www.elsevier.com/locate/yexcr

Experimental Cell Research 295 (2004) 448–459

Identification of the protein Zibra, its genomic organization,

regulation, and expression in breast cancer cells

H. Garrett R. Thompson, Joseph W. Harris, Limin Lin, and James P. Brody*

Department of Biomedical Engineering, University of California-Irvine, Irvine, CA 92697-2715, USA

Received 29 October 2003, revised version received 14 January 2004

Abstract

The mRNA that encodes zibra (zinc, in-between-ring finger, ubiquitin-associated domain), previously known as hypothetical protein

FLJ10111, or RNF31 is expressed in several distinct cancers. Little is known about the genomic organization, expression, or regulation of

zibra. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we cloned the full-length zibra cDNA from a

transformed breast cell line. We identified a novel exon, the 5V untranslated region including the +1 start site, and three alternatively spliced

zibra transcripts. The zibra protein contains three zinc ring-finger motifs, an ubiquitin-associated domain, and an in-between-ring-finger

domain, characteristic of ubiquitin ligases. We obtained an antibody to zibra and confirmed the presence of translated zibra protein for the

first time. Promoter studies localized a core element responsible for basal activity to a 14-bp region in the 5V untranslated region. Although

there are numerous consensus Ets factor binding sites in the zibra promoter, we found no affect on promoter activity from Ets-1, PDEF, or

PEA-3/E1A-F. Treatment of cells with the proteasome inhibitor I (PSI) decreased zibra protein to an undetectable level after 8 h. Zibra

remained undetectable even after 32 h, while mRNA levels remained essentially unchanged. In conclusion, zibra is a translationally regulated

putative ubiquitin ligase that is frequently overexpressed in different forms of cancer.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Breast cancer; Cell lines; E3; FLJ10111; Gene regulation; Hypothetical protein; Proteasome; PSI; RNF31; Zibra

Introduction

Several unifying principles are shared by most, if not

all, cancers. Genetic changes lead to abnormal regulation

of physiological processes, which in turn lead to uncon-

trolled proliferation of the cancer, bypassing mechanisms

that govern normal cells. These abnormally regulated

physiological processes have been described as the hall-

marks of cancer. They include evasion of apoptosis,

angiogenesis, tissue invasion and metastasis, unlimited

replication, and independence from both positive and

negative growth signaling [1]. One strategy for identifying

the molecular basis for these hallmarks of cancer is to

search for common molecular signatures among dissimilar

cancers.

We identified a module of genes that are expressed in

several distinct types of cancers using publicly available

0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2004.01.019

* Corresponding author. Department of Biomedical Engineering,

University of California-Irvine, 204 Rockwell Engineering Center, Irvine,

CA 92697-2715. Fax: +1-949-824-9968.

E-mail address: jpbrody@uci.edu (J.P. Brody).

large-scale gene expression data [2]. Zibra, previously

known as the hypothetical protein FLJ10111, or Ring-finger

protein 31 (RNF31) is one member of this module. Other

members include several breast cancer and proteasome-

related gene products [2]: PSMD7, a 19S proteasome lid

non-ATPase subunit; SQSTM1, encoding the ubiquitin-

binding protein p62 overexpressed in breast tumors [3];

and PDEF, an Ets family transcription factor whose mRNA

is overexpressed in breast cancer [2–4].

Zibra was first identified by the Nedo Full-length cDNA

Sequencing Project in Japan. Although several similar mes-

sages for the gene have been found, most compiled from

individual expressed sequence tags (ESTs) [5,6], no protein

product has yet been identified. Transcripts of zibra mRNA

are increased in some breast cancer cell lines compared to

untransformed cells [2] (this report) and represent a high

percentage of transcripts in various cancer tissues, including

oligodendroglioma, and bronchioalveolar, papillary serous,

borderline ovarian, and breast carcinomas based on EST and

Unigene normalized expression data (http://genome-www3.

Stanford.edu/cgi-bin/SMD/source/sourceResult?option=

Number&criteria=NM_017999&choice=Gene).

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459 449

Several lines of evidence hint that the zibra protein is

involved in the ubiquitin–proteasome pathway. Zibra is

located in a tightly packed region of chromosome 14q11.2

that contains other proteasome-related genes including

PA28a and PA28h [5], which bind the 20S proteasome

and enhance the generation of major MHC class I-binding

peptides [5,7]. Transcription of these genes as well as the

ISGF3 gene located immediately downstream of zibra is

induced by interferon g [5], although zibra itself is not

regulated by interferon g [5] (this report). The products of

other genes in this region are not well characterized with the

notable exception of NEDD8, an ubiquitin-like molecule.

Furthermore, zibra codes for both the in-between-ring finger

(IBR) motif, common among E2 and E3 enzymes in the

ubiquitination of targeted substrates, and the ubiquitin-

associated (UBA) domain, responsible for binding multi-

ubiquitin chains noncovalently.

The RING finger is a zinc-binding protein domain that

was initially characterized as a protein interaction domain

[8]. More recently, it has become clear that a large number

of proteins containing RING finger motifs function as E3

ligases, with the RING finger motif itself serving to recruit

specific E2s [9]. In addition, the RING finger has been

demonstrated to be necessary and sufficient to specifically

catalyze the ubiquitin ligase reaction [10].

