Yonago Acta medica 2002;45:1–8

Genomic Organization of the Human Arpp Gene
Akiko Miyazaki*†, Yoshiyuki Tsukamoto*, Kenzo Sato*, Shigetsugu Ohgi† and
Masatsugu Moriyama**Department of Molecular Biology, and †Second Department of Surgery, Tottori University
Faculty of Medicine, Yonago 683-8503 Japan

A novel ankyrin-repeated protein, Arpp, is specifically expressed in skeletal and cardiac
muscles. Arpp protein is homologous, in its amino acid sequences (52.7% identity), to
Carp protein which is proposed to be a putative genetic marker for cardiac hypertrophy.
In this study, we isolated the human Arpp gene by screening a human genomic library
and analyzed the genomic structure and its 5' flanking region. The Arpp gene wasfound to encompass a sequence of 11 kb and to consist of 9 exons. The translational
initiation site and the stop codon were found to be located at exon 1 and exon 9, respectively. Each exon from 5 to 8 was found to encode 1 of the 4 ankyrin-like domains,respectively. The 2.7 kb upstream of exon 1 was sequenced. The TATA box was identified 29 bp upstream of the transcriptional start site, and multiple putative regulatory
elements including the E box and upstream stimulating factor-1 were distributed within
the proximal promoter regions. Since these elements were also found in the promoter
region of the mouse Arpp gene, they may play an important role in the transcriptional
regulation of both human and murine Arpp genes.
Key words: Arpp; Carp; E box; heart; skeletal muscle

2001), suggesting that the expression of hArpp
may be regulated developmentally or tissuespecifically. To elucidate the molecularmechanism for the tissue-specific and agedependent expression of hArpp, it is necessary
to isolate and characterize the structure of thehArpp gene and its promoter. In this study, we
analyzed the structure of the hArpp gene and its
5' flanking region.

We recently isolated a novel human gene, Arpp,
encoding a 43 kDa protein that is characterized
by the presence of 4 ankyrin-repeated domains
in its central portion (Moriyama et al., 2001).
Arpp has homologies in its amino acid sequence(52.7% identity), as well as in its size and structural feature, to cardiac ankyrin-repeatedprotein (Carp) which is highly expressed in theheart but rarely expressed in skeletal muscles(Zou et al., 1997). In contrast to Carp, humanArpp (hArpp) transcripts were found to be expressed both in skeletal and cardiac muscles(Moriyama et al., 2001). Interestingly, expression of hArpp is restricted to the ventricles, but
it is very low or undetectable in the atria or large
vessels (Moriyama et al., 2001). Furthermore,
hArpp expression in the heart is high in theadult but very low in the fetus (Moriyama et al.,Materials and Methods
Screening of the human genomic library
A human genomic library constructed fromnormal placenta (Clontech, Palo Alto, CA) wasused for screening. Full-length hArpp cDNA

Abbreviations: bp, base pair; Carp, cardiac ankyrin repeat protein; cDNA, complementary DNA; DDBJ,DNA Data Bank of Japan; hArpp, human Arpp; kb, kilobase; mArpp, mouse Arpp; mRNA, messenger RNA;
MyHC, myosin heavy chain; PCR, polymerase chain reaction; pfu, plaque-forming unit; SDS, sodium dodecil
sulfate; SSC, sodium chloride/sodium citrate; USF, upstream stimulating factor

1

A. Miyazaki et al.

(1169 bp) labeled with [32P]-dCTP was used asa probe. For the 1st screening, 3.0 × 108 pfu
were incubated with K802 host cells, followedby plating on NZY-agar plates. After incubation for several hours, nylon membranes(Hybond-N extra; Amersham Pharmacia
BioTech, Tokyo, Japan) were put on the plate.
The resulting replica membranes were hybridized overnight at 42˚C with the radio labeledprobe. Membranes were washed twice for 30
min with a 0.2-fold amount of sodium chloride/sodium citrate (SSC) containing 0.1% sodium
dodecil sulfate (SDS) at 65˚C, followed byautoradiography for 12 h at –80˚C with intensifying screens. The 2nd and 3rd screenings were
performed in a similar way. Out of 21 positive
clones obtained in the 1st screening, 5 clones
survived after the 3rd screening. Phage DNA
was extracted and purified using a Qiagen
Lambda Kit (Qiagen, Tokyo) which was also
used for further studies. The inserted DNA fragments were digested appropriately with restriction enzymes and subcloned into pBlueScriptsk(–) phagemid vector (Stratagene, La Jolla,
CA). The resulting plasmids were used for sequencing.

