From the Division of Immunogenetics, Department of
Pediatrics, Diabetes Institute, Rangos Research Center, Children's
Hospital of Pittsburgh, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15213 and § Department of Human
Genetics, Graduate School of Public Health, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
Received for publication, October 4, 2002, and in revised form, October 25, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Islet cell autoantigen 69-kDa
(ICA69), protein product of the human ICA1 gene, is one
target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional
promoters within the 5'-regulatory region of ICA1. 5'-RNA
ligase-mediated rapid amplification of cDNA ends evaluation of
ICA1 transcripts expressed in human islets, testis, heart,
and cultured neuroblastoma cells reveals that three 5'-untranslated
region exons are variably expressed from the ICA1 gene in a
tissue-specific manner. Surrounding the transcription initiation sites
are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters,
including consensus Sp1/GC boxes, an initiator element, cAMP-responsive
element-binding protein (CREB) sites, and clusters of other putative
transcription factor sites within a genomic CpG island. Luciferase
reporter constructs demonstrate that the first two ICA1
exon promoters reciprocally stimulate luciferase expression within
islet- (RIN 1046-38 cells) and brain-derived (NMB7) cells in culture;
the exon A promoter exhibits greater activity in islet cells, whereas
the exon B promoter more efficiently activates transcription in
neuronal cells. Mutation of a CREB site within the ICA1
exon B promoter significantly enhances transcriptional activity in both
cell lines. Our basic understanding of expression from the functional
core promoter elements of ICA1 is an important advance that
will not only add to our knowledge of the ICA69 autoantigen but will
also facilitate a rational approach to discover the function of ICA69
and to identify relevant ICA1 promoter polymorphisms and
their potential associations with disease.
Islet cell autoantigen 69 kDa (ICA69) is identified with a
group of Type 1 diabetes-related islet autoantigens considered to be
specific protein targets of the diabetogenic autoimmune response.
By using sera from pre-diabetic individuals, Pietropaolo and
co-workers (1) first identified ICA69 through immunoscreening of a
human islet cDNA expression library. The 1785-bp nucleotide sequence of the full-length clone and its deduced 483-amino acid protein coding region demonstrated no overt homology to known molecules
at the time of its discovery, and nucleic acid and protein analyses
revealed that the molecule is primarily expressed in neuroendocrine
tissues (1, 2). More recent subcellular fractionation studies of murine
brain tissue have shown that the majority of ICA69 protein is cytosolic
and soluble, although a subfraction appears to be membrane-bound and
associates with synaptic-like microvesicles (3). Although of unknown
significance, smaller isoforms of the protein may be expressed from at
least three human transcript variants (1, 4-6), representing truncated
cDNAs that arise from alternative splicing of coding region exons
(4).
ICA69 is encoded on human chromosome 7p22 by the ICA1 gene
(7), which is composed of 14 coding exons and three 5'-untranslated region (UTR)1 sequences, each
of which splices with exon 1 in a mutually exclusive manner (4). In
addition, multiple cDNA coding region splice variants from human,
mouse, and rat have been identified within islet and brain
bacteriophage Recently a protein from the nematode Caenorhabditis elegans,
termed ric-19, was reported to exhibit amino acid homology with ICA69
(3). Based on functional studies of ric-19 in C. elegans, these authors have proposed that ICA69/ric-19 participates in the
process of neuroendocrine secretion through an association with
secretory vesicles. These data suggest that ICA69 may be involved in
the insulin secretory pathway in islet Most basic and clinical research investigations concerning ICA69 have
focused on the importance of the molecule as an autoimmune target in
Type 1 diabetes. Three lines of evidence from at least four independent
groups substantiate a role for ICA69 autoimmunity in diabetes. 1) First
degree relatives of diabetic patients who developed the disease during
follow-up have detectable serum levels of ICA69 autoantibodies (1, 9).
2) T-cells isolated from newly diagnosed diabetic patients and from
non-obese diabetic (NOD) mice demonstrate reactivity against the
recombinant ICA69 molecule (10-12). 3) T-cells specific for the ICA69
peptide Tep-69 play a driving role in the acceleration of islet cell
destruction in the NOD mouse model of Type 1 diabetes (13), whereas
intraperitoneal injection of Tep-69 is associated with apparent immune
toleration and decreased diabetes incidence in NOD mice (14). It has
been reported that a majority of patients with recent onset Type 1 diabetes shows evidence of autoreactive T-cells and/or autoantibodies with immune specificity for the ICA69 molecule (11), but it must also
be acknowledged that some investigators have questioned the
significance of ICA69 autoantibodies based on their own studies (15).
Motivated by an interest in understanding how autoantigen expression in
key body tissues relates to autoimmunity and as a prerequisite to
searching for functional polymorphisms in the promoter region of the
gene encoding ICA69, we have defined the basic structure and functional
characteristics of the ICA1 promoter. Sequences adjacent to
the multiple ICA1 transcription initiation sites contain
motifs typical of a non-TATA, non-CAAT, GC-rich regulatory region,
including consensus Sp1/GC box sites, Inr (initiator) elements, and
CREB sites. The major alternative transcription initiation sites
associate with independent 5'-UTR exons, and a detailed analysis of
ICA1 transcripts from different tissues provides evidence
for a tissue-specific utilization of the distinct initiation sites
consistent with the observed 5'-UTR heterogeneity of mature
protein-coding ICA69 transcripts. In vitro luciferase reporter gene assays of promoter function correlate with the observed preferential transcription initiation site usage within different tissues, whereas site-directed mutagenesis of promoter reporter constructs demonstrate the importance of an Sp1/GC box site and a CREB
site to the regulation of expression from exons A and B, respectively.
The significance and potential implications of the ICA1
promoter structure and function are discussed in the context of
understanding ICA69 biology and its role as a Type 1 diabetes autoantigen.
Cell Lines--
Two adherent cell lines were maintained in
culture in order to provide RNA for transcript analysis and for testing
promoter activity of cloned ICA1 5'-flanking sequences in a
firefly luciferase reporter assay. The human neuroblastoma cell line
NMB7 was grown in RPMI containing 10% fetal bovine serum, supplemented
with L-glutamine and penicillin/streptomycin. Rat
insulinoma (RIN 1046-38) cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum,
L-glutamine, and penicillin/streptomycin. Cells were
incubated at 37 °C in a 5% CO2 atmosphere. Passage of
cells was conducted at 70-80% confluence as necessary. All cell
culture reagents were obtained from Invitrogen and lot-certified in-house. NMB7 cells were provided by Dr. Ira Bergman and Judi Griffin (Children's Hospital of Pittsburgh, Pittsburgh).
Nucleic Acid Purification--
Human total genomic DNA was
purified from whole blood using the Qiagen Genomic DNA Extraction Kit
(Qiagen, Inc., Valencia, CA). Briefly, 10 ml of heparinized blood was
successively subjected to cellular lysis, nuclear isolation, nuclear
lysis, and anion-exchange chromatography using the buffers and prepared
columns supplied with the Qiagen kit. Typical yields ranged from 150 to
350 µg of total genomic DNA per 10 ml of whole blood.
Bacterial plasmid DNA was isolated by one of two methods, depending
upon the quantity and concentration desired. For DNA sequencing and
restriction enzyme analysis of subcloned DNA, the QIAprep Spin Miniprep
Kit (Qiagen) was used to isolate 10-20 µg of plasmid DNA from 3 to 5 ml of an overnight bacterial culture. Alternatively, plasmid DNA used
for transfection of cultured cells was prepared from 100 to 200 ml of
overnight bacterial culture using the Qiagen Maxiprep DNA Isolation
Kit. Typical DNA yields ranged from 150 to 450 µg.
For BAC clone DNA, a Qiagen Maxiprep protocol modified for use with
BACs was followed (protocol available from manufacturer). Major
modifications to the basic protocol included the use of a larger
culture volume (500 ml) and elution of BAC clone DNA from the column
with buffer warmed to 50 °C. Yields of BAC clone DNA ranged from 100 to 150 µg.
