Organization and promoter analysis of the zebrafish(Danio rerio) chemokine gene (CXC-64) promoter

Li-Chen Chen Æ Jen-Leih Wu ÆChyuan-Yuan Shiau Æ Jyh-Yih Chen

Received: 17 November 2008 / Accepted: 24 March 2009 / Published online: 18 April 2009

� Springer Science+Business Media B.V. 2009

Abstract Zebrafish CXC-64, a chemokine repre-

senting a superfamily of chemotactic cytokines

present in fish, is involved in recruitment, activation,

and response to inflammatory stimulation. We cloned

and sequenced the genomic DNA of the zebrafish

CXC-64 chemokine; it was most similar to CXCL11

from humans and CXCL10 from a catfish. The

zebrafish CXC-64 gene is approximately 4.0 kb long

and has a four-exon, three-intron structure common

to the human CXCL11 gene. However, the promoter

region includes a typical TATA box and multi-

transcription factor-binding sequences. To understand

the roles of lipopolysaccharide (LPS), poly I:poly C,

and tumor necrosis factor (TNF)-a in regulating

zebrafish CXC-64 expression, serial deletions were

made in the promoter region of this clone. Different

fragments of the zebrafish CXC-64 50-flanking region

were transfected into RAW264.7 (mouse macro-

phage; Abelson murine leukemia virus transformed)

and zfl (zebrafish liver) cells and then treated with 0,

10, 50, 100, and 200 ng/ml LPS, poly I:poly C, or

TNF-a. The results showed that the promoter activity

presented dose-dependent effects in LPS-treated

RAW264.7 cells, TNF-a-treated RAW264.7 cells,

and LPS-treated zfl cells. These results reveal that the

zebrafish CXC-64 chemokine gene promoter region

can be induced by LPS in both human and fish cell

lines, which suggests that it plays an important role in

regulating LPS.

Keywords Zebrafish � Chemokine �Gene expression � Functional analysis

Introduction

Chemokines are a superfamily of approximately 40

different cytokines that play important roles in

immunity (Secombes et al. 2001). These chemokines

can be divided into four distinct structurally related

subgroups [CXC (a), CC (b), C (c), and CX3C (d)]

according to the arrangement of the first two cysteine

residues (Laing and Secombes 2004). Chemokines

have been reported in catfish, haddock, carp, rainbow

trout, flounder, and zebrafish (Lee et al. 2001;

Peatman and Liu 2006; Kim et al. 2007). Specific

biological functions of chemokines in fish have not

L.-C. Chen � C.-Y. Shiau

Department of Food Science, National Taiwan Ocean

University, Keelung 202, Taiwan

L.-C. Chen

Department of Food Science, National I-Lan University,

Ilan 260, Taiwan

J.-L. Wu (&)

Institute of Cellular and Organismic Biology, Academia

Sinica, Nankang, Taipei 115, Taiwan

J.-Y. Chen (&)

Marine Research Station, Institute of Cellular and

Organismic Biology, Academia Sinica, 23-10 Dahuen

Road, Jiaushi, Ilan 262, Taiwan

e-mail: zoocjy@gate.sinica.edu.tw

123

Fish Physiol Biochem (2010) 36:511–521

DOI 10.1007/s10695-009-9321-y

yet been identified by promoter activity analyses. In

mammalian species, CXCL9, CXCL10, and CXCL11

share significant levels of sequence similarity and

share the same CXCR3 receptor (Baoprasertkul et al.

2004). CXCL9, CXCL10, and CXCL11 are involved

in interferon (IFN)-induced chemokine recruitment-

activated T-cells through mobilization of calcium and

chemotaxis (Baggiolini et al. 1997).

We recently characterized and analyzed five

zebrafish chemokine genes named CXC-46 (acces-

sion no.: XM_685640), CXC-56 (no.: XM_690954),

CXC-64 (no.: XM_687326), CXC-66 (no.:

XM_001339337), and scyba (no.: AF279919) (Chen

et al. 2008) following our previous publication. All

five zebrafish CXC chemokines are expressed in a

wide range of tissues at different expression levels.