GTACC-3') and R8 (5'-AGGACAGCACCT
CCACTGTA-3'), F9 (5'-TGAAAGCCTTCAGGACAGTT-3') and R9 (5'-TCCCTCCTCCCCAAACCAGC-3'), respectively. Ten nanograms of the cloned phage DNA were used as atemplate for the PCR. PCR conditions were
94˚C for 2 min, followed by 35 cycles of 94˚C
for 1 min, 55˚C for 45 s and 72˚C for 1 min andfinally incubated at 72˚C for 5 min. Subsequently, the PCR products were subcloned intoa pGEM-T Easy Vector (Promega, Madison,
WI), followed by sequencing.
Determination of the transcriptional startsite
The cap site hunting method was used to determine the transcriptional start site of the hArpp
gene. The Cap Site cDNA constructed from thehuman skeletal muscle was purchased from
Nippon Gene (Toyama, Japan), and used as atemplate for the following PCR. The procedure
to generate cap site cDNA was described indetail previously (Moriyama et al., 2001). The
primer pair used for the 1st round of PCR was asense primer complementary to r-oligo, 1RDT
(5'-GATGCTAGCTGCGAGTCAAGTC-3')
and a reverse primer complementary to hArpp
mRNA, Rev1 (5'-GTGCTTCTCATCCTCCAG
CAC-3'). The primer pair for the nested PCR
was a sense primer complementary to r-oligo,
2RDT (5'-CGAGTCAAGTCGACGAAGTGC3') and a reverse primer complementary to
hArpp messenger RNA (mRNA), Rev2 (5'GCAAGTCCATGGGCAGCTTC-3'). PCR
was performed according to the manufacturer’s
instructions.

Determination of exon/intron boundaries
The exon/intron boundary and the size of eachexon and intron were determined by polymerase chain reaction (PCR). Primer pairs usedwere designed within the introns adjacent to theexon/intron boundaries. Consequently, the resulting DNA fragments contained the whole
length of each exon and both its exon/intron andintron/exon boundaries. Primer pairs used toamplify the boundaries were as follows: F1 (5'CAGTGAGCTCATGGCCAAAGG-3') and R1(5'-GTAGCA GACAGGATCTCCCT-3'), F2(5'-TTGGGGGAGAGGCTGCCTGT-3') andR2 (5'-GCCATGGCTACCTGGCTGGT-3'),F3 (5'-GGTCTCCCAAGCCCCCCCAAA-3')
and R4 (5'-TGACCCAGGAGCCGAACGGA3'), F5 (5'-GCTTGGGTGGGAGAT GGGCT3') and R5 (5'-GTTGGCACAGGTGGCAGG
GA-3'), F6 (5'-ACCCCCGAATACTTTCTT
CC-3') and R6 (5'-CAGCCAGGGGAATAT
CTGTT-3'), F7 (5'-CCCGCCTTCCCAGGG

Results and Discussion
Genomic structure of the hArpp geneWe 1st screened the human genomic library(Clontech, Palo Alto, CA) using hArpp cDNA
as a probe and isolated 21 independent positive
clones from a total of 3.0 × 108 pfu. Out of the
21 clones, 9 clones were subjected to a 2nd
screening and then 5 out of the 9 clones were2

Genomic organization of the hArpp

B NE
B
BK

B

E BN

12

10 97 199

K NK

3 45

358

466

565

664

NH2

763

67

89

862

1009

1 kb

1169

COOH

AI AII AIII AIV
1 29 63

116

152

185

218

251

284

100 bp333

50 aa
Fig. 1. Structure of the hArpp gene, its transcripts and the protein product. λ-arpp1 covers the entire hArpp
gene. Exons (1 to 9) of the hArpp gene are indicated by closed boxes with restriction sites; BamHI (B), EcoRI(E), KpnI (K) and NheI (N), respectively. In the schematic representation of the hArpp mRNA, each exon isindicated by an open box and the 5' and 3' untranslated regions are indicated by hatched boxes. In theschematic representation of hArpp protein, the 4 ankyrin repeat domains (AI to AIV), lysine-rich region,
PEST sequence and proline-rich region are indicated by open, checked, dotted and striped boxes, respectively. bp, base pair; hArpp, human Arpp; kb, kilobase; mRNA, messenger RNA.