YAC clone DNA was co-purified along with yeast genomic DNA according to
a standard protocol (16) with some modifications. Briefly, following
2000 × g centrifugation of 100 ml of fresh yeast cell
culture at room temperature for 10 min, the cell pellet was resuspended
in SCE buffer (0.9 M sorbitol, 0.1 M sodium
citrate, 0.06 M EDTA) with freshly added 0.3 M
PCR Amplification--
Due to the nature of the sequences being
amplified, the PCR technology used in an experiment was adapted to each
DNA target and template. Reaction components were used in amounts and
concentrations as recommended by the manufacturer unless otherwise
noted. Suggested annealing temperatures for each PCR kit were adjusted
based on the sequence identity of the amplification primers and the
sequence composition of the amplification target with the assistance of Oligo 4.0 (Molecular Biology Insights, Inc., Cascade, CO) primer design
software. Reactions were cycled 30-35 times unless otherwise specified.
For PCR amplification of simple target DNAs (i.e. <5 kb
with moderate to low GC content) AmpliTaq DNA polymerase
enzyme and buffers (PerkinElmer Life Sciences/Applied Biosystems) were
used. GC-rich regions of ICA1 sequence from human genomic,
YAC, and BAC DNA samples were amplified using reagents from the
Advantage GC Genomic PCR kit (Clontech, Palo Alto,
CA), whereas the Advantage GC cDNA Enzyme
(Clontech) facilitated amplification of GC-rich plasmid inserts and cDNA templates generated for 5'-RACE analysis. In cases of long PCR (>5 kb), or when other PCR methods failed, the
eLONGase long PCR enzyme mix (Invitrogen) was employed.
Oligonucleotides were synthesized in the DNA Sequencing and Synthesis
Core Facilities of the Diabetes Institute, Children's Hospital of Pittsburgh.
YAC and BAC Library Screening--
PCR primers designed from the
ICA1 exon 2 intron-exon boundary sequences were used to
screen the Centre d'Etude du Polymorphisme Humain (CEPH) Mega-YAC
Human DNA Library by systematic amplification of YAC clone DNA pools
(primer sequences: 5'-CCTGGGACTTACAGGATCGA-3' and
5'-GACAGCAATAAAGAGCTCAC-3', annealing temperature 55 °C, 178-bp PCR
product). The California Institute of Technology BAC Library (CITB
Release IV, Research Genetics, Inc., Pasadena, CA) was similarly screened using a PCR approach. The PCR amplimer used for BAC library screening was a microsatellite (CA repeat) centered 1830 bp upstream of
the ICA1 translation initiation codon (primer sequences:
5'-TATGAAACAGTGTTATTCTGGACCT-3' and 5'-GTACAGTATAGTAGTGCTAACA-3',
annealing temperature 55 °C, 540-bp PCR product). Stab vials or
frozen aliquots of each PCR-positive YAC and BAC library clone
identified through screening were obtained, and purified DNA extracted
from their respective cultures was retested under PCR conditions
similar to those used for library screening to verify that the target
ICA1 sequences were present.
Subcloning of PCR Products--
If necessary, amplified products
from GenomeWalker-PCR, RT-PCR, and 5'-RACE experiments were
gel-purified using the Qiagen Gel Extraction Kit (Qiagen) before
subcloning, or they were subcloned by direct ligation of an aliquot of
the PCR. The gel-purified or neat PCRs were subcloned into the pCR 2.1 vector (Original TA Cloning Kit, Invitrogen). When PCR primers were
designed to include restriction sites, they were digested with the
appropriate restriction enzyme(s) and ligated into an
overhang-compatible aliquot of the pGL3 basic luciferase reporter
vector. All ligation reac-competent Escherichia coli
(Invitrogen) and plated on 100-mm LB agar plates containing 50 µg/ml
ampicillin or kanamycin and 50 µg/ml
5-bromo-4-chloro-3-indoyl-
In situations where T/A PCR product ligation proved to be inefficient
because of 3' 5'-Rapid Amplification of cDNA Ends (5'-RACE)--
The
FirstChoice RLM-RACE Kit (Ambion, Austin, TX) was used for RNA
ligase-mediated (RLM) RACE analysis of ICA1 transcripts, because it permits selective amplification of capped RNA molecules from
non-poly(A)-selected RNA. Briefly, total cellular RNA is treated with
calf intestinal alkaline phosphatase to remove the 5'-phosphate group
from uncapped mRNA precursors, tRNA, rRNA, and small nuclear RNA
molecules, followed by phenol/chloroform extraction and recovery of the
dephosphorylated RNA by ethanol precipitation. Dephosphorylated RNA is
then incubated with tobacco acid pyrophosphatase to remove
m7Gpp from the cap structure of the 5' end of capped
RNAs, leaving a single 5'-terminal phosphate group. Ligation of a
synthetic RNA adapter of known sequence to the calf intestinal alkaline phosphatase- and tobacco acid pyrophosphatase-treated RNA proceeds in
the presence of E. coli RNA ligase. Adapter-ligated RNA is reverse-transcribed into cDNA using Moloney murine leukemia
virus-reverse transcriptase enzyme in the presence of random decamer
primers. The resultant single-stranded cDNA then serves as template
in nested PCRs using adapter sequence-specific primers (provided with
the RLM-RACE kit) and gene-specific primers (GSP1 and GSP2) designed
from ICA1 exons 1 and 2. The sequences of these latter primers are as follows: GSP1 (antisense exon 2),
5'-TGCATCTTATTTACAACTGACTTATCTTGA G-3' and GSP2 (antisense exon 1/2
boundary), 5'-TGTAAGTCCCAGGGATAACTGCATTTGTGT CCTGA-3'. The Advantage GC
cDNA enzyme was used in all nested RLM-RACE PCRs.
Cloning of ICA1 Sequences into pGL3 Basic--
To clone segments
of the ICA1 5'-flanking region and UTR exons, a 1028-bp
genomic segment spanning the entire region was amplified from
CITB-503D2 DNA via PCR, followed by T/A ligation of the product into
the pCR2.1 vector (amplification primers, 5'-TAGGAAGCAGCTATGCCAACACT-3' and 5'-CAGAGAAGGCAGCTCCTACCA-3'). Excision of various segments of the
cloned PCR product using pairs of restriction endonucleases recognizing
sites found in the pCR2.1 vector arms, internal restriction sites of
the insert, or a combination of the two allowed for directional cloning
of defined ICA1 sequences into a pGL3 Basic vector having compatible overhangs. A second strategy for cloning ICA1
sequences into pGL3 Basic involved the design of
ICA1-specific primers with restriction endonuclease sites
added at the 5' end. After spin column chromatography purification of a
PCR product amplified with these primers, the product was digested with
one or more restriction enzymes to create overhangs compatible with
those generated on an aliquot of the pGL3 Basic vector. Heat
inactivation and gel purification or spin column purification of the
digested PCR product was then followed by ligation into pGL3 Basic.
Site-directed Mutagenesis--
Two pGL3 promoter reporter
constructs, ExA Luciferase Assays--
For luciferase transfection experiments,
NMB7 and RIN 1046-38 cells were plated at a density of 0.8 × 105 cells/well of a 12-well plate the day before
transfection. Growth in the appropriate complete medium for 20-24 h
generally resulted in 50-70% cellular confluence in each well at the
time of transfection. Transient transfection of luciferase constructs
and mutants thereof into the various cultured cell lines using
Effectene Transfection Reagent (Qiagen) was followed by incubation of
the transfected cells at 37 °C and cellular lysis 35-45 h after
transfection. Luminescence assays of cellular lysates allowed for a
semi-quantitative measure of luciferase production driven by each
cloned segment of the ICA1 5'-flanking region. Within a
given assay, plate wells were set up in triplicate for each transfected
construct or control vector. The amount of DNA transfected was held
constant for each construct and cell line, with a total amount 0.3 µg/well of a 12-well plate. Each Effectene reagent was used in the
amount recommended by the manufacturer's protocol in proportion to the
amount of DNA applied to each well. The strength of the promoting
activity for each construct was assessed by comparison to basal
luciferase expression from the promoterless pGL3 Basic vector
transfected into triplicate samples of the same cell type within the
same assay. To allow for normalization of firefly luciferase values based on transfection efficiency, a co-reporter vector expressing Renilla luciferase from the thymidine kinase promoter
(pRL-TK) was included at a ratio of 1:10 of co-reporter plasmid to
experimental promoter construct (or control vector) in the transfection
mixture. Careful optimization of transfection conditions to maximize
transfection efficiency provided an assay system yielding consistent
results from repeated experiments.