Specifically, phylogenetic analyses revealed that

zebrafish CXC-64 is closest to catfish CXCL10 and

human CXCL11. Zebrafish CXCL-64 showed a

significant difference after LPS treatment for 6 and

72 h (Chen et al. 2008). Those results indicated that

the zebrafish CXCL-64 chemokine may play an

important role in fish immunology. The molecular

structure of CXCL11 was initially determined after

isolation from humans (Tensen et al. 1999). The

CXCL11 complementary (c)DNA sequences of many

vertebrate species have now been published (Widney

et al. 2000; Laing et al. 2002), yet no reports on

molecular regulation or promoter analysis are avail-

able for fish.

With the exception of reports of zebrafish genomic

DNA sequences from the Sanger Institute (http://

www.sanger.ac.uk/), research on fish chemokines has

been limited to functional analyses. Most studies

have been restricted in their scope to examining

genomic DNA sequences. Only one study of catfish

attempted to analyze the expressions and exon

structures of 23 CC chemokine genes within a teleost

species (Bao et al. 2006). From our previously

described experimental results, the lack of a draft

genomic DNA sequence of CXC-64 in zebrafish

rendered functional analysis more difficult than that

faced by those with published genomes, because a

CXC-64 genomic DNA sequence included in geno-

mic clones is required as a first step. In this study, we

used a zebrafish CXC-64 cDNA fragment as a probe

to screen a zebrafish genomic DNA library and

obtained a few clones of CXC-64. Our studies pro-

vide new insights suggesting that lipopolysaccharide

(LPS), poly I:poly C, and tumor necrosis factor

(TNF)-a are involved in regulating CXC-64 promoter

activity.

Materials and methods

Cloning, sequencing, and construction

of the zebrafish CXC-64 promoter

Screening of 1 9 106 recombinant bacteriophages

from a zebrafish genomic DNA library (Mobitec

Molecular Biotechnology, Germany) was carried out

with 32P-labeled CXC-64 cDNA as a probe. Follow-

ing our cloning procedure from the zebrafish cDNA

library, we obtained ten clones that were identified by

sequencing to be a zebrafish CXC-64 clone. Ten

fragments were used for the promoter analysis of the

DNA constructs in this study. These constructs were

produced by ligating 0.4, 0.8, 1.2, 1.6, and 2.1-kb

DNA fragments into a pGL3-basic vector with the

SacI and SmaI restriction endonuclease sites. The

constructs were divided into two types, one contain-

ing the 50 untranslated region (UTR; 400A, 800A,

1200A, 1600A, and 2100A) and the other not

containing the 50UTR region (400U, 800U, 1200U,

1600U, and 2100U).

Promoter activity analysis

RAW264.7 (mouse macrophage; Abelson murine

leukemia virus transformed) and zebrafish liver (zfl,

ATCC CRL-2643) cell-line culture conditions fol-

lowed those of a previous report (Chen et al.1998).

Briefly, zfl cells were grown in 50% Leibovitz’s L-15

medium with 2 mM L-glutamine (Vitacell 30-2008),

35% Dulbecco’s modified Eagle’s medium (DMEM)

with 4.5 g/l glucose, 4 mM L-glutamine (Gibco

12100, Grand Island, NY, USA), and 15% Ham’s

F12 with 1 mM L-glutamine (Gibco 21700; none with

sodium bicarbonate), supplemented with 0.15 g/l

sodium bicarbonate, 15 mM HEPES 0.01 mg/ml

insulin (Sigma no. I-1882, St. Louis, MO, USA),

50 ng/ml endothelial growth factor (EGF), and 5%

DMEM (4.5 g/l glucose) containing 10% newborn

calf serum. Approximately 5 9 105 cells were seeded

in each well 12 h before transfection. Liposome DNA

transfection was carried out as described previously

(Chen et al. 1998). Typically, transfection lasted

512 Fish Physiol Biochem (2010) 36:511–521

123

12 h. Promoter activity was measured 24 h after

transfection and was quantified as described previ-

ously (Chen et al. 1998). Different fragments of the

zebrafish CXC-64 50-flanking region were transfected

into RAW264.7 and zfl cells and then treated with 0,

10, 50, 100, and 200 ng/ml LPS, poly I:poly C, or

TNF-a. For other experiments, a treatment group was

treated with or without LPS (100 ng/ml), poly I:poly

C (50 ng/ml), or TNF-a (50 ng/ml). After completing

transfection, cells were either treated with each drug

or left untreated, and were then incubated. After 24 h,

luciferase expression by the cells was analyzed using

the 10-Pack dual-luciferase reporter assay system

(Promega, Madison, WI, USA). Cells were lysed,

after which the resulting lysate was transferred to a

96-well plate and measured in a Fluoroskan Ascent

FL luminometer (Thermo Labsystems, Ramsey, MN,

USA).