Methods and materials

Cell culture

Normal breast epithelial cells MCF10-2A, MCF10-2F,

MCF12A, and MCF12F were cultured in 1:1 DMEM/

Ham’s F12 medium supplemented with 20 ng/ml EGF,

100 ng/ml cholera toxin, 0.01 mg/ml bovine insulin, and

500 ng/ml hydrocortisone. These four cell lines were

cultured with 5% horse serum, and in the case of MCF10-

2F and MCF12F, the serum was chelexed before use. The

transformed cell lines MCF-7, HCC1806, HCC1428,

DU4475, and BT-20 were cultured with 10% FBS supple-

mented EMEM (MCF-7 and BT-20) or RPMI 1640

(HCC1806, HCC1428, and DU4475). Cells were routinely

cultured in monolayer in 10 cm2 culture dishes in the

recommended media until <80% confluent and passaged

at 1:4 dilution two to three times per week. NIH3T3 cells

were cultured in DMEM with 10% CS. All cell lines were

purchased from ATCC (Manassas, VA).

Proteasome inhibitor I (PSI) (Calbiochem, San Diego,

CA) at a final concentration of 10 AM or 0.075% DMSO

(v/v) vehicle was added to cultured HCC1806 cells for the

times indicated, and the cells were harvested for RNA or

protein collection. INFg (Biosource International, Camarillo,

CA) was added to cultured HCC1806 cells at a final

concentration of 200 Units/ml for 2–72 h before harvesting

RNA. 17, h-estradiol (Sigma, St. Louis, MO) at a final

concentration of 10�7 M was added to HCC1806 cells

cultured in phenol red-free DMEM (Invitrogen, Carlsbad,

CA) with 10% charcoal stripped FBS (Gemini Bioproducts,

Woodland, CA) for 24 h. Media were changed to serum-free

overnight for NIH3T3 cells transfected with the zibra pro-

moter after 48 h, and then treated for 8 h with either 10 ng/ml

IL-1 or 20 ng/ml TNF-a in serum-free DMEM.

Recovery of mRNA from cultured cells

Total RNA was harvested from cells solubilized in TRI

Reagent (Molecular Research Center, Cincinnati, OH)

according to manufacturer’s instructions. RNA extraction,

precipitation, and solubilization were performed as de-

scribed [2]. A measure of 250 Ag total RNA was used for

recovery of polyA mRNA from each cell line cultured

using a spin-column format (Qiagen, Valencia, CA).

Message RNA was quantitated by fluorescence using

the RiboGreen RNA Quantitation Reagent (Molecular

Probes, Eugene, OR) assayed in triplicate using the

Fluoroskan Accent FL combination luminometer/fluorom-

eter (LabSystems, Finland).

RT-PCR

Reverse transcription was performed using the Thermo-

Script RT-PCR system according to manufacturer’s instruc-

tions (Invitrogen). In the case where cDNA was prepared

by RLM-RACE (Ambion, Austin, TX), 2 Al of 50 AMrandom decamers was substituted in place of oligo (dT).

RLM-RACE PCR

5V RLM-RACE PCR was performed according to

Ambion’s recommendations and general PCR conditions.

The forward primers used were specific to the 5V RACEadapter that was ligated to the RNA before the RT-PCR

reaction. Both Taq Polymerase (Promega, Madison, WI) and

Pfu Turbo polymerase (Stratagene, La Jolla, CA) were

utilized for the 5V RLM RACE PCR reactions. The first

round of PCR used ‘‘outer primers’’; the second round used

‘‘inner primers’’ (Table 1) to specifically amplify the first

reaction. 3V RLM-RACE did not involve a second round

reaction as the inside primer designed with the cloning site

worked well in a single round.

Cloning Ets factor genes

Genes of interest were cloned in the usual way either

using prostate cDNA as a template (Clontech, Palo Alto,

CA) or cDNA prepared in house from various cell lines as

described [2]. Primers were designed from sequences avail-

able through the NCBI database (Table 2). Genes were

cloned using Pfu Turbo DNA polymerase (Stratagene).

Subsequently, the genes were cloned into pcDNA3.1 (Invi-

trogen) for mammalian expression. Usable amounts of the

constructs were made by Maxi prep (Qiagen).

Table 1

RLM-RACE primers

Primer name Sequence AK000973

(1804 bp)

BC012077

(3496 bp)

AY256461

(zibra: 3211 bp)

Tm (jC)

5V OR-RACE1 5V-TGAGCGTTGCAGCGGACAAG-3V 384 354 No binding 79

5V IR-RACE1 5V-CGCAAGCTT-AAACGCCTGCCCGGAAT-3V 286 256 No binding 75

5V OR-RACE2 5V-ATCTGGTTGCTCCTCTGTGTAG-3V No binding 559 219 69

5V IR-RACE2 5V-CGCAAGCTT-CTTGACACCACGCCAGTA-3V No binding 466 126 67

5V OR-RACE3 5V-TCTCCTGCAGCAGTGACAGT-3V 530 2174 1898 71

5V IR-RACE3 5V-CGCAAGCTT-CTGACCAGGCTCTGCTTGT-3V 459 2103 1827 70

3V OF 5V-CTCGAGACTGCCTCTTCTACCT-3V 1278 2949 2673 70

3V IF 5V-CGCAAGCTT-CGGCTGCCGAGTGATAGA-3V 1399 3070 2794 72

Zibra-specific primers and their relative binding sites on GenBank sequences. OR (outside reverse), IR (inside reverse), OF (outside forward), IF (inside

forward) are indicated for 5V or 3V RLM-RACE primers. RACE1, RACE2, and RACE3 correspond to graphical representation in Fig. 1. HindIII restriction site

is bolded.