Transfected cells were lysed by adding 100 µl of Passive Lysis Buffer
(Promega, Madison, WI) to each well of a 12-well plate, followed by
vigorous pipetting of the detached cells. Cell lysates were subjected
to two freeze-thaw cycles (liquid N2 and 20 °C H2O) and either immediately assayed for luciferase activity
or stored at
In order to compare inter-construct firefly (FF) luciferase activity
values, the raw data relative light unit (RLU) readings were corrected
by normalizing each sample according to transfection efficiency. One
Renilla luciferase RLU (R-RLU) measurement from the pGL3
Basic control transfectant samples in a given experiment was selected
and used to normalize each measured FF luciferase value as follows:
((normalizing R-RLU) Oligonucleotide Synthesis--
All oligonucleotides used as
primers in the various PCR-based methods were synthesized on an ABI 394 DNA Synthesizer (Applied Biosystems, Inc.) using solid phase synthesis
and phosphoramidite nucleoside chemistry, unless a primer was provided
with a particular molecular biology kit.
Automated Fluorescent Sequencing--
Automated fluorescent
sequencing of plasmid DNA or purified PCR products was performed using
an ABI 377 Automated DNA Sequence Analyzer (Applied Biosystems, Inc.)
with either the dRhodamine or BigDye Terminator Cycle Sequencing Kits
(Applied Biosystems, Inc.). Typically, TA vector-cloned PCR products
were sequenced using the universal Sequencing of YAC and BAC Clone Ends--
To sequence YAC clone
ends, the junctions between the two YAC vector arms and the insert
sequence were first amplified and subcloned from a YAC fragment library
constructed using the Universal GenomeWalker (GW) kit
(Clontech Laboratories, Inc.). A gene-specific primer set (GSP1 and GSP2) was designed for each of the YAC vector arms
to be used in combination with the set of nested GenomeWalker adapter
primers provided with the GenomeWalker kit: HYAC-C,
5'-GCTACTTGGAGCCACTATCGACTACGCGAT-3' and LS-2,
5'-TCTCGGTAGCCAAGTTGGTTTAAGG-3' (left YAC arm); HYAC-D, 5'-GGTGATGTCGGCGATATAGGCGCCAGCAAC-3' and RA-2,
5'-TCGAACGCCCGATCTCAAGATTAC-3' (right YAC arm). Reaction
conditions suggested by the GenomeWalker kit were employed without
modification. Any PCR products amplified from the five YAC GenomeWalker
library reactions were subcloned into the pCR2.1 vector for
plasmid-based automated fluorescent sequencing.
The ends of each BAC clone were sequenced directly using 1 µg of
purified BAC clone DNA in each of two automated fluorescent sequencing
reactions extended from the two universal primers Sequence Homology Analyses--
Homology searches of nucleic
acid and protein amino acid sequences were conducted through the Basic
Local Alignment Search Tool (BLAST) server available on the National
Center for Biotechnology Information (NCBI) internet website
(www.ncbi.nlm.nih.gov).
GenBankTM Sequence Submissions--
Novel
ICA1 cDNA and promoter function-associated genomic
regions were submitted to the GenBankTM data base using the
BankIt submission tool available through the NCBI website. YAC and BAC
clone end sequences were submitted to the GSS data base via electronic
mail to the address: batch-sub{at}ncbi.nlm.nih.gov.
Transcription Factor Binding Site Analysis of 5'-Flanking
Sequences--
To assess the 5'-flanking and UTR regions of
ICA1 for potential regulatory sequences, genomic DNA
sequences of interest were analyzed using the public domain
MatInspector version 2.2 software program available on the internet
(genomatix.gsf.de/cgi-bin/matinspector/matinspector.pl). A core
similarity of Genomic Organization of the ICA1 5'-UTR Exons--
We identified a
single ICA1-containing CEPH Mega-YAC clone (CEPH-813G2) in a
PCR-based YAC library screen. By using DNA purified from this YAC
clone, we then initiated a cloning strategy involving primer-based
genome walking in the 5' direction from ICA1 exon 1, but we
failed to identify any ICA1 5'-untranslated sequence within
~12 kb of genomic DNA sequence upstream of the translation initiation
codon (data not shown). We did, however, identify a microsatellite
(18-20 adjacent CA dinucleotides) within this interval (~1830
bp upstream of the ATG), for which we designed flanking primers that
uniquely amplify this marker locus from CEPH-813G2 DNA as well as from
human genomic DNA. The microsatellite flanking primers were used in a
successful PCR-based screen of the California Institute of Technology
BAC (CITB) library. Two BAC clones, CITB-426N6 and CITB-503D2, were
identified as PCR-positive for the expected microsatellite amplimer band.
Sequence from a Human Genome Project (HGP) PAC clone (RP11-560C1,
GenBankTM accession number AC007009, R.H. Waterston, Genome
Sequencing Center, Washington University School of Medicine, St. Louis,
MO) that encompasses all three of the known ICA1 5'-UTR
sequences became publicly available shortly after we had identified the two ICA1-positive BAC clones. We confirmed these data (Fig.
1) by amplifying and directly sequencing
PCR products spanning the region of interest from our YAC and BAC clone
DNA using flanking primers. It must be noted that we have labeled the
ICA1 5'-UTR exon sequences as exons A, B, C rather than
adopt the
The ICA1 gene locus within its chromosomal context is
summarized in Fig. 2, demonstrating that
the ICA1 gene is transcribed from 7p22 in a Tel Analysis of cDNAs and ESTs from the ICA1 5'-UTR--
To
characterize the ICA1 gene transcription initiation site(s),
we first examined ICA69 cDNA and EST sequences available in the
NCBI GenBankTM data bases for messages with potentially
full-length 5' ends. As shown in Fig. 3,
publicly available human ICA1 transcripts generally do not
agree with respect to the sequence content and/or length of their
5'-untranslated leader regions. Three species of mRNA differing in
5' end sequence content are found, corresponding to the splicing of the
known exon A, B, and C sequences downstream to either of two splice
acceptor sites in ICA1 exon 1 common to all transcripts.
Splicing to exon 1 is variable, with some exon B transcripts using a
splice acceptor farther downstream than the splice site found in all
exon A transcripts.
Additionally, there is great variability in the 5' termini of sequences
from each of the exons depicted in Fig. 3. Notably, the 5' exon B
untranslated sequences from cDNA clone IS4 and ESTs AW583029,
BG484463, and BI754058 are short by comparison with the ESTs truncating
at the NotI restriction site, whereas the exon A clones
variably terminate over a range of 56 bp. The extension exon B
transcripts to the NotI restriction site is likely to be an
artifact of the EST library preparation method, which commonly employs
this rare cutter to generate sticky ends for cloning, so the 5' termini
of these clones have been cleaved off.
RLM-RACE Localization of the ICA1 Transcription Initiation
Sites--
We identified transcription initiation sites and determined
the lengths of ICA1 exons A-C by analyzing islet, testis,
heart, and NMB7 total cellular RNA via RNA ligase-mediated 5'-RACE
(RLM-RACE). Gene-specific primers (GSPs) for amplification of
ICA1 5'-UTRs were designed from the sequences of exons 1 and
2. Because these exons are common to all ICA69-encoding transcripts,
different ICA1 5' end sequence clones could be sequenced
individually regardless of the amplified UTR flanked by the known
RLM-RACE adapter and exon 1/2 sequence primers.
The results of our RLM-RACE analysis are summarized in Fig.
4. For each RNA sample analyzed at least
20 RACE-PCR-generated clones were independently sequenced. Overall,
significant agreement of the first nucleotide from the sequenced exon A
and B transcripts is noted both within and among the different tissues.
Although every sequenced clone beginning with exon A or exon B did not start at exactly the same nucleotide, a cluster of start sites within a
2-4-bp interval was consistently detected for the major species from
each exon. Additional variability in the exon A starting nucleotide was
observed for testis transcripts, as two clones extended beyond the
major start site and three clones proved to be shorter. The alternative
starting nucleotides for exon B transcripts appear to be utilized in a
somewhat tissue-specific manner. Most notably, every RLM-RACE clone
derived from heart tissue transcripts agrees with regard to 5'-UTR exon
length and starting 5'-nucleotide, extending ~35 nucleotides upstream
of the common exon B start site identified from testis, islet, and
neuroblastoma RNA samples.