subjected to the 3rd screening. The inserts ofthese clones were analyzed by restriction mapping. As shown in Fig. 1, a clone designated as
λ-arpp1 carrying a 13 kb insert was further

characterized by hybridization with a complementary DNA (cDNA) sequence. By the analysis of 1.1 kb of the cDNA sequence using the
DNA Data Bank of Japan (DDBJ) data base, we
3A. Miyazaki et al.

Table 1. DNA sequences at the exon/intron boundaries of the hArpp gene
Exon
No. Size(bp)

3' Splice site(acceptor)

5' Splice site
(donor)Intron(bp)

1

97

ctgtggcctgcAGAGGCGGTTATGGAGAGGAGGAGAATGAGgtgcgagcaggggtg

4925

2

102

tctttggatcaccagCAACTCCGAGGAGACCTGCAGAAGGTGAAGgtaagcctgggagga

338

3

159

ggccatcccgcgcagGGCCAAGAGCGCGTG

GAGCCCGAGGAGATCgtaagggtcctgggg

99

4

108

cgtccacatctgcagACTGGCCCTGTGGATGACACGTGCGACCAGgtgatgctcctagcc

2249

5

99

cctctctggggacagTTCCGTCGGACAGCAGACTTCCAGGATCGGgtgagtgagagggca

362

6

99

ctcatttctttctagCTGGACTGCACAGCC

AATGTGAGGGATAAGgtgaggcaaaaacac

900

7

99

attcctcccacccagCTGCTGAGCACCCCG

AATGCCAGAGACAGGgtgagtgctagcctg

190

8

99

ccacactgactctagGAAGGGGATACTGCC

ATGACCAAGAACCTGgtaagctcattccct873

tcccgccccctccagGCAGGAAAGACCCCG
9 307
hArpp, human Arpp.

AAAGCTGTTTTTGCTaattgcgatgttcat

99 bp to 4925 bp (Table 1). Although the Carp
gene contains a couple of destabilizing motifs(ATTTA) in the 3' untranslated region (Shawand Kamen, 1986), these were absent in the 3'
untranslated region of the hArpp gene. Further
studies will be required to determine whether
the presence or absence of this sequence may be
involved in the stability of hArpp and CarpmRNA.
We recently reported that hArpp is homologous to Carp in its amino acid sequences(52.7% identity) and is specifically expressed in
the heart and skeletal muscles. Since Carp has
been proposed to be a putative genetic marker
for cardiac hypertrophy, we can easily hypothesize that hArpp may also be involved in thehypertrophy pathway of skeletal muscles aswell as cardiac muscles. Interestingly, we
found the CAGA motif (CAGAGC) at position+386 to +398. The CAGA motif has beenreported to be 1 of the target motifs for transforming growth factor β signaling, which is
known to play an important role in cardiac
hypertrophy (Kanai et al., 2001).
In addition, we recently isolated a mouse
Arpp (mArpp) gene and found that the struc-found that the cDNA sequence was colinearwith the human genomic sequences publishedin the DDBJ data base (accession number: AL
359388). Based on these data, we determined
the precise exon/intron boundaries by PCR using the λ-arpp1 DNA as a template. Primers
used for the PCR are shown in Materials andMethods. Following subcloning of the PCR
products, sequence analysis revealed that thehArpp gene was split into 9 exons distributed
over 11 kb (Fig. 1). The nucleotide sequencesof the exon/intron boundaries are shown in
Table 1. Concerning the structure of the exon/
intron junctions, all of them except for exon 9
were consistent with the AG/GT rule (Table 1).
Exon 1 is short (97 bp), although it contains a 5'
untranslated region and a translational initiation
codon. On the other hand, intron 1 is relatively
long (4925 bp). Exons 2 to 5 are relatively short,
ranging in size from 99 to 159 bp. Each exon
from 5 to 8 encodes 1 of the 4 ankyrin-likedomains, respectively. Exon 9 (307 bp) contains a stop codon and a 3' untranslated regionof mRNA. It also contains the polyadenylation
signal AATAAA 136 bp downstream of thestop codon. The sizes of the introns varied from4