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459450

Cloning of the Zibra promoter region

Genomic DNA was extracted, precipitated, and quanti-

tated as previously described from HCC1806 [3].

HCC1806 genomic DNA (100 ng) was used as a template

for cloning the 2.1 kb zibra promoter and enhancer region.

We targeted approximately 2.3 kb upstream of the putative

+1 transcriptional start site based on sequences available in

NCBI databases (BC017376, AK000973, and BC012077).

Nested PCR was performed to obtain the specific product

using the outside forward primer 5V-CCTGAGTGCCT-CACCTCATC-3V and the outside reverse primer 5V-GTCACTCAGACCACGGTCAC-3V. The 2nd round used

the forward primer 5V-GGGGTACC-GAGGGAAGT-

CAAGTCAGCCACAT-3V and the reverse primer 5V-GTGGTACC-CAGGGTTAGCCGAGAACAGGAA-3V,where the bolded sequences are KpnI restriction sites. The

zibra sense and antisense constructs were subsequently

cloned into the KpnI site of pGL3basic vector (Promega),

screened for direction, and sequenced. All zibra promoter

constructs were made from these parent clones using the

primers in Table 3.

Transfections

Transfections of plasmid DNA were as previously de-

scribed [3], with the following additions and modifications.

Table 2

Ets factor cloning primers

Name Sequence

PDEF-F 5V-CCCAAGCTT-ACCAGGCAGCTAACAGACACA-3VPDEF-R 5V-CGCGGATCC-ATGCCAGGTGTGGTGCAGAAT-3VPEA3-F 5V-CTTAAGCTT-CTCCTGGGAGCAGGTC-3VPEA3-R 5V-GTGGATCC-GCTGGGGGCTAGTAAGA-3VEts1-F 5V-GTTAAGCTT-CCATCCCGTACGTCCC-3VEts1-R 5V-ACGGTACC-TCACTCGTCGGCATCTG-3V

Gene-specific primers for PDEF, PEA3/E1A-F, and Ets1 are shown. Bolded seq

primers were generated from publicly available sequence data of the GenBank ac

NIH3T3 cells were plated in 12-well culture plates at a

density of 80,000 cells/well. After 24 h, cells were trans-

fected with appropriate DNA constructs using the Effectene

Transfection Reagent (Qiagen) in triplicate transfections for

48 h. Control transfections included empty vectors for both

the pGL3-basic and pcDNA3.1(+). In total, 0.5 Ag empty

vector, wild-type zibra promoter, and various deletion

constructs were cotransfected with 0.2 Ag PDEF construct

or the empty pcDNA3.1(+) vector. In some cases, 0.2 AgpSV40 hgal (Promega) was cotransfected. Values normal-

ized to hgal activity used the FluoReporter LacZ/Galacto-

sidase Quantitation kit (Molecular Probes), according to the

manufacturer’s instructions, measured in triplicate. An NF-

nB reporter construct (Stratagene) containing five tandem

NF-nB consensus sequences to drive luciferase expression

was used as a positive control to demonstrate cell response

to TNF-a and IL-1 as well as inducible NF-nB activity.

Where specified, samples were also normalized to total

protein content.

Generation of peptide antibody

Rabbits were immunized with zibra C-terminal peptide

(C)EEIPLGQSIPRRRK conjugated with KLH through the

N-terminus cysteine residue and emulsified in Freund’s

adjuvant at 3-week intervals. The antiserum was harvested

after the third booster (Zymed Labs, South San Francisco,

Binding

position

Restriction

enzyme

Tm (jC) GenBank

accession no.

371 HindIII 74 NM_012391

1813 C BamHI 78 NM_012391

7 HindIII 65 NM_001986

1533 C BamHI 64 NM_001986

249 HindIII 69 NM_005238

1604 C KpnI 68 NM_005238

uences are restriction sites listed. ‘‘C’’ denotes complement strand. These

cession number shown.

Table 3

Zibra promoter primers

Primer sequence (restriction enzyme sites are in bold) Binding position Restriction enzyme