Complete Genomic Organization of the ICA1 Gene--
By having
defined the sizes of the ICA1 5'-UTR exons, our knowledge of
ICA1 exon-intron organization is now complete (Table I). The genomic interval from the
RLM-RACE determined transcription initiation site for exon A through
coding exon 14 spans >148.7 kb as calculated from available chromosome
7 HGP data. Several of the intron distances determined from these data
differ significantly in comparison to the PCR-amplified size data
reported by Gaedigk et al. (4). Of note, the length of the
intron between exon C and exon 1 as well as the lengths of introns 2, 6, and 8 were previously unknown. Remarkably, intron 6 is 59,651 kb
long based on clone RP11-560C1 data, comprising 40.1% of total
ICA1 gene length. Significant refinements of intron lengths
over the previous report are noted for introns 3, 7, 12, and 13. For
example, the length reported for intron 3 by Gaedigk et al.
(4) was 6.5 kb; however, we confirmed the length of this intron to be
only 749-bp by amplifying and directly sequencing the intron with
primers designed from exons 3 and 4 (data not shown).
Defining ICA1 Promoter Function--
Our approach to analyzing the
ICA1 5'-flanking sequences for promoter activity was
instructed by the confirmation of initiation sites for transcription of
the ICA1 gene and assisted in part by a computer analysis of
the flanking sequences for potential transcription activator sequences
and transcription factor (TF)-binding sites. The sequence of this
region and the locations of TF clusters are presented in Fig.
5. Fig. 5 includes the entire sequence of interest from the functional ICA1 5'-flanking region, with
the UTR exon sequences and major transcription initiation sites
identified. Clusters of high scoring sequence matches to TF-binding
sites (MatInspector version 2.2 results) are arrayed schematically in Fig. 6A to
emphasize the high density of potential regulatory elements in
proximity to the three major ICA1 transcription initiation sites. A close inspection and analysis of the sequence also reveal that
it meets criteria to define a CpG island, specifically being a sequence
tract of >200 bp with GC content of >50% and an observed:expected ratio for the occurrence of the dinucleotide CG [O:E(CpG)] of >0.6
(17). For a 1000-bp segment of ICA1 5'-UTR flanking region extending downstream from base
The delineation of independent ICA1 5'-UTR exons required
that our promoter cloning strategy address the possibility that independent promoter activities are associated with the three separate
exons. Thus, we constructed four basic luciferase promoter reporter
plasmids (Fig. 6B). The first includes upstream flanking sequence contiguous with a downstream sequence interval encompassing all three 5'-UTR exons (ExABC
The results of our ICA1-luciferase promoter reporter assays
are summarized in Fig. 6C. Transfection of construct ExABC
Functional Impact of Site-directed Mutagenesis of ICA1 Promoter
Elements--
The results of luciferase assays involving the mutated
ExA
For exon B mutants, there are some very dynamic changes seen in both
cell lines, particularly with mutation of the CREB site (Fig.
7D). There is a 6.3-fold increase in luciferase activity in
RIN 1046-38 cells when transfected with the ExB-GC mutant, whereas a
similar increase is not seen when ExB-GC is transfected into NMB7 cells
(Fig. 7D). Interestingly, however, mutation of the exon B
CREB site results in a marked augmentation of luciferase expression in
both RIN 1046-38 and NMB7 cells, with 14.7- and 22.9-fold increases in
luciferase activity over pGL3 Basic, respectively, corresponding to
7.7- (p < 0.05) and 2.0-fold increases in activity over the parent ExB In the present study, we have explored the complex structure and
functional characteristics of the diabetes-related autoantigen gene
ICA1. Initiation of ICA1 transcription is found
to originate from any of three distinct 5'-untranslated exons having
independent transcription initiation signals characteristic of
non-TATA, non-CAAT, GC-rich promoters. Transcripts utilizing each of
the three 5'-UTR exons coexist in many ICA1-expressing
tissues, although the 5'-UTR exon sequences are never included together
in the same transcript. We present evidence, however, that exon A
transcripts predominate in islets and testis RNA samples as compared
with exon B or C transcripts, whereas exon B transcripts are the major
expressed form in neuronal and cardiac tissue RNA samples. No
additional ICA1 5'-UTR sequences were detected. An earlier
report (4) had suggested that the three 5'-UTR exons are alternatively
spliced, but our data conclusively demonstrate that the identified 5'
termini of these exons lack the appropriate splice acceptor consensus sequences (Fig. 5). Furthermore, the 5'-RLM-RACE procedure identified the same 5' end initiating nucleotides among ICA1
transcripts amplified from different tissues, suggesting that these
initiation sites are common starting points for transcription rather
than artifacts of the amplification procedure.
Parallel functional studies to screen cloned ICA1
5'-flanking sequences for transcription promoting activity further
support the conclusion that utilization of ICA1 5'-UTR exons
for transcription initiation is tissue-specific. Specifically, a
promoter reporter construct containing only exon A and its upstream
flanking sequence showed greater activity in islet-derived RIN 1046-38 cells than in neuroblastoma cells, whereas a second construct designed
from exon B and its upstream flanking sequence was preferentially
active in the neuron-derived cells rather than islets. Based on
sequence surrounding the identified transcription initiation sites, we originally hypothesized that the Sp1/GC box-Inr element paring was
likely to play a role in the activation of transcription from exon A,
given the common association of these consensus sequences reported in
the literature (18). However, when promoter sequences were mutated at
these sites, there was an associated increase in the activity of
luciferase transcription, at least as measured in islet-derived RIN
1046-38 cells. Although these experiments do not provide definitive
evidence to suggest a precise molecular mechanism to account for this
observation, they imply that the Sp1/GC box site plays an inhibitory
role in controlling exon A transcription. It seems reasonable to
postulate that Sp3, the Sp transcription factor family member
associated with transcription inhibition (19-21), rather than Sp1, is
the major transcription factor recognizing and binding to the GC box
site in RIN 1046-38 cells. With mutation of the exon A GC box, Sp3
cannot bind as well to the sequence and, therefore, has less chance to
inhibit expression from exon A. Similarly, a modest increase in
transcriptional activity within RIN 1046-38 cells is noted when the
exon B Sp1/GC box site is mutated, perhaps additional evidence that Sp3
plays a role in controlling ICA1 transcription within RIN
1046-38 cells, in contrast to NMB7 (neuronal) cells where these
mutations have no effect. Further experimentation, such as
electrophoretic mobility shift assays, designed to explore the
potential for Sp1/3 binding to the GC box sites will be necessary to
provide support for these hypotheses (22).
An understanding of the role for CREB site binding proteins in
controlling transcription from ICA1 exon B will also benefit from additional studies of transcription factor binding to
ICA1 promoter sequences. Mutation analysis of the exon B
CREB site, resulting in marked increases in luciferase activity for
both RIN 1046-38 and NMB7 cells, suggests a negative regulatory role for CREB on gene expression through binding to regulatory elements in
the ICA1 promoter (23-26). Likewise, Reusch et
al. (23, 27) reported that CREB plays a pivotal role in adipocyte
survival likely regulating the expression of specific pro- and
anti-apoptotic genes such as Akt. Thus, our data suggests that this
CREB-related influence may be less cell type-specific than the effects
of mutation at the GC box sites, but the effect on expression in
pancreatic islet-derived cells does appear to be of a greater magnitude
than that noted for neuron-derived cells.
The transcription factor CREB and its wide profile of inducibility has
mainly been implicated in glucose homeostasis, growth factor-dependent cell survival, and T-cell receptor
signaling (28). CREB was the first transcription factor for which it
was demonstrated that phosphorylation regulates its activity; the molecule is activated by cAMP and a variety of other signals. Its
family members consist of the activating transcription factor 1 (ATF1)
and the cAMP-response element modulator. CREB is a substrate for a host
of cellular kinases including AKT (29), p38/Ras (30), MAP-KAP-2 (31),
protein kinase C (32), pp90rsk (33), and
calcium-calmodulin kinases II (34) and IV (35). Although CREB is
perhaps one of the most studied phosphorylation-dependent transcription factors, relatively little is known about the
physiological role of this protein in different cellular
microenvironments. There is still discussion on how signal
discrimination is achieved within the CREB system. Even though several
signals have been shown to promote phosphorylation of CREB at Ser-133,
it is assumed that CREB can distinguish cAMP from non-cAMP signals at
the level of co-activator CREB-binding protein recruitment (28). The
characterization of cofactors modulating CREB-binding protein
commitment to a specific signaling pathway from a wide array of
cellular stimuli is currently under investigation.