Genomic organization of the hArpp

A
gatgctagctgcgagtcaagtcgacgaagtgcagg

1RDT

A GAG GCG GTT ATG GAC GGC ACC ATG GAG GAC TCC GAG GCG GTG
M
D
G
T
M
E
D
S
E
A
V

2RDT
CAG AGG GCC ACA GCG CTC ATC GAG CAG CGG CTG GCA CAG GAG GAG GAG AAT GAG AAA CTC CGA
Q
R
A
T
A
L
I
E
Q
R
L
A
Q
E
E
E
N
E
K
L
R

GGA GAC ACA CGC CAG AAG CTG CCC ATG GAC TTG CTG GTG CTG GAG GAT GAG AAG CAC CAC GGG
G
D
T
R
Q
K
L
P
M
D
L
L
V
L
E
D
E
K
H
H
G

Rev2

B

Rev1

(bp)
174
142

102
87
80

Fig. 2. Determination of the transcriptional start site by the cap site hunting method. A: Positions of theprimers (1RDT and Rev1, 2RDT and Rev2) are shown by arrows, respectively. The sequence of the synthetic
r-oligo ligated to the 5' end of the mRNA is shown in small letters. The putative transcriptional start site isindicated by the vertical arrowhead. B: Electrophoresis of the nested-PCR products. Following the 1st and2nd nested PCRs, the resulting PCR products were electrophoresed. A single band at a size of 150 bp isshown to be detected (arrow). bp, base pair; mRNA, messenger RNA; PCR, polymerase chain reaction.

tural feature of the hArpp gene is well conserved in the mArpp gene.* Comparison of thenucleotide sequences corresponding to theankyrin repeated domain revealed that hArpp is
88.9 % identical to mArpp, suggesting thatthese regions are likely to represent a functional
domain and play an important role both in humans and mice.

as described in Materials and Methods. Briefly,
the primary PCR and the secondary nested PCR
were performed using 1RDT and 2RDT as thesense primers complementary to r-oligo and
Rev1 and Rev2 as the hArpp gene-specificreverse primers (Fig. 2A). The resulting PCR
products at a size of 150 bp were subcloned(Fig. 2B), followed by sequencing. We sequenced 6 clones and found that all of themterminated at the same nucleotide at their 5'
ends of the hArpp cDNA, clearly showing that
it is the major transcriptional start site of thehArpp gene.

Determination of the transcriptional startsite
The transcriptional start site of the hArpp gene
was determined by the cap site hunting method* Tsukamoto Y. Unpublished observation.