5V-GGGGTACC-GAGGGAAGTCAAGTCAGCCACAT-3V �1457 KpnI

5V-GTGGTACC-CAGGGTTAGCCGAGAACAGGAA-3V +640 C KpnI

5V-GGCGATATC-CTGTGGCAAGAATCTGTAGAG-3V �1147 EcoRV

5VGCGGATATC-ATGGCAGAACGAAGGTTAGG-3V �958 EcoRV

5V-CCGGATATC-TGGAAGCTCCTGCTCACT-3V �527 EcoRV

5V-GCGGATATC-GGCCATGCTGCTTCAGT-3V �311 EcoRV

5V-GGCGATATC-AAGGCTCGACCGCAAGTA-3V +77 EcoRV

5V-GGCGATATC-ACCGGACTGGTCCAGCTA-3V +321 EcoRV

5V-GTCGATATC-GCGCCTGCGGCTAT-3V +375 EcoRV

5V-GTCGATATCAGAGCGCTTGACTGAGG-3V +426 EcoRV

5V-GTCGATATC-TGACACCGCTCCAACCT-3V +540 EcoRV

5V-GGTGATATC-GCCCGTTCCTCCGAAA-3V +571 EcoRV

5V-GGTGATATC-TCCGAAATTGGGTCGCAGTC-3V +580 EcoRV

5V-GTCGATATC-CTTCCTGTTCTCGGCTAAC-3V +618 EcoRV

5V-AGGTTGGAGCGGTGTCA-3V +556 C None

5V-CCGGATATC-TCTGGCACCTGCACTTG-3V 4801 C pGL3basic EcoRV

5V-GTCGCTAGATCTCTGGTCTC-3V �296 None

5V-AGCCAGTTGCCACTAGGA-3 �140 None

5V-CAAGTAGCGGACGGAGA-3V +89 None

5VATGGCAGAACGAAGGTTAGG-3V �958 None

5V-CCGCTCGAG-TCTTCTAAGAGCTGCTAGTCCT-3V +178 C XhoI

5V-CCGCTCGAG-ATGGCAGAACGAAGGTTA-3V �958 XhoI

5V-TCTTCTAAGAGCTGCTAGTCC-3V +178 C None

Promoter-specific primers for the various promoter constructs seen in Fig. 2 are shown. Bolded sequences are restriction sites listed. ‘‘C’’ denotes

complement strand.

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459 451

CA). This peptide was used as competitive inhibitor to

demonstrate antibody specificity.

SDS-PAGE and Western blots

PBS-washed cells were lysed on ice in RIPA buffer (50

mM Tris pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5%

sodium deoxycholate, 0.1% SDS, 0.1 mM sodium ortho-

vanadate) supplemented with protease inhibitor cocktail

(Sigma). Protein concentration was determined by addition

of Bradford dye modified for use with detergents (BioRad,

Hercules, CA) [11]. Protein (15 or 40 Ag) was subjected to

SDS-PAGE and transferred to nitrocellulose. Western blot

analyses were carried out in the usual way using appro-

priate primary and horseradish peroxidase (HRP)-conju-

gated secondary antibodies. Two primary anti-zibra

antibodies were used. ‘‘Large’’ zibra was generated from

the same peptide with the first isoleucine of ‘‘small’’ zibra

replaced by valine, as predicted by the zibra mRNA

sequence (Abcam, Cambridge, UK). Anti-large zibra was

diluted 1:250 and anti-small zibra was diluted 1:2000.

HRP-conjugated donkey anti-rabbit, donkey anti-goat, and

sheep anti-mouse IgG secondary antibodies were pur-

chased from Abcam and diluted 1:2500, 1:3000, and

1:1500, respectively.

Specific competitive peptide inhibition was carried out

using 200 Ag unconjugated peptide preincubated with the

primary antibody for 2 h before addition to the membrane.

Noncompetitive control peptide RPPGFSPFR (Peptide

Institute, Inc. Osaka, Japan) was incubated under identical

conditions.

Results

Analysis of zibra’s location on chromosome 14q11.2

shows three previously sequenced full-length transcripts

from cDNA libraries. GenBank accession number

BC017376 and AK00973 list 1975- and 1804-bp (‘‘short’’)

sequences cloned from ovarian adenocarcinoma and the

head of a human 10-week embryo, respectively. Both nearly

perfectly overlap the 3496-bp (‘‘long’’) sequence BC012077

cloned from human placenta choriocarcinoma (Fig. 1), but

when translated results in the deletion of 560 and 10 amino

acids. The 10 amino acid deletion would result in the

clipping of the IBR domain, which would presumably affect

the function of the molecule where it is expressed. Another

notable exception is a single nucleotide difference near the

3V end which allows a conservative amino acid substitution

from isoleucine to valine. Other sequences are available

through GenBank, but are comprised of overlapping

expressed sequence tags (ESTs) and the composite sequen-

ces may or may not represent true transcripts.

Identification of zibra mRNA structure

To determine the +1 transcription start site of the zibra

gene, we performed 5V RLM-RACE on HCC1806-trans-

Fig. 1. Alternative transcripts identified by 5V RLM-RACE. Genomic alignment of three alternative zibra transcripts identified by RLM-RACE on chromosome

14q11.2. Genomic numbering is noted as base position. Adjacent regions to the +1 were sequenced. RLM-RACE2 was amplified by PCR using a reverse

primer generated from 3V RLM-RACE data, subsequently cloned, and sequenced. The full-length sequence is shown (zibra). Two other fully sequenced

transcripts, AK000973 and BC012077, are shown for reference. Alignment was performed using the UCSC Genome Browser located at http://

www.genome.ucsc.edu/ using the April 2003 freeze [42,43]. The location of a predicted CpG island is indicated. The hollow arrow indicates a novel 21-bp

upstream start site. The filled arrow indicated a novel 64-bp region in the full-length transcript previously unreported. Zibra sequence data have been deposited

in GenBank under accession number AY256461. Full 5V sequence data for these transcripts are provided with the exon splice sites bold and underlined.

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459452

formed breast epithelial cells in which mature 5V-cappedmRNA is selectively reverse transcribed, amplified, and

sequenced. Using a reverse primer that was expected to

bind both the long and the short zibra sequences, we

identified a novel 21-bp sequence with the +1 site approx-

imately 650 bp upstream of the previous clones (‘‘RLM-

Fig. 2. Identification of the zibra domain structure. Analysis of the deduced ami

ubiquitin associated domain (UBA), zinc finger (ZF_RING_1, zf-RanBP, ZF_RA

(IBR) domain. Each domain type is represented separately for clarity due to the h

refers to a database entry for the domain, accessible at http://www.ebi.ac.uk/interpro

method. Entries beginning with PS, PF, and SM refer to ProSmart, ProFam, or S

protein sequence used for this analysis is the sequence from our sequenced full

Q86VI2.