Data are presented as mean ± standard error of

the mean (SEM) from at least three independent

experiments. A significant difference (*P \ 0.05)

was determined by comparing treated groups with the

untreated group.

Results

Characterization of the zebrafish chemokine

CXC-64 gene structure

In the zebrafish CXC-64 chemokine gene, the first

exon encodes a mature peptide and 50 UTR

sequences. The mature peptide is encoded by exons

1, 2, 3, and 4. The nucleotide sequence of the

zebrafish CXC-64 chemokine gene is shown in

Fig. 1. The gene contains four exons, and the exon

structure is dissimilar to that of mouse CXCL11

(from Sanger Institute information). All four exons

and three introns were sequenced, and the genomic

DNA sequence was compared with the zebrafish

chemokine CXC-64 cDNA sequence. The compara-

tive results showed that all introns possessed the

classic 50 GT/AG 30 exon-intron splice motif (Fig. 1).

Analysis of the zebrafish CXC-64 chemokine gene

promoter region using the TFSEARCH program

(http://molsun1.cbrc.aist.go.jp/research/db/TFSEARC

H.html) revealed several conserved sequence

elements that are likely to be binding sites for

transcription factors. These putative transcription

Fig. 1 Nucleotide sequence of the cloned and sequenced

zebrafish CXC-64 chemokine gene containing part of the 50

untranslated region (UTR) and promoter region. Consensus

transcription factor binding sites and PCR primers are

indicated by underlining. Uppercase letters represent the

50UTR

Fish Physiol Biochem (2010) 36:511–521 513

123

factor-binding sites suggest their importance in zeb-

rafish CXC-64 chemokine gene regulation. The zeb-

rafish CXC-64 chemokine gene showed several

putative transcriptional factor-binding sequences,

for example GATA-1, C/EBP, Oct-1, and TATA.

Figure 1 shows the sequence of approximately 2.3 kb

50 upstream of the first methionine sequence con-

taining the zebrafish CXC-64 chemokine promoter

sequence as determined in this paper. Computer-

assisted inspection of the 50-region immediately

upstream of the zebrafish CXC-64 chemokine coding

regions and comparisons with the promoter regions of

human and mouse CXC chemokines revealed no

consensus sequence motifs in the zebrafish CXC-64

promoter region.

Fig. 1 continued

Fig. 1 continued

514 Fish Physiol Biochem (2010) 36:511–521

123

Determination of promoter activity

of the zebrafish CXC-64 chemokine gene

To understand the promoter activity after treatment

with LPS, poly I:poly C, and TNF-a in RAW264.7

(ATCC TIB-71) and zfl cells (ATCC CRL-2643), we

constructed sequential 50 deletions of the putative

promoter region including or not including the

flanking transcription start sites described above

(Fig. 1) ligated with the luciferase coding sequence.

In the first experiment, we attempted to determine

appropriate conditions for the promoter analysis by

treating RAW264.7 (Fig. 2) and zfl cells (Fig. 3) with

the 2100U fragment. Data in Figs. 2 and 3 reveal that

the promoter activities of the 2100U fragment after

being treated with LPS (Figs. 2a, 3a), poly I:poly C

(Figs. 2b, 3b), and TNF-a (Figs. 2c, 3c) presented

dose-dependent effects in LPS-treated RAW264.7

cells (Fig. 2a), TNF-a-treated RAW264.7 cells

(Fig. 2c), and LPS-treated zfl cells (Fig. 3a). The

results described above reveal that the 2100U frag-

ment of the zebrafish CXC-64 chemokine gene

promoter region could be induced by LPS in both

human and fish cell lines, suggesting its importance

in regulating LPS.