Knowing that mutation at the CREB site within the ICA1 exon
B promoter enhances ICA1 transcriptional activity will be of
importance in completing ongoing experiments to augment expression of
ICA69 in islet cell lines that, in turn, may aid in elucidating the role for ICA69 in trafficking between the trans-Golgi network and
immature secretory granules of pancreatic Two additional elements of ICA1 promoter structure are
likely to provide important clues toward understanding the
tissue-specific aspects of ICA1 promoter function. First,
the high density of potential TF-binding sites surrounding the three
UTR exons provides fodder for mechanisms of transcription activation or
repression based on TF availability in different tissues and on the
potential involvement of known tissue-specific factors with potential
binding sites in the ICA1 promoter region, like GKLF
(gastrointestinal tract), OLF1 (olfactory neuroepithelium), MyoD
(myogenic cells), or MZF1 (myeloid cells). Second, existence of the
ICA1 promoter within a genomic CpG island hints at a role
for DNA methylation/demethylation in the control of ICA1
expression. The methylation state of genomic DNA, most often within CpG
islands, plays a central role in the epigenetics of gene imprinting
(38-41) and has been implicated, although perhaps not proven (37), as
an on/off switch for cell type-specific gene expression during cellular
differentiation (41-47). The observed differences in ICA1
promoter activity between RIN 1046-38 and NMB7 cells will be better
understood from studies investigating TF recognition and modulation of
the ICA1 promoter and through an analysis of DNA methylation
within native genomic DNA of the ICA1 CpG island sequence.
The variability of ICA1 transcripts, with respect to both
the identity of 5'-UTR sequences and coding region splicing events, is
likely to impact ICA69 protein expression. The potential contribution of alternative ICA1 splice forms to ICA69 translation is
obvious (4), although no studies to date have provided definitive
evidence for the existence of different ICA69 protein isoforms. On the other hand, 5'-UTR sequence identity could influence in vivo
ICA69 mRNA stability and translation efficiency, depending upon the secondary structure of the 5'-UTR (48) and upon the number of ATGs
contributed by a given 5'-UTR upstream of the accepted ICA69 translation start site in exon 1 (49). Although some variability of the
exon 1 splice acceptor for 5'-UTRs has been detected, all human
ICA1 transcripts known to us splice the 5'-UTR to one of a
few closely related exon 1 consensus acceptors upstream of the translation initiation codon (Ref. 4 and data not shown). Therefore, it
is unlikely that truly alternative translation initiation can occur.
Identifying which of these factors further enhances the tissue- and
cell type-specific differences in ICA1 expression that we
have observed and to what extent they influence ICA69 protein
translation remain important unanswered questions in our quest to
understand the biology of this molecule.
The sum of available ICA1 and ICA69 structural and
functional genetic data point to a complex process of transcript
generation from an expansive gene locus. Of note, the large introns
(largest almost 60 kb) and overall gene size (>150 kb) by nature
require a high fidelity of RNA processing to command the expression of a comparatively small protein (483 amino acids). From the standpoint of
understanding basic biology, the significance of introns and intron
size continues to be a subject of discussion in the literature (50-52). Available cDNA sequences suggest that opportunities for alternative intron/exon processing of ICA1 transcripts may
contribute to isoform variation of ICA69 protein expression (53),
although the particular influence that extremely large introns would
contribute to this process is unknown. That these large introns contain
other genes or genetic regulatory elements is a strong possibility (41, 51), especially considering that two ICA1 introns measure
~26 and ~60 kb, larger than many whole genes and easily large
enough to envelop genetic regulatory elements of importance to
ICA1 gene function. More simply, considering the remarkable
cross-species conservation of the ICA69 coding region, perhaps large
introns merely confer a relative level of protection against coding
region mutations and disruptive recombination events because these
processes would be more likely to affect non-coding intron sequences on a statistical basis (54, 55). It is difficult to draw any firm
conclusions regarding the significance of such huge introns to the
ICA1 gene; we anticipate that additional collective gene structure data from the various ongoing genome projects will enhance our understanding of these observations.
The basic structure and function of the ICA1 core promoter
units contributed by this work and the suggestion of candidate regions
harboring additional ICA1 5'-regulatory sequences will facilitate the targeted screening of genomic regions for polymorphisms that could alter ICA1 promoter function. There is already
evidence that a polymorphic variable number of tandem repeats in
the insulin promoter is functionally important to variations of insulin
expression and correlates with diabetes susceptibility (56-60). In
addition, multiple research groups have recently reported the
expression of ICA69, insulin, and the other major diabetes autoantigens
glutamic acid decarboxylase (GAD65) and IA-2 within cells of the thymus (60-65) and in peripheral lymphoid organs (66). The centrality of
these lymphoid tissues to the maintenance of immune self- tolerance suggests that perhaps at least one determinant of which proteins are
targeted as autoantigens is the level or nature of expression in
tissues other than where it has its characteristic biological effect.
Immunohistochemical and molecular analyses of autoantigen-containing cells within lymphoid tissues have identified features of a dendritic cell phenotype (65-67), implicating these powerful antigen presenting cells in the process of establishing and maintaining immune tolerance via de novo expression of self-protein antigens. These
observations lead us to postulate that inheritance of functional
polymorphisms within promoters controlling the expression of identified
autoantigens will ultimately be correlated with the occurrence of
autoimmunity and autoimmune diseases. With our fundamental
understanding of the structure and function of the ICA1 gene
promoters in hand, a rational approach to identify relevant
ICA1 promoter polymorphisms and to investigate disease
associations is now feasible.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cDNA libraries by immunoscreening with human
serum or by DNA probe hybridization (1, 4-6). Intron-exon boundaries
were established for the human and murine ICA1 genes using a
combination of
phage genomic DNA library screening and PCR
experiments (4). Collectively, these data argue for a high level of
evolutionary conservation of the ICA1 gene, not only upon
comparison of the human protein to the rat (6) and mouse (5) homologues
but also in terms of exon/intron partitioning (4).
cells, as the molecule is
known to be specifically expressed within islets (8) and by
insulin-producing cell lines maintained in culture (2). However, the
true cellular function of ICA69 and its importance to normal mammalian
pancreatic islet physiology remain unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol and lyticase enzyme (Sigma). Formation of yeast
spheroplasts was allowed to proceed for 1-2 h at 37 °C with gentle
shaking. Spheroplasted cells were then pelleted by centrifugation at
1000 × g for 10 min. Yeast cell lysis was achieved by
suspension of spheroplasts in lysis buffer (0.5 M Tris-Cl,
pH 8.0, 3% N-lauroylsarcosine, 0.2 M EDTA, 1 mg/ml proteinase K) buffer and incubation for 30-45 min at 65 °C in
a water bath. Overnight treatment of cell lysate with RNase PLUS (5 Prime
3 Prime, Inc., Boulder, CO) at 37 °C adequately removed
contaminating yeast RNA from the sample. Isolation of the DNA fraction
was achieved through two successive organic extractions with an equal
volume of 50:50 phenol/chloroform, followed by 3-4 extractions of the
aqueous phase with chloroform only. YAC and yeast DNA were
co-precipitated from the aqueous phase with 0.1 volumes of 3 M NaCl and 2.5 volumes of ice-cold 100% ethanol. After
gentle spooling, the precipitated DNA was washed in 70% ethanol and
air-dried. Purified DNA was resuspended in TE buffer, pH 8.0, with a
typical preparation yielding 300-700 mg of nucleic acid as measured by
A260 measurement.
-D-galactopyranoside (X-gal) for blue-white color selection of transformants (if applicable).
5'-exonuclease activity from the PCR enzyme or enzyme
mix used for amplification, the PCR product(s) were "tailed" with
dA-overhangs prior to ligation. Briefly, 1 unit of AmpliTaq
DNA polymerase was added to the completely cycled PCRs, incubated for
15 min at 37 °C, and immediately extracted with an equal volume of
phenol/chloroform. The tailed products were then ethanol-precipitated,
resuspended in a small volume (~1/2 of original reaction
volume), and directly used in the T/A ligation step.
957 and ExB
440, were modified using the QuikChange
Site-directed Mutagenesis Kit (Stratagene, Cedar Creek, TX) to
introduce mutations at suspected key sites within these promoters.