RACE1,’’ Fig. 1). Using a reverse primer that was

expected only to bind the long zibra sequence, we identi-

fied a second distinct transcript with the same +1 site

(‘‘RLM-RACE2,’’ Fig. 1). Unexpectedly, when we used a

reverse primer that bound midway in the gene, an addi-

tional +1 start site was identified (‘‘RLM-RACE3’’, Fig. 1),

no acid sequence revealed the presence of several domains, including the

NBP2_1, ZF_RANBP2_2, and ZnF_RBZ), and an in-between-ring finger

omology of zibra with several zinc finger domain types. The Interpro entry

/ISpy?mode=single&ac=Q86VI2. Method accession refers to the predictive

MART, respectively. Vertical bars denote 10 amino acid blocks. The zibra

-length zibra, GenBank accession number AAP12522, TrEMBL accession

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459 453

with the immediate downstream sequence identical to the

long zibra sequence.

The finding of the RLM-RACE3 transcription start site

midway in the zibra gene raises the possibility that this is an

artifact. We are unable to rule this possibility out as

Northern blot analysis is not sensitive enough to detect the

relatively low level of gene expression, and this sequence

cannot be differentiated from other transcripts by real-time

PCR as it is entirely contained within the zibra sequence and

is not unique. However, the RLM-RACE method is

designed to exclusively amplify the mature message that

has been 5V-capped, and we were unable to detect any

product in multiple negative control RLM-RACE reactions.

The DNA corresponding to RLM-RACE2 was cloned,

sequenced, and submitted as GenBank accession number

AY256461 (‘‘zibra,’’ Fig. 1). In addition to the novel +1

start site (hollow arrow, Fig. 1), an additional 64-bp tran-

Fig. 3. Zibra mRNA and protein expression levels in normal and transformed

HCC1806, and MCF-7) breast epithelial cells were used for these studies. (A) Zibr

lines were measured by real-time PCR. Samples were assayed three times in dup

SEM. (B) Zibra protein expression of the same cell lines. Identical duplicate blots

show antibody specificity. Molecular weight markers are indicated. Arrow shows

scribed sequence previously unreported is identified (filled

arrow, Fig. 1). The 3V polyA tail of the mRNA was also

determined by 3V RLM-RACE with only one identified end

sequence.

Because the RLM-RACE data demonstrate the presence

of at least two different transcripts with the novel 21 bp

upstream region, we expected multiple PCR products using

a forward primer specific to the novel 21 bp sequence and a

reverse primer designed from the 3V RLM-RACE data.

However, the full-length zibra sequence was the only

detectable transcript. Similarly, primers designed to amplify

both the long and short zibra sequences (BC012077

andAK000973) generated only the long product from

HCC1806 total cDNA. Using these same primers, the long

zibra product could be amplified by means of cDNA from

several other cultured breast epithelial cell lines, including

HCC1428, MCF-7, MCF10-2A, and MCF10-2F (data not

breast epithelial cells. Normal (MCF10-2A) and transformed (HCC1428,

a mRNA levels from one normal and three transformed breast epithelial cell

licate. Data are represented as a percentage of total mRNA. Error bars are

assayed, without or with competitive peptide (cp), indicated by � or +, to

the specific immunoreactive band.

Fig. 4. Genomic sequence of zibra promoter–enhancer. The 1.5-kb

promoter–enhancer construct is presented with putative transcription factor

binding sites identified. The newly identified 21-bp 5V UTR sequence is

bold. Asterisks at some Sp1 sites denote that sequence is identical to an ER

binding site, although estradiol had no effect on promoter activity. Positions

of various promoter transcripts are identified with their appropriate number

for reference. The +1 start sites of the two other FLJ10111 clones (Fig. 1)

are represented with down arrowheads, and the up arrowhead indicates the

beginning of a putative CpG island. The transcription start site of PSME2 is

also identified.

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459454

shown). Furthermore, a forward primer designed to span the

21 bp exon and the unique exon 2 of RLM-RACE1 poorly

amplified cDNA weakly and nonspecifically from each of

these cell lines, and thus only the sequence of the reported

transcript could be determined.

Zibra protein sequence similarities and expression analysis

The deduced translated protein product of the zibra

transcript is 921 amino acids with a predicted molecular

weight of 102 kDa. The overall structure of the protein is

presented in Fig. 2. Because the zinc finger regions of zibra,

between amino acids 147 and 282, match several different

zinc finger classes, each class is listed individually. A

composite of the overall domain structure is indicated

directly below.

Although zibra mRNA is not detectable by Northern blot

(data not shown), we could quantitatively measure tran-

script levels by real-time PCR. Because we previously

found zibra mRNA to be generally increased in transformed

breast epithelial cells [2] and six of the top 10 zibra-

expressing tissue types are carcinomas based on Unigene

and EST expression information (http://genome-www3.

Stanford.edu/cgi-bin/SMD/source/sourceResult?option=

Number&criteria=NM_017999&choice=Gene), we mea-

sured relative RNA expression in normal and transformed

breast epithelial cell lines. In addition, we analyzed zibra

protein expression by Western blot. To demonstrate antibody

specificity, a competitive peptide generated against the small

zibra sequence was preincubated with the primary antibody

before assay. One immunospecific band is identified at

approximately 145 kDa (Fig. 3B). Curiously, zibra protein

was undetectable in MCF-7 cells which express the highest

level of zibra mRNA.