Deletion of the fragment from 2100U to 400U

increased the luciferase activity after LPS treatment

for 24 h in this test with RAW264.7 cells. In contrast,

minimal promoter activity was generated with the

promoter region including the 50UTR region of the

800A, 1200A, 1600A, and 2100A fragments

(Fig. 4a), although the promoter activity of 400A

was much higher. After treatment with poly I:poly C,

the promoter activity in each fragment showed

elevated activity with 6 h of treatment compared

with treatment for 24 h (Fig. 4b). However, the

promoter activity of the promoter fragment which

included the 50UTR was lower than that of the

promoter fragment which did not include the 50UTR,

Fig. 1 continued

0

1

2

3

4

5

6

7

0 10 50 100 200

0 10 50 100 200

0 10 50 100 200

LPS (ng/ml)

Act

ivit

y F

old

b b

a aa

0

2

4

6

8

10

poly I:C (ng/ml)

Act

ivit

y F

old

cbc

d

ba

0

2

4

6

8

10

TNF-α (ng/ml)

Act

ivit

y F

old

bcba

c d

a

a

b

c

Fig. 2 Analysis of zebrafish CXC-64 chemokine promoter

activity in RAW264.7 cells. Cells were treated with 0 (basal),

10, 50, 100, and 200 ng/ml lipopolysaccharide (LPS) (a); 0

(basal), 10, 50, 100, and 200 ng/ml poly I:poly C (b); or 0

(basal), 10, 50, 100 and 200 ng/ml tumor necrosis factor

(TNF)-a (c). The data, which were normalized for transfection

efficiency against the secreted Renilla luciferase activities

using the Dual-Glo luciferase assay reagent (Promega), are

expressed as multiples of change in the activity of the pGL3

control vector. The control was the pGL3-basic vector group.

Each bar represents the mean value from three determinations,

with the standard error (SE). Data (mean ± SE) with differentnumbers differ significantly (P \ 0.05) among treatments

Fish Physiol Biochem (2010) 36:511–521 515

123

except for the activities of fragment 400A treated

with poly I:poly C and TNF-a (Fig. 4b, c).

The promoter activity of fragment 400U in zfl cells

was lower than that of the 400A promoter region after

treatment with LPS, poly I:poly C, and TNF-a(Fig. 5a–c). After treatment with LPS, the promoter

activity in each fragment showed elevated activity

with time-dependent effects (Fig. 5a). Therefore, LPS

treatment results differed from those with poly I:poly

C treatment. poly I:poly C treatment presented

reduced promoter activity compared with the

untreated group after 6 and 24 h of treatment with

1200U and 1600U in zfl cells (Fig. 5b). TNF-atreatment with 800U, 1200U, and 1600U in zfl cells

produced decreases following 6–24 h of treatment

(Fig. 5c).

Discussion

In this study we sequenced the zebrafish CXC-64

chemokine gene. This is the first fish chemokine gene

to be characterized at the genomic level with a

promoter analysis. We previously conducted phylo-

genetic analyses of fish chemokines and concluded

that the zebrafish CXC-64 chemokine may be

involved in LPS regulation (Chen et al. 2008). As

shown in Fig. 1, the zebrafish CXC-64 chemokine is

composed of four exons and three introns. The typical

ag/gt rule was followed for the acceptor and donor

sites. In the genomic structure of carp chemokine that

has three exons, the exon number and intron numbers

were not similar to those in zebrafish. Although we

used a carp chemokine to examine the genomic

database of zebrafish which seemed similar to that of

a previously published carp chemokine gene structure

(Savan et al. 2003), our sequence analysis showed

that the genomic structures were not similar between

carp and zebrafish. Our sequence analysis revealed

that the structure of the zebrafish CXC-64 chemokine

coding region was very similar to that of the human

CXCL11 gene (Tensen et al. 1999), suggesting that

both genes came from a common ancestral gene.