Sequence mutations were introduced to the Sp1/GC box, Inr, (Sp1/GC box + Inr), and CREB sites within ExA
957 using the following
oligonucleotides, respectively (mutant name and bases mutated are in
boldface and in parentheses):
5'-CCTGCCGGAGAGCAGGGtattGGTCACTCTGGGCGGCG (ExA-GC,
564 to
561), 5'-CGGAGAGCAGGGGCGGGGTggaggTGGGCGGCGGATCCG (ExA-Inr,
557 to
553),
5'-CCTGCCGGAGAGCAGGGtattGGTggaggTGGGCGGCGGATCCGAGC (ExA-GC/Inr, mutations of
564 to
561 and
557 to
553), and 5'-CCTGTCCGCCAGGTCATcggcACGCAAACGCTATGGCCACGTGG (ExA-CREB,
612 to
609). For the ExB
440 construct, the Sp1/GC box and CREB
sites were modified with the following oligonucleotides, respectively:
5'-CCGGTTCCTGCGCTCCCCaataCCCTTTCCCTCGCCTTCG (ExB-GC,
196
to
193) and
5'-CCCTTTCCCTCGCCTTCGatccACGCTGACGTCGGATGAGTG (ExB-CREB,
174 to
171). The mutation strategy of the QuikChange protocol was adhered to for all site-directed mutation reactions, using
the above oligonucleotides in combination with a reverse complement
sequence primer in each PCR-based mutagenesis reaction. After digestion
of the reactions with DpnI to remove non-mutated, methylated
DNA, each mutated plasmid reaction was used to transform XL1-Blue
supercompetent cells. Resultant colonies were then miniprepped and
screened via automated fluorescent sequencing for successful mutation incorporation.
70 °C for analysis the following day. Firefly and
Renilla luciferase activities of each lysate were measured
sequentially via manual reagent injection in a Monolight 2010 luminometer using the Dual-Luciferase Reporter Assay System
(Promega).
(sample R-RLU)) × (sample FF-RLU) = (Nml sample FF activity). The normalized (Nml)
triplicate values for each construct were then averaged to arrive at a
relative measure of luciferase activity for that ICA1
promoter reporter construct. Fold increases in promoter activity over
the pGL3 Basic vector were calculated from the following formula: (Avg
Nml sample FF activity)
(Avg Nml pGL3 Basic control) = (sample fold increased activity over pGL3 Basic); where Avg is
average. These calculations were performed independently for each
transfection experiment data set (n = 3-5), and the
average of all results obtained for a given ICA1 promoter
reporter construct was used as a measure of relative promoter strength.
Where indicated, statistical analysis of luciferase reporter data was
performed using the Mann-Whitney U test.
21 M13 and M13 reverse primers or
internal primers designed from insert sequences. Direct sequencing of
PCR products involved centrifugal filtration purification of the
amplified DNA on Amicon Microcon YM-50 columns (Millipore Corp.)
followed by sequencing with the same primers used for amplification.
Inserts contained within the pGL3 Basic vector were sequenced with
primers designed from regions flanking the multiple cloning site of the vector (pGL3-upstream, 5'-AGTGCAAGTGCAGGTGCCAG AA-3' and
pGL3-downstream, 5'-CTTTATGTTTTTGGCGTCTTCCAT-3') or with primers
internal to the cloned ICA1 sequence.
21 M13 and M13 reverse.
0.900 from the MatInspector analysis was used as a
cut-off for consideration of potential query sequence matches with
known transcription factor recognition sequences.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3,
2,
1 exon notation proposed by Gaedigk et
al. (4). This modification to the exon identifiers was made
because the genomic alignment of the UTR exons reported by these
authors is in error, as they had localized the true leading exon (exon
A or exon
2) between exons B (exon
3) and C (exon
1) in their
report of the exon-intron boundary data. We feel that maintaining the
negative integer notation for these exons would lead to confusion
regarding the overall genomic organization and promoter structure of
the ICA1 gene.
View larger version (16K):
[in a new window]
Fig. 1.
Organization of the ICA1
5'-UTR exons. Flanking PCR primers were used to amplify the
genomic region containing ICA1 5'-UTR exons from YAC and BAC
clone DNA. Because of the high GC content of the region, specialized
GC-rich PCR conditions were employed. Amplified PCR products were
directly sequenced using the indicated amplification primers,
confirming the UTR exon arrangement shown. The three 5'-UTR exons
localize >26 kb upstream of the translation initiation codon in exon 1 yet span a genomic interval of <600 bp. The three exons are not found
spliced to one another in any combination within isolated
ICA1 cDNAs or ESTs, indicating that the splicing to exon
1 is mutually exclusive.
Cen
orientation. The BAC clones CITB-426N6 and CITB-503D2 that we
identified share significant overlap with respect to their genomic
sequence content (according to BAC end sequencing results), and
together they span a gap that had once existed between HGP clones
RP11-560C1 and RP4-594A5. It is also notable that the RP11-560C1 PAC
clone insert begins ~10 kb to the 5' side of ICA1 exon A
and extends downstream to include every ICA1 exon with the
exception of exon 14. Sequence from the YAC clone ends has allowed us
to map our data onto HGP sequence data, with the YAC insert spanning
1.09 Mb of the HGP chromosome 7 working draft sequence (Fig. 2). The
entire ICA1 locus is contained within this YAC clone, along
with nearly 1 Mb of downstream sequence extending centromeric from
7p22.
View larger version (17K):
[in a new window]
Fig. 2.
Chromosomal context of isolated YAC and BAC
clones at the ICA1 locus. CEPH(Mega YAC)-813G2
intercepts a 1.1-Mb sequence mapping to HGP chromosome 7 clone
NT_007844.6. The two PAC clones RP4-733B9 (accession number AC005532)
and RP4-594A5 (accession number AC007128) define this interval, as they
yielded positive BLAST hits for left (L) and right
(R) CEPH-813G2 end sequences, respectively. The relationship
of the two BAC clones from our library screen (CITB-426N6 and
CITB-503D2) to PAC clones RP11-560C1 (accession number AC007009) and
the ICA1 locus was determined by BAC end sequencing and is
also illustrated. The only other defined gene encompassed by the YAC
sequence is RPA3 (replication protein A3), although C1GALT1
(UDP-galactose:N-acetylgalactosamine- -R
1,3-galactosyltransferase) lies within 1 Mb telomeric to the start
of ICA1 immediately beyond the end of the YAC clone insert.
The direction of transcription for each of these genes is indicated by
an arrow below the chromosome 7 clone NT_007844.6
sequence. Hypothetical genes derived from computer analysis of genome
data are not included in the diagram; however, it should be noted that
a high concentration of putative protein coding segments flanks the
left end of the YAC clone, whereas the remainder of the interval is
rather sparsely populated by potential genes or ESTs found in the data
base.
View larger version (35K):
[in a new window]
Fig. 3.
cDNA clones and ESTs containing
ICA1 5'-UTR exon sequence. A BLAST search of the
non-redundant (NR) and human EST data bases using genomic sequence from
the 5'-UTR region and exon 1 returned 19 matches for sequences
demonstrating splice patterns consistent with ICA1
transcripts as indicated by downstream splicing to exon 2 (not shown).
Alignment of these cDNA and EST clones does not clearly define a
consensus for initiation sites among the three UTR exons. Although
three of the exon A clones approach the size for this exon as
determined from our data (as per Figs. 4 and 5), these sequences were
not obtained with the purpose of defining transcription initiation
sites for the gene. For exon B containing transcripts, the apparent
agreement of sequences terminating at 230 is likely an artifact
related to the existence of a NotI restriction site at this
location. The remaining exon B clones exhibit little agreement in their
5' termini. The numerical annotation adopted for this figure is
consistent with that used in the text and is arbitrarily defined by
designating the last base of exon C as
1. All of the untranslated
sequences are numbered negatively from this point upstream along the
genomic sequence continuum. The first base of exon 1 is numbered +1. An
internal exon 1 alternative splice acceptor site occurs around +66, and
an asterisk denotes the location of the translation
initiation codon at +80 relative to this splice acceptor site.
View larger version (26K):
[in a new window]
Fig. 4.