Zibra migrates at a molecular weight greater than pre-

dicted based on the sum of amino acid residues. Our

sequence data showed that the expressed mRNA transcript

contains a single nucleotide difference allowing for the

conservative amino acid change. Thus, the antibody would

have higher affinity for the short form, and also for the

protein product predicted from the transcript that was the

source of the cDNA sequenced from HCC1806 cells. While

it is possible that we could have observed the short form

(AK000973, predicted 57.6 kDa), we did not observe this

band. Instead, the only specific band (e.g., that could be

competed off with a specific peptide) was the observed band

at approximately 145 kDa. While migrating slower than

expected in the polyacrylamide gel, the presence of the

larger MW protein is in accordance with the full-length

transcript that we were able to sequence from these cells.

The retarded migration by polyacrylamide gel electro-

phoresis could be explained by several possibilities, includ-

ing overall hydrophobicity, posttranslational modifications,

of which there are several high-probability amino acid sites

including those for phosphorylation [12], O-Gln [13], and

sumoylation based on the tetrapeptide motif B-K-x-D/E (B

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459 455

is a hydrophobic residue, K is the lysine conjugated to

SUMO, x is any amino acid, and D or E is an acidic

residue), and propensity of these amino acids to maintain

similar hydrophobicity.

Analysis of zibra promoter elements

Because of the observed differences in zibra gene ex-

pression between normal and transformed tissues and cells,

we analyzed the promoter elements responsible for in-

creased mRNA expression. Based on the newly discovered

upstream +1 transcription start site, the approximately 1.1-

kb region from �958 to +178 was screened for putative

Fig. 5. Transfection of zibra promoter–enhancer constructs. (A) The 1.1-kb region

was transfected in NIH3T3 cells. (B) A larger promoter region containing a stron

construct from A is shown for reference. Filled arrows denote relative location of z

PSME2 gene on the antisense strand. Relative light units denoting luciferase–h-ga�958 to +640. Values are the means of three independent experiments performed

SEM.

transcription factor binding sites using the Transfac database

version 6 (Fig. 4), the most distal 104 bp of which overlap

the PSME2 gene sequence. This same region was cloned

into the pGL3Basic-luciferase reporter plasmid and assayed

for promoter activity (Fig. 5A). Basal activity was deter-

mined to be 2- to 3-fold the antisense control and more than

10-fold the level of empty luciferase vector. However,

subsequent deletions from the 5V end reveal that the stron-

gest promoter does not contain the early upstream region

from �958 to �297, as the �296 to +178 construct was 4-

fold greater than basal activity, with subsequent deletions

abrogating the maximal effect. The +89 to +178 construct,

which contains two Sp1 sites, gives similar promoter

surrounding the newly identified +1 as well as deletion constructs as shown

g enhancer element was transfected into NIH3T3 cells. The �958 to +178

ibra +1 transcription start site. Open arrows denote transcription start site of

l are presented for each construct, normalized to the largest sense construct

in triplicate wells, with each well assayed in triplicate. Error bars represent

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459456

activity as the much larger �958 to +178 promoter, suggest-

ing that these Sp1 sites contribute to the basal promoter

activity. Deletion of the �296 to �140 region eliminates

more than half of the maximal promoter activity. Analysis of

this region reveals the presence of two consensus sequences

for TFIID/TBP/Sp1 (Fig. 5A).

Next, we studied a larger approximately 1.5 kb promoter-

luciferase construct using the same 5V end with an additional

462 bp downstream that encompassed the first base of the

previously reported short zibra sequence. The largest pro-

moter, �958 to +640, drove luciferase expression approx-

imately 40-fold higher than the �958 to +178 construct

(Fig. 5B), suggesting regulatory elements between +178 and

+640. Because of the proximal location of zibra to PSME2

(encoding PA28h) in a head-to-head configuration less than

1 kb away, the antisense promoter construct was assayed for

activity. While this construct possessed significant promoter

activity, it is interesting to note that deletion of the final 84

bp of the sense construct abolishes activity to levels lower

than antisense. These data suggest that the genomic region

between PSME2 and zibra confers some activity in both

orientations.

Subsequent deletions of the large promoter construct

from the 5V end reveal essentially full promoter activity

with the +426 to +640 construct. This is in accordance with

previous finding that most promoter activity is located

between +178 and +640, as seen when the �958 to +178

and �958 to +640 constructs are compared. Deletion of the

next 114 bp from the 5V end dramatically reduces promoter

activity by half, and deletion of the next 31 bp nearly

abolishes all activity. We see a similar knockdown of

promoter activity with the nearly full-length �958 to +556

construct. Taken together, these data provide evidence of a

core element of basal promoter activity that lies between

+557 and +570. The only identified transcriptional ele-

ments, based on the TRANSFAC database V6.0, that may

be responsible for this activity is a TFIID/TBP/Sp1 consen-

sus site located +566 to +575 (Fig. 4). The location of this

Fig. 6. Transfection of zibra promoter–enhancer constructs and treatment with TN

as a deletion construct eliminating a strong consensus NF-nB binding site was tra

positive control. After transfection, cells were treated with TNF-a or IL-1 for 8 h a

the means of three independent experiments performed in triplicate wells, with e

sequence 48 bp upstream of the +1 reported for the short

zibra sequence and 78 bp upstream of the +1 reported for

the long zibra sequence is suggestive that this element may

play an important role in the basal regulation of these

transcripts.