The 50 flanking region of the zebrafish chemokine

CXC-64 gene contains multiple consensus sequences

for transcription factor-binding sites such as Oct-1,

C/EBP, Nkx-2, etc.; this structure is very common in

promoter regions of immunity-related genes with

constitutive expression in several tissues (Garcıa-

Moruja et al. 2005). In comparison with other

promoters harboring a non-canonical TATA box,

C/EBP may be critically involved in initiating and

sustaining zebrafish CXC-64 chemokine gene tran-

scription. In our results of the zebrafish CXC-64

chemokine promoter activity analysis, the functional

activity of the promoter fragments seemed to be

upregulated by LPS stimulation and downregulated

by TNF-a in RAW264.7 cells. But in liver cells

(zebrafish zfl cells), only zebrafish CXC-64

0

0.05

0.1

0.15

0.2

0.25

0 10 50 100 200

0 10 50 100 200

0 10 50 100 200

LPS (ng/ml)

Act

ivit

y F

old

a

a

b

c

a

cbc b

0

0.2

0.4

0.6

0.8

1

poly I:C (ng/ml)

Act

ivit

y F

old b

ab

c

ab a

0

0.2

0.4

0.6

0.8

1

TNF-α (ng/ml)

Act

ivit

y F

old

bb

c

ab

Fig. 3 Analysis of zebrafish CXC-64 chemokine promoter

activity in zfl cells. Cells were treated with 0 (basal), 10, 50,

100, and 200 ng/ml lipopolysaccharide (LPS) (a); 0 (basal), 10,

50, 100, and 200 ng/ml poly I:poly C (b); or 0 (basal), 10, 50,

100, and 200 ng/ml tumor necrosis factor (TNF)-a (c). The

data, which were normalized for transfection efficiency against

the secreted Renilla luciferase activities using the Dual-Glo

luciferase assay reagent (Promega), are expressed as multiples

of change in the activity of the pGL3 control vector. The

control was the pGL3-basic vector group. Basal levels are

presented by the untreated group. Each bar represents the mean

value from three determinations, with the standard error (SE).

Data (mean ± SE) with different numbers differ significantly

(P \ 0.05) among treatments

516 Fish Physiol Biochem (2010) 36:511–521

123

0

10

20

30

40

50

60

400U

800U

1200

U

1600

U

2100

U40

0A80

0A

1200

A

1600

A

2100

A

Act

ivat

ion

fo

ld

No treatment

LPS treatment-6hr

LPS treatment-24hr**

1 1

2

1 1 1 1 1 11 1 1 1

22

2

2 2

0

10

20

30

40

50

60

400U 800U 1200U 1600U 2100U 400A 800A 1200A 1600A 2100A

400U 800U 1200U 1600U 2100U 400A 800A 1200A 1600A 2100A

Promoter length (bp)

Promoter length (bp)

Promoter length (bp)

Act

ivat

ion

fo

ld

No treatment

poly I:C treatment-6hr

poly I:C treatment-24hr

*

*

1 12

13

12

12

1 2 1 2

2

12 2 1

3

1

1 3

2

2

*

*

*

*

**

* ***

**

*1 2 1

1

2

2

0

5

10

15

20

25

Act

ivat

ion

fo

ld

No treatment

TNF-α treatment-6hr

TNF-α treatment-24hr

**

1

321 1 1

1

1

2

1 2 1 2

2

2

2

3 2

3

1

1 3

2

2

**

*

*

*** *

*

*

*

**

1 2 3

a

b

c

Fig. 4 Analysis of

zebrafish CXC-64

chemokine promoter

activity in RAW264.7 cells

on lipopolysaccharide

(LPS) (a), poly I:poly C

(b), or tumor necrosis factor

(TNF)-a (c) treatment.

RAW264.7 cells were

transfected with the various

constructs, and luciferase

activity was determined as

described in ‘‘Materials and

methods’’. The constructs

were divided into two types,

one containing the 50

untranslated region (UTR)

(400A, 800A, 1200A,

1600A, and 2100A) and the

other not containing the

50UTR region (400U, 800U,

1200U, 1600U, and

2100U). Each barrepresents the mean value

from three determinations,

with the standard error (SE).

Data (mean ± SE) with

different numbers differ

significantly (P \ 0.05)

among treatments

Fish Physiol Biochem (2010) 36:511–521 517

123

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

400U

800U

1200

U

1600

U

2100

U40

0A80

0A12

00A

1600

A

2100

A

Act

ivat

ion

fo

ld

No treatment

LPS treatment-6hr

LPS treatment-24hr

*

1

2

1 2

12

11 1

3

2

21

22

12

2

1

21 1

2

11

2

0

0.5

1

1.5

2

2.5

3

3.5

4

400U 800U 1200U 1600U 2100U 400A 800A 1200A 1600A 2100A

Promoter length (bp)

400U 800U 1200U 1600U 2100U 400A 800A 1200A 1600A 2100A

Promoter length (bp)

Promoter length (bp)

Act

ivat

ion

fo

ld

No treatment

poly I:C treatment-6hr

poly I:C treatment-24hr

*

*

1

221 1 1

31

2 1 222 3

3

3

1

13

2

2

*

***

***

***

** 1

2

31

2

1

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Act

ivat

ion

fo

ld

No treatment

TNF-α treatment-6hr

TNF-α treatment-24hr

** 1

3

21

3 13

1 22

2 3

2

1

1 3

1

2

* * ***

*

**

* ***1 2 3

a

b

c

Fig. 5 Analysis of

zebrafish CXC-64

chemokine promoter

activity in zfl cells on

lipopolysaccharide (LPS)

(a), poly I:poly C (b), or

tumor necrosis factor

(TNF)-a (c) treatment. zfl

cells were transfected with

the various constructs, and

luciferase activity was

determined as described in

‘‘Materials and methods’’.

The constructs were divided

into two types, one

containing the 50

untranslated region (UTR;

400A, 800A, 1200A,

1600A, and 2100A) and the

other not containing the

50UTR region (400U, 800U,

1200U, 1600U, and

2100U). Each barrepresents the mean value

from three determinations,

with the standard error (SE).

Data (mean ± SE) with

different numbers differ

significantly (P \ 0.05)

among treatments

518 Fish Physiol Biochem (2010) 36:511–521

123

chemokine promoter activity was upregulated by LPS

stimulation. In contrast, recent work has revealed that

the murine gene, which is homologous with the

human T cell, attracted chemokine CXC receptor

ligand 11, suggesting that macrophage-like

RAW264.7 cells and stimulants (interferons, LPS,

IL-1 beta, and TNF-a) can increase CXCL9,

CXCL10, and CXCL11 mRNA levels by up to

10,000-fold according to a real-time PCR analysis

(Meyer et al. 2001). From chemokine expressions in

the liver, chemokines, chemotactic cytokines that

attract leucocytes to inflammatory sites, may be

important in the development of intrahepatic inflam-

mation (Zeremski et al. 2007). Our results confirmed

previous observations described above which specu-

lated on zebrafish CXC-64 chemokine promoter

activity in RAW264.7 and zfl cells. The results were

confirmed by LPS and TNF-a treatments at different

concentrations in both RAW264.7 and zfl cells. The

activity of the negative control in LPS (0 ng/ml)

stimulation was quite low compared with the results

of poly I:poly C or TNF-a stimulation. These results

suggest that zebrafish CXC-64 chemokine promoter

activities in RAW264.7 and zfl cells were induced by

LPS treatment and suggest that the zebrafish CXC-64

gene can serve as a pathogenic infection indicator

according to our promoter activity assay and mRNA

expression analysis of LPS induction (Chen et al.

2008).

One of the interesting features of the CXCL11

gene is that it is produced in transplants by infiltrating

macrophages and by donor endothelial cells in rats.

Infiltrating macrophages secrete CXCL11, which has

chemotactic properties for CD4? T cells. IFN-c and

TNF-a are secreted by CD4? T cells, and these

cytokines induce macrophages to secrete CXCL11

(Mitsuhashi et al. 2007). Subsequently, human

monocytes secrete significantly higher levels of

CXCL10 and CXCL11 when stimulated by wild-

type Neisseria meningitidis organisms, and the LPS

in the outer membrane of N. meningitidis plays a

dominant role as an inflammation-inducing molecule

in meningococcal disease (Ovstebø et al. 2008).

Supporting this, LPS-induced CXCL11 gene expres-

sion and may stimulate CXCL11 gene promoter

activity. When gastric epithelial cell lines were used

to characterize the constitutive and regulated expres-

sion of 3 CXC chemokines (IP-10, I-TAC, and Mig)

in response to IFN-c, TNF-a, and different

Helicobacter pylori preparations, results suggested

that IFN-c synergizes with TNF-a to induce upreg-

ulation of CXC chemokine secretion in gastric

epithelial cells (Kraft et al. 2001).

Some of the previously described promoters for

CXC chemokines (I-TAC, IL-8, IP-10, and GCP-2)

contain active binding sites for nuclear factor (NF)-

jB and NF-IL6 associated with activation in response

to inflammatory stimuli (TNF-a) (Smale 1997;

Garcıa-Moruja et al. 2005), but this was not the case

for the zebrafish CXC-64 chemokine 50 flanking

region, in which none of these transcription factor-

binding sequences was detected. These experimental

results were confirmed and extended by our results;

treatment with LPS, poly I:poly C, and TNF-amodified zebrafish CXC-64 chemokine basal pro-

moter activity (400U and 400A promoter fragments),

and when treatment times were extended from 6 to

24 h higher promoter activity was observed in

transfected cell lines from RAW264.7 and zfl cells.

We knew that the constructs were divided into two

types, one containing the 50 untranslated region

(UTR) (400A, 800A, 1200A, 1600A, and 2100A)

and the other not containing the 50UTR region (400U,

800U, 1200U, 1600U, and 2100U). Both the 400A

and 400U promoter fragments showed high promoter

activity in each treatment, suggesting that an

enhancer exists in the 400A and 400U promoter

regions. Transfection experiments using a collection

of deleted fragments of the zebrafish CXC-64

chemokine promoter showed that sequences located

between -1 and -400 are especially important for

promoter activity, because a deletion from -2100 to

-400 dramatically upregulated promoter activity. In

particular, this region contains binding elements for

GATA-1, IRF-1, AP-1, and DEF cis-acting proteins

(Fig. 1). The GATA-1 transcription factor is impli-

cated in regulating CCR3 gene expression (Zimmer-

mann et al. 2005), and interferon regulatory factor

(IRF)-1, as assayed by chromatin immunoprecipita-

tion assays, which demonstrated that IFN treatment

of Daudi and DRST3 cells induced STAT3 binding to

the CXCL11 promoter region (Yang et al. 2007).

Our data clearly show that zebrafish CXC-64

chemokine promoters have similar luciferase activi-

ties in zfl cells and RAW264.7 cells after LPS

treatment (Figs. 4a, 5a). Differences in luciferase

activities in RAW264.7 cells after poly I:poly C

treatment were only significant for the 400U, 800U,

Fish Physiol Biochem (2010) 36:511–521 519

123

1200U, and 400A fragments after 6 h of treatment

(Fig. 4b). But in zfl cells, we did not observe similar

results. Interestingly, promoter activities of 1200U

and 400A were markedly low in zfl cells after TNF-atreatment compared with in RAW264.7 cells, except

for the respective 400U fragments (Figs. 4c, 5c). This

indicates that zebrafish CXC-64 chemokine promoter

regulation by transcription factors might not be under

the control of lymphoma cell transcription factors,

although RAW264.7 cells can express antimicrobial

peptides of LL-37/hCAP-18, and protein products

may induce chemokine expressions after LPS treat-

ment (Yang et al. 2003). One possible explanation of

these results is that the zebrafish CXC-64 chemokine

may be selectively produced in the zebrafish liver but

not lymphoma cells, suggesting that regulation of

gene expression by zebrafish CXC-64 chemokine

differs across cell lines. However, the relative

importance of zebrafish CXC-64 chemokine pro-

moter activity and the putative interaction between

cis or trans elements described herein remain to be

determined.

The physiological function of the zebrafish CXC-64

chemokine in immunity and its promoter activity are

not fully understood. In addition, the regulatory

mechanism of transcription factor interactions in fish

tissues remains to be determined. Furthermore, it will

be interesting to see whether any differences between

humans and fish can be identified in immunity

reactions in CXC-64 chemokine promoter regulation.

Acknowledgments This work was supported by a grant from

the Marine Research Station, Institute of Cellular and

Organismic Biology, Academia Sinica.

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