Summary of RLM-RACE results for
ICA1 initiation. Four different sources of RNA
were assayed to determine the frequency of use for the 5'-UTR exons in
ICA1 transcription initiation. Four major transcript
variants (exons A, B1, B2, and C) that include sequence from the 5'-UTR
exons were identified. Exon A and exon B transcripts were more highly
represented overall as compared with exon C transcripts. Notably, exon
A transcripts were absent from the set of neuroblastoma and heart
ICA1 transcripts sequenced, whereas the heart transcript set
was dominated by the expression of the longer exon B2 transcript
variant. The asterisk indicates that for transcripts
appearing to begin in exon 1, there was no one dominant starting
nucleotide and that this region is the same region in which alternative
splice acceptance of 5'-UTR exons occurs. The signifies that the
"Total n" reflects a larger number of sequences than
those included in the table. The remaining few sequences variably
terminated at points within exons A and B but without an apparent
pattern.
Summary of ICA1 exon-intron lengths and genomic organization
680, the sequence is composed of
73.9% GC bases and has an O:E(CpG) ratio of 0.81.
View larger version (58K):
[in a new window]
Fig. 5.
Sequence of the ICA1 5'-UTR
exons and TF-binding sites in the 5'-flanking region. The entire
sequence of the ICA1 5'-UTR exons and flanking regions is
depicted with exon sequences displayed in capital letters
and intron sequences in all lowercase letters. The consensus
Inr sequence at the major initiation site for exon A is in
boldface, as are the major initiating nucleotides for exons
B and C. Although the exon A Inr is an ideal match to the pyrimidine
(Py)-rich consensus Inr sequence (PyPyA+1N(T/A)PyPy), the
initiating nucleotides for exon B transcription also exist within
pyrimidine-rich tracts that associate closely with Sp1-binding site
motifs. Other close matches to TF consensus sequences are indicated as
underlined segments. The strand polarity of TF recognition
sequences is indicated by either a + for sense or for antisense
orientation.
View larger version (21K):
[in a new window]
Fig. 6.
A, TF-binding site sequence matches
identified by the MatInspector version 2.2 program are illustrated in a
schematic depiction of the ICA1 5'-UTR exons and flanking
region. Clustering of potential TF-binding sites surrounding exon A and
around 900 is indicated by dotted outlines on the figure.
Each of the three exons is preceded by an Sp1/GC box site, whereas exon
A has two additional Sp1/GC box sites at its 3' end. Of the remaining
TF indicated on the figure, many are constitutively expressed in cells,
but others are noted to be somewhat more tissue-specific
(i.e. MyoD and OLF1). B, summary of luciferase
reporter vector constructs used to assess ICA1 5'-flanking
region promoter activity. Shown are the basic structures of the four luciferase reporter vectors
constructed using portions of ICA1 flanking region sequence
and 5'-UTR exons. Dashed lines indicate where sequence is
missing, such that the joined segments would be directly juxtaposed in
the plasmid vector. The ExA
957 and ExB
440 were specifically
designed to omit the native exon A and B splice donors, respectively,
so that cryptic splicing events would be less likely to affect
luciferase gene translation. C, results of luciferase
reporter assays for ICA1 promoter constructs. Each of the
four experimental constructs and the control (no insert) vector were
transfected separately into each of the two cell lines indicated. Fold
increase in luciferase activity was calculated from the raw data set as
described in the text. Transfection of the exon A and B only constructs
(ExA
957 and ExB
440) resulted in opposite expression profiles in
the islet- versus neuron-derived cell lines. This
observation is consistent with a tissue-specific pattern of expression
from the ICA1 exons A and B. Expression from the exon C
construct was less efficient, perhaps due to the inclusion of the
native exon C splice donor in the luciferase reporter vector. Results
are expressed as mean ± S.E. of 3-5 independent sets of
transfection experiments performed in triplicate.
1012, total plasmid insert length 1031 bp). The three remaining reporter plasmids contain sequences from only
one of the 5'-UTR exons, along with a reasonable amount of upstream
flanking sequence truncated so as not to include any portion of the
preceding exon (ExA
957, 453 bp; ExB
440, 293 bp; and ExC
90, 109 bp). The ExA
957 and ExB
440 constructs were engineered to exclude
the splice donor site at their 3' termini so that cryptic splicing
events would be minimized during transcription of the luciferase
reporter gene in vivo. The two cell lines used in our
promoter reporter assays were chosen based on evidence for cellular
ICA69 expression assessed by RT-PCR and on their similarities with
tissues having the highest levels of ICA69 expression, namely
pancreatic islets (rat insulinoma cells, RIN 1046-38) and neuronal
tissue (human neuroblastoma cells, NMB7).
1012 resulted in 1.3- and 3.9-fold increases in reporter gene
(luciferase enzyme) activity in RIN 1046-38 and NMB7 cells,
respectively, as measured in a dual luciferase assay. Interestingly,
however, transfection of cells with the independent exon A and B
constructs (ExA
957 and ExB
440) demonstrated a difference in
promoting activity that was dependent upon the transfected cell type.
The ExA
957 construct was more active in RIN 1046-38 (rat insulinoma) cells, exhibiting a 2.5-fold increase in luciferase activity, whereas
the ExB
440 construct showed a greatly enhanced, 12-fold level of
activity in NMB7 (neuroblastoma) cells. The augmentation of luciferase
activity for the isolated exon A and B constructs over the ExABC
1012
construct is thought to result from the exclusion of splice donors from
the exon 3' ends that may be facilitating cryptic splicing in the
luciferase transfection system. The construct designed to isolate exon
C from the other 5'-UTR exons (ExC
90), includes the exon C splice
donor signal and 19 bp of 3'-flanking sequence. Transfection of this
plasmid did not result in any significant increase in luciferase
activity as compared with the empty pGL3 Basic control vector in either
of the cell lines tested.
957 (Fig. 7A) and ExB
440 (Fig. 7C) constructs are summarized in
Fig. 7, B and D. In NMB7 cells, it is again
obvious that there is very little transcriptional activity from exon A,
as the parent ExA
957 construct and all four mutants showed
essentially no activity over background pGL3 Basic transcription (Fig.
7B). This finding correlates with the results of our
transcript analysis detailed above. For RIN 1046-38 cells, however, the
ExA
957 mutants exhibit increases in promoter activity for mutation
of the Sp1/GC box and the Inr independently, as well as for the double
mutant combination of the two (Fig. 7B). The ExA-CREB mutant
shows no difference in promoting activity over the parent ExA
957
construct (Fig. 7B).
View larger version (23K):
[in a new window]
Fig. 7.
Site-directed mutations of promoter elements
in transiently transfected RIN 1046-38 and NMB7 cells.
A, alignment of the parent exon A (WT) and mutant
sequences (Mut) introduced to the Sp1/GC box (ExA-GC, 564
to
561), Inr (ExA-Inr,
557 to
553), and CREB sites within exon A
(ExA-CREB,
612 to
609). B, for RIN 1046-38 cells, the
ExA
957 mutants exhibit increases in promoter activity for mutation of the Sp1/GC box and the Inr
independently, as well as for the double mutant combination of the two
[Sp1/GC box + Inr]. The ExA-CREB mutant shows no difference in
promoting activity over the parent ExA
957 construct. C,
alignment of the parent exon B (WT) and mutant sequences
(Mut) introduced to the Sp1/GC box (ExB-GC,
196 to
193),
and CREB sites within exon B (ExB-CREB
174 to
171). D,
mutation of the exon B CREB site leads to a significant increase in
luciferase expression in both RIN 1046-38 and NMB7 cells, corresponding
to 7.7- (p < 0.05) and 2.0-fold enhancement in
activity over the parent ExB
440 vector. Results are expressed as
mean ± S.E. of at least 3 independent sets of transfection
experiments performed in triplicate. By using Mann-Whitney test, *,
p < 0.05 comparing the mean of results for the
mutation of the exon B CREB site with the mean of results for the
parent exon B. p values < 0.05 were deemed
statistically significant.
440 vector (Fig. 7D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells that has been
proposed (36).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. William Rudert, Robert Ferrell, Timothy Wright, and Michael Gorin for helpful discussions and insights; Dr. Ram Menon and Angel Shaufl for assistance with luciferase assays; Dr. Alessandro Doria (Joslin Diabetes Center, Boston) for providing facilities and instruction in BAC library screening; Dr. David Patterson (Eleanor Roosevelt Institute, Denver, CO) for YAC library screening; Dr. Christopher Newgard (Duke University, Durham, NC) for providing the RIN 1046-38 cell line; and Chip Scheide for computer support.