5

A. Miyazaki et al.

: CACTTACCAAGAGAGGAGACAGATTGGATAAGGTTTGTTCATGACTAAAAGTCACCCAGC-615

CAAATGCAGTGATGCTTAACGGGGAAGCATGTGGGACCCCGGAGAGCCGAAGGCCAGCGT-555

GAGCTGTGATCAGAGAGGGAGACAGCTGCCCTTCCTGCTACCACGGCCCTGGCCTGGACA-495

AGTAGAGTGTGACCCTCCTCACAGCCAGCAGGGCCTGTCTGCAGGACAGCCTGGGAGTAC-435

AGAACTGTCCTGGGTCCCCCGTTTGGTTTTCAGGCCCTCTTCCTGGAGCTGGCTGAGCAG-375

GGTTTCCAGGCATCCAGCAGGTGGCACTGTCATCTGGGCTGGAGCATGCAGCCAGCAGTT-315

CCCTTTTGCAACTCAGTTGCCTTGGCTAAGGCCCAACCAGTCCCAAGTGCTCAGGACAGA-255

GGCTGGACGGGCTCCTTCCTGCACCCCTGCTCTTGGGACACAGTGCCCTCCGGCTCTAAT-195

AGGCCAGGAGTTGGGGGGGCAACTGGCTCTGCTCCCTGGCCCTGGCTCCCCCTGCTCCCT-135

GGGGCTGCCCGAGGTGAAGGTGACAGGTGGGGGAGGCAGGTGGAGAATTGGGCCAGTGAG-75

CTCATGGCAAAGGCGCCCAGCTGGGCAGGGGTGGGTGCTCTGGCCTATAAAGCCCCCGAG-15

GCCCTGTGGCCTGCAGAGGCGGTTATGGACGGCACCATGGAGGACTCCGAGGCGGTGCAG+45

AGGGCCACAGCGCTCATCGAGCAGCGGCTGGCACAGGAGGAGGAGAATGAGGTGCGAGCA+105

Fig. 3. Sequence alignment of the 5' flanking region of the hArpp gene. The putative binding sites fortranscription factors are underlined. The transcriptional start site is shown by the arrow. AP-4, activator
protein 4; GATA-X, GATA binding site; hArpp, human Arpp; TCF11, transcription factor 11; USF, upstream
stimulating factor.

6

Genomic organization of the hArpp

mans and mice. In addition to the E box, 4 consensus binding sites for USF-1 (–14 to –5, –99
to –92, –112 to –104 and –357 to –351) werealso found in the upstream of the hArpp gene.
Furthermore, all of the USF sites were completely conserved between humans and mice. It
has been reported that the ventricular myosin
light chain-2v promoter contains multiple USF
sites and is actually transactivated by USF-1(Harvey and Rosenthal, 1999), leading us to
speculate that the USF sites may be involved in
the transcriptional regulation of the hArpp genein the human heart.
Since recent reports have stressed that Carp
expression is activated during cardiac hypertrophy and that Carp is a genetic marker for
cardiac hypertrophy (Kuo et al., 1999; Aihara etal., 2000), we can easily hypothesize that Arppmay also participate in muscle hypertrophy ofthe heart and skeletal muscles. Further studies
will be required to address this possibility.

Sequences of the 5' flanking region of thehArpp gene
Next, to isolate the 5' flanking region of thehArpp gene, a BamHI/BamHI fragment (–2682
to –775) at a size of 1907 bp and a BamHI/
BamHI fragment (–775 to +4771) at a size of5546 bp were subcloned into pBluescript sk(–)
vector. Sequencing of the isolated clones revealed that the TATA box and multiple consensus sites for transcription factors were identified in the 5' flanking region of the hArpp gene(Fig. 3). The TATA box was found at 29 bp
upstream of the transcriptional start site (position –29). Several putative regulatory elements
including the E box, upstream stimulating
factor (USF), c-Myb, sex-determining region Y
gene product, GATA and Nkx2-5 site wereidentified. Four E box consensus motifs(CNNTG) were identified at –100 to –90, –113
to –103, –359 to –350 and –535 to –524,
respectively. The former 3 sites were well conserved in the 5'-flanking region of the murine
Arpp gene, but the latter did not appear inmice.* It is well known that promoters of muscle specific genes often contain multiple E
boxes, and that expressions of these genes are
actually regulated by transcription factors thattarget the E box. Accordingly, it is possible that
Arpp may also be regulated by transcription
factors that target the E box.
Skeletal muscles are known to be classified
based on the expression patterns of myosin
heavy chain (MyHC) isoforms. Furthermore,
the expression of the MyHC isoforms is shown
to be regulated under the control of the cisregulatory elements of the MyHC promoter.
We recently found that Arpp is preferentiallyexpressed in type 1 skeletal muscle fibers.†
This expression pattern was observed not onlyin humans but also in mice,* suggesting that
cis-elements responsible for the specification of
the type 1 muscle fiber may exist within the
promoter sequences conserved between hu-

Acknowledgments: The authors are grateful to Professor H. Ito of the First Department of Pathology
and Professor J. Hasegawa of the Department ofClinical Pharmacology, Tottori University Facultyof Medicine, for their helpful discussion.

References
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Received October 12, 2001; accepted October 23, 2001
Corresponding author: Dr. Masatsugu Moriyama

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