Because PDEF and zibra mRNA are coordinately in-

creased in transformed breast epithelial cells in culture [2],

and several Ets factor family members shown to be upregu-

lated in cancer [14–17] are potent regulators of promoter

activity [18–21], cotransfection experiments with the zibra

promoter and three Ets factors were performed. Initially, it

appeared as though Ets-1, PDEF, and PEA3 all dramatically

stimulated the zibra promoter, in order of increasing poten-

cy, as measured by luciferase activity. However, it was

determined that each of these transcription factors affected

h-gal expression via the SV40 enhancer. When luciferase

activity was divided by total protein, no promoter regulation

was observed alone or in combination (data not shown). In

addition, several consensus NF-nB and estrogen receptor

binding sites are present in the upstream 0.5-kb region (Fig.

4). Therefore, we assessed the possibility that NF-nB or

estrogen regulates zibra expression. While addition of TNF-

a or IL-1 increased NF-nB activity as measured by the NF-

nB reporter construct, neither cytokine affected the zibra

promoter (Fig. 6) or protein concentration (data not shown).

In addition, neither 200 U/ml INFa˜nor 10�7 M estradiol

had any effect.

Previous studies have assessed the role of proteasome

inhibitors on cellular death in several different cell types

[22–25]. An appropriate inhibitor and dose was determined

with cell proliferation assays [3]. Previous findings demon-

strate that PSI treatment of cultured HCC1806 cells, as well

as other normal and transformed breast epithelial cells,

results in a rapid increase of ubiquitinated proteins and a

time-dependent increase of p62 protein and stabilization of

p62 mRNA. The ability of p62 to bind ubiquitin through its

UBA domain, its coexpression with zibra mRNA, and its

ability to bind several signaling proteins suggest that p62

F-a and IL-1. The 1.1-kb region surrounding the newly identified +1 as well

nsfected in NIH3T3 cells. A NF-nB reporter construct was transfected as a

nd promoter activity measured by luciferase–h-gal. Relative light units areach well assayed in triplicate. Error bars represent SEM.

Fig. 7. Effect of PSI on zibra mRNA and protein expression. HCC1806

cells were cultured in the presence of 10 AM PSI for 8, 16, or 32 h.

Untreated and 32-h DMSO-treated cells served as controls. (A) Total cell

lysate was assayed for zibra protein by Western blot. h-actin is included as aloading control. (B) mRNA levels were measured by real-time PCR. Values

are the means of three independent experiments performed in duplicate and

represented as a percentage of total mRNA. Error bars represent SEM.

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459 457

may play a regulatory role in the ubiquitin-proteasome

pathway. To assess the effects of PSI on zibra expression,

HCC1806 cells were treated with 10 AM PSI for 0, 8, 16, or

32 h. A 32 h DMSO vehicle treatment was included as a

control. A measure of 40 Ag total cell lysate, extracted in

RIPA buffer, was subjected to Western blot analysis for

zibra and h-actin and visualized by chemiluminescence. PSI

dramatically decreased zibra protein expression after 8 h and

was still undetectable after 32 h (Fig. 7A). To show equal

loading of cell lysate, h-actin is included as a control.

To determine whether the decrease of zibra protein in

PSI-treated cells was because of decreased mRNA, mRNA

expression level of zibra from HCC1806 cells treated with

10 AM PSI was measured by real-time PCR over 32 h (Fig.

7B). DMSO-treated vehicle values are shown for reference.

The addition of PSI to cultured HCC1806 cell had essen-

tially no effect on mRNA levels of the zibra transcript at any

time point. Cells treated with DMSO for 32 h served as

control.

Discussion

Because both the long and short submitted zibra sequen-

ces were obtained from cDNA libraries, it is possible that

these sequences are not full-length or mature transcripts.

Thus, we determined both the 5V and 3V end by RLM-RACE.

As such, we identified a novel 21-bp transcribed region

upstream of the previously reported sequence as well as an

additional 64-bp region at the end of exon 3 (filled arrow,

Fig. 1). In an effort to confirm the presence of the short zibra

sequence, we designed a reverse RLM-RACE primer at the

5V end of exon 11. While, we could not verify the presence

of the short transcript, we identified an additional transcript

(‘‘RLM-RACE3’’, Fig. 1) that begins within exon 9 of zibra,

but contains all of exon 10, as in the full-length zibra

sequence. Interestingly, a recent paper predicted and vali-

dated 845 significantly tumor-associated alternative splic-

ings, including FLJ10111 (zibra) with a 23-bp insertion at

position 1026 of the NM_017999 (short zibra) sequence

[26]. However, we did not observe this insertion.

Recent analysis of publicly available mRNA expression

data revealed a module of genes that are coexpressed in

several tissue types, including breast cancer [2]. Member

genes of this module include the Ets factor transcription

factor PDEF, SQSTM1/p62, PSMD7, and zibra. Because

EST and UniGene data suggest that zibra mRNA is over-

expressed in several cancers and our previous findings in

breast cell lines, we analyzed zibra mRNA and protein

expression in breast cancer cells. In this study, we demon-

strate that while zibra mRNA expression may in fact be a

marker of breast cancer, zibra protein levels may not parallel

mRNA levels. For instance, we observe that MCF-7 cells in

which zibra mRNA is approximately 3-fold greater express

less protein than other cell lines studied. Of the members of

the module of coexpressed genes in breast cancer, zibra is

not alone in this regard. PDEF mRNA is expressed up to

three orders of magnitude greater in breast cancer cells

compared to normal cells. However, no studies to date have

identified PDEF protein in breast cancer, and one study

finds that PDEF protein is reduced in human invasive breast

cancer and is absent in invasive breast cancer cell lines [27].