![]() |
FOOTNOTES |
---|
* This work was supported by the University of Pittsburgh M.D.,Ph.D. Program (to R. P. F.), the Henry Hillman Endowment Chair in Pediatric Immunology (to M. T.), National Institutes of Health Grants R01 DK53456 and R01 DK56200 (to M. P.), and an American Diabetes Association Career Development award (to M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF146364, BZ286433, BZ286436, BAC503D2, BZ286434, BZ286435, YAC813G2, BZ286437, YAC813G2, and BZ286438.
¶ Present address: Medical Services-GRB 740, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114-2696.
To whom correspondence should be addressed: Division of
Immunogenetics, Diabetes Institute, Rangos Research Center, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, 3460 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-692-6491; Fax: 412-692-8131; E-mail: pietroma+@pitt.edu.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M210175200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: UTR, untranslated region; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; RLM, RNA ligase-mediated; CITB, the California Institute of Technology BAC Library; CEPH, the Centre d'Etude du Polymorphisme Humain; Inr, initiator; CREB, cAMP-responsive element-binding protein; NOD, non-obese diabetic; FF, firefly; RLU, relative light unit; NCBI, National Center for Biotechnology Information; HGP, Human Genome Project; GSP, gene-specific primers; TF, transcription factor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Pietropaolo, M., Castaño, L., Babu, S., Buelow, R., Kuo, Y.-L., Martin, S., Martin, A., Powers, A. C., Prochazka, M., Naggert, J., Leiter, E. H., and Eisenbarth, G. S. (1993) J. Clin. Invest. 92, 359-371[Medline] [Order article via Infotrieve] |
2. | Karges, W., Pietropaolo, M., Ackerley, C., and Dosch, H.-M. (1996) Diabetes 45, 513-521[Abstract] |
3. |
Pilon, M.,
Peng, X. R.,
Spence, A. M.,
Plasterk, R. H.,
and Dosch, H.-M.
(2000)
Mol. Biol. Cell
11,
3277-3288 |
4. | Gaedigk, R., Karges, W., Hui, M. F., Scherer, S. W., and Dosch, H.-M. (1996) Genomics 38, 382-391[CrossRef][Medline] [Order article via Infotrieve] |
5. | Karges, W., Gaedigk, R., Hui, M. F., Cheung, R. K., and Dosch, H.-M. (1996) Biochim. Biophys. Acta 1360, 97-101 |
6. | Miyazaki, I., Gaedigk, R., Hui, M. F., Cheung, R. K., Morkowski, J., Rajotte, R. V., and Dosch, H.-M. (1994) Biochim. Biophys. Acta 1227, 101-104[Medline] [Order article via Infotrieve] |
7. | Gaedigk, R., Duncan, A. M. V., Miyazaki, I., Robinson, B. H., and Dosch, H.-M. (1994) Cytogenet. Cell Genet. 66, 274-276[Medline] [Order article via Infotrieve] |
8. | Stassi, G., Schloot, N., and Pietropaolo, M. (1997) Diabetologia 40, 120-122[CrossRef][Medline] [Order article via Infotrieve] |
9. | Martin, S., Kardorf, J., Schulte, B., Lampeter, E. F., Gries, F. A., Melchers, I., Wagner, R., Bertrams, J., Roep, B. O., Pfutzner, A., Pietropaolo, M., and Kolb, H. (1995) Diabetologia 38, 351-355[CrossRef][Medline] [Order article via Infotrieve] |
10. | Roep, B. O. (1996) Diabetes 45, 1147-1156[Abstract] |
11. | Roep, B. O., Duinkerken, G., Schreuder, G. M. Th., Kolb, H., DeVries, R. R. P., and Martin, S. (1996) Eur. J. Immunol. 26, 1285-1289[Medline] [Order article via Infotrieve] |
12. |
Miyazaki, I.,
Cheung, R. K.,
Gaedigk, R.,
Hui, M. F.,
Van der Meulen, J.,
Rajotte, R. V.,
and Dosch, H.-M.
(1995)
J. Immunol.
154,
1461-1469 |
13. |
Winer, S.,
Gunaratnam, L.,
Astsatourov, I.,
Cheung, R. K.,
Kubiak, V.,
Karges, W.,
Hammond-McKibben, D.,
Gaedigk, R.,
Graziano, D.,
Trucco, M.,
Becker, D. J.,
and Dosch, H.-M.
(2000)
J. Immunol.
165,
4086-4094 |
14. | Karges, W., Hammond-McKibben, D., Gaedigk, R., Shibuya, N., Cheung, R., and Dosch, H. M. (1997) Diabetes 46, 1548-1556[Abstract] |
15. | Lampasona, V., Ferrari, M., Bosi, E., Pastore, M. R., Bingley, P. J., and Bonifacio, E. (1994) J. Autoimmun. 7, 665-674[CrossRef][Medline] [Order article via Infotrieve] |
16. | Chaplin, D. D., and Brownstein, B. H. (1992) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , John Wiley and Sons, New YorkUnit 6.10 |
17. | Gardiner-Garden, M., and Frommer, M. (1987) J. Mol. Biol. 196, 261-282[Medline] [Order article via Infotrieve] |
18. | Carey, M., and Smale, S. T. (2000) Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
19. |
Braun, H.,
Koop, R.,
Ertmer, A.,
Nacht, S.,
and Suske, G.
(2001)
Nucleic Acids Res.
29,
4994-5000 |
20. |
De Luca, P.,
Majello, B.,
and Lania, L.
(1996)
J. Biol. Chem.
271,
8533-8536 |
21. | Hagen, G., Muller, S., Beato, M., and Suske, G. (1994) EMBO J. 13, 3843-3851[Abstract] |
22. |
LeVan, T. D.,
Bloom, J. W.,
Bailey, T. J.,
Karp, C. L.,
Halonen, M.,
Martinez, F. D.,
and Vercelli, D.
(2001)
J. Immunol.
167,
5838-5844 |
23. |
Reusch, J. E.,
and Klemm, D. J.
(2002)
J. Biol. Chem.
277,
1426-1432 |
24. | Choi, R. C., Siow, N. L., Zhu, S. Q., Wan, D. C., Wong, Y. H., and Tsim, K. W. (2001) Mol. Cell. Neurosci. 17, 732-745[CrossRef][Medline] [Order article via Infotrieve] |
25. | Cibelli, G., Jungling, S., Schoch, S., Gerdes, H. H., and Thiel, G. (1996) Eur. J. Biochem. 236, 171-179[Abstract] |
26. | Della Fazia, M. A., Servillo, G., and Sassone-Corsi, P. (1997) FEBS Lett. 410, 22-24[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Reusch, J. E.,
Colton, L. A.,
and Klemm, D. J.
(2000)
Mol. Cell. Biol.
20,
1008-1020 |
28. | Mayr, B., and Montminy, M. (2001) Nat. Rev. Mol. Cell. Biol. 2, 599-609[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Du, K.,
and Montminy, M.
(1998)
J. Biol. Chem.
273,
32377-32379 |
30. | Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K. A., Boxer, L. M., Heasley, L. E., and Reusch, J. E. (2000) J. Biol. Chem. 14, 10761-10766[CrossRef] |
31. | Tan, Y., Rouse, J., Zhang, A., Cariati, S., Cohen, P., and Comb, M. J. (1996) EMBO J. 15, 4629-4642[Abstract] |
32. | Yamamoto, K. K., Gonzalez, G. A., Biggs, W. H., III, and Montminy, M. R. (1998) Nature 334, 494-498 |
33. | Xing, J., Ginty, D. D., and Greenberg, M. E. (1996) Science 273, 959-963[Abstract] |
34. | Sun, P., Enslen, H., Myung, P. S., and Maurer, R. A. (1994) Genes Dev. 8, 2527-2539[Abstract] |
35. | Matthews, R. P., Guthrie, C. R., Wailes, L. M., Zhao, X., Means, A. R., and McKnight, G. S. (1994) Mol. Cell. Biol. 14, 6107-6116[Abstract] |
36. | Solimena, M., Spitzenberger, F., Pietropaolo, S., Verkade, P., Habermann, B., Lacas-Gervais, S., Mziaut, H., Trucco, M., and Pietropaolo, M. (2002) Diabetes Metab. Rev. 18 Suppl. 4, 32 (abstr.) |
37. |
Jones, P. A.,
and Takai, D.