

5% glycerol, 80 µg ml–1 poly(dI–dC) and 100 µg ml–1 bovine serum

albumin (BSA)]. The reaction solution was incubated at 22°C for

15 min, and then free and bound complexes were resolved by electro-

phoresis through 1 mm thick 6% native polyacrylamide gels (80 : 1

acrylamide : bisacrylamide) in 0.25 TBE buffer at 8 V cm–1 for

60 min.

Yeast one-hybrid analysis

Yeast one-hybrid analyses were carried out based on a MATCH-

MAKER One-Hybrid System (Clontech). To make the reporter

construct, pBS_pCPC(CWBx8) was digested with XbaI and blunted,

then digested with SalI, and subcloned into the SmaI and SalI sites

of pLacZi (Clontech). To make the effector construct, the WER and

CPC cDNA fragments were amplified by PCR using primers

WERNco1F (5′-GGGCCATGGGGATGAGAAAGAAAGTAAGTA-3′)

and WERXhoR (5′-CCCCTCGAGTCAAAAACAGTGTCCATCTAT-

3′) or CPCNcoF (5′-ATACCATGGGGATGTTTCGTTCAGACAAGG-

3′) and CPCXhoR (5′-CCCCTCGAGTCATTTCCTAAAAAAGTC-

TCT-3′). The fragments obtained by PCR were digested with NcoI and

XhoI and cloned into yeast expression vector pACT2 (Clontech),

designated pACT2_WERFL or pACT2_CPC, respectively. To make

pACT2_WERN and pACT2_WERC, pACT2_WERFL was used as

a PCR template with primers WERR3R1 and pACT2stopXho1 for

pACT2_WERN, and pACT2NcoR and WER 127aaF1 for

pACT2_WERC. PCR products were self-ligated and sequenced. To

make the expression plasmid which was constructed to express both

WER and CPC, the pADH1::GAL4AD:CPC:termADH1 fragment was

released from pACT2_CPC with NotI and Tth111I and blunted at both

ends, and ligated into pACT2_WER at a blunted SalI site. One-hybrid

experiments were performed according to the Clontech Yeast Proto-

cols Handbook using yeast strain YM4271 (Clontech).

Computer program for searching transcription factor-binding sites

For searching transcription factor-binding sites, the TRANSFAC

database (Heinemeyer et al. 1998) was used through the TFSEARCH

program (Y. Akiyama, ‘TFSEARCH: Searching Transcription Factor

Binding Sites’, http://mbs.cbrc.jp/papia/).


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http://mbs.cbrc.jp/papia/



Acknowledgments

We would like to acknowledge funding from the Japanese Minis-

try of Education, Culture, Sports, Science and Technology and from

the Core Research of Science and Technology (CREST) Research

Project. K.O. and Y.K. were supported by The Grant for the Biodiver-

sity Research of the 21st Century COE (A14). Y.K. was supported as a

Junior Research Associate by RIKEN from 2000 to 2004.

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(Received February 21, 2005; Accepted March 23, 2005)


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Full Title of the paper Regulation of CAPRICE Transcription by MYB