The apparent discrepancy between zibra mRNA and

protein levels suggests several possibilities. The 5V end of

the transcript contains a putative CpG island, and the

methylation status of this region may confer epigenetic

control on the expression of the gene. In cancer cell lines,

we noted increased expression of the zibra gene, which may

suggest decreased methylation. In addition, capped mRNA

can also be methylated, although the effects on translation

are not yet clear. Alternatively, feedback mechanisms, the

simplest of which is that lack of protein, either by reduced

translation or increased degradation, may signal an increase

in zibra transcription. This is not likely the case, as addition

of proteasome inhibitor rapidly reduces zibra protein con-

centration within the cell without any change in mRNA.

Even after 32 h, the protein is decreased, with only minor

changes on mRNA. Another more likely scenario supported

by our data is that zibra protein concentration is tightly

regulated, and mRNA expression is a function of the

individual cell type. Transformed breast epithelial cells

may, in general, express more zibra mRNA due to altered

RNA stability or transcription mechanisms.

Thus, in an effort to determine the functional regulation

of zibra gene expression, we cloned the promoter encom-

H.G.R. Thompson et al. / Experimental Cell Research 295 (2004) 448–459458

passing both the previously reported transcription start site

and the newly identified +1 site. The �958 to +178

construct, based on the new start site, has approximately

3-fold promoter activity compared to its antisense. Remov-

ing 662 bp from the 5V end increases basal promoter activity

another 4-fold (Fig. 5A). However, adding 462 bp to the 3Vend increased promoter activity greater than 30-fold (Fig.

5B). Thus, most basal promoter activity lies between +178

and +640.

While analysis of the promoter identified a Kozak

consensus sequence at +39 (Fig. 4), identical activities are

observed between the �311 construct and the +77 construct

which does not contain this sequence (Fig. 5B). Therefore,

we conclude that this site has little or no effect on the

transcription of the reporter gene. Various deletion con-

structs found that half of the maximal activity is regulated

between +426 and +540, and the remaining between +540

and +571. Strikingly, the �958 to +556 construct had

minimal activity (compared to �958 to +640), indicating

that nearly all basal promoter activity lies between +557 and

+570.

This 14-bp sequence of DNA contains a short region

predicted to be homologous to only TFIID, TATA Binding

Protein (TBP), Sp1, and estrogen receptor (denoted by the

asterisk on Sp1 in Fig. 4) binding sites. Sp1 is a basal

promoter transcription factor and Sp1 activation of TATA-

less promoters, such as this one, requires interaction with

TFIID [28] and multiple Sp1 sites are synergistic and

required for maximal stimulation [29]. In TATA-less pro-

moters, Sp1 also interacts with other proteins other than

TFIID to stimulate transcription, including E2 [30,31], NF-

nB [32], Oct-1 [33], SREBP-1 [34,35], and the Ets factors

Ets-1 [36,37] and PU.1 [38].

To determine the mechanism of zibra gene regulation, we

first looked at Ets factors because of the large number of Ets

factor consensus sequences in the promoter, synergistic

activation with Sp1, and the predicted and confirmed coex-

pression of zibra with PDEF. We cotransfected PDEF, Ets-1,

and PEA-3/E1A-F with various zibra promoter constructs

either alone or in combination. Each of these factors is

overexpressed in cancer models and directly implicated in

tumorigenesis. Surprisingly, we observed no changes in

promoter activity with the addition of these transcription

factors.

We next assessed the effect of estrogen, as the zibra

promoter contains a large number of estrogen receptor

binding sites including one in the critical short basal region,

and many breast cancer genes are dysregulated by estrogens.

We treated promoter-transfected cells with physiologically

relevant concentrations of 17-h-estradiol but observed no

changes in promoter activity. Lastly, because NF-nB has

been shown to regulate genes involved in cancers, its link to

Sp1, its observed synergistic transcriptional activation with

Ets-1 [39], and the finding that biological morphogens, such

as TNF-a, upregulate the expression of Ets-1 transcription

factor [40,41], we treated cells with either TNF-a or IL-1 to

increase endogenous NF-nB activity, again with no effect on

promoter activity or zibra protein expression.

Zibra maps to a region of chromosome 14 in a locus

rich in genes involved in the structure and function of the

proteasome. Because the amino acid motifs are consistent

with E3 ubiquitin ligases in the proteasome degradation

pathway and our previous finding that zibra is coex-

pressed with other genes likely involved in the ubiqui-

tin-proteasome pathway, we assessed whether chemically

inhibiting the proteasome would exert a regulatory effect

on gene expression. We found that while there was

essentially no change in mRNA levels, protein levels

dropped by 8 h and were still undetectable after 32 h.

These data taken together suggest that the zibra protein is

regulated posttranscriptionally. This is in accordance with

our previous and current findings that zibra mRNA levels

are increased in transformed breast epithelial cells com-

pared to normal cells, but protein levels are essentially the

same or lower.

In conclusion, zibra mRNA is expressed at high levels in

several distinct cancers, but its protein expression appears

to be uncorrelated to mRNA levels. It has at least three

alternative transcripts. Its domain structure, location on

chromosome 14, and responsiveness to proteasome inhibi-

tion suggest that one cellular function is as an ubiquitin

ligase. Zibra’s promoter contains a small 14-bp region that

is important for basal transcription, and protein levels can

be regulated posttranscriptionally.

Acknowledgment

This work was supported by the National Institutes of

Health Human Genome Research Institute grant number

1K22HG00047-01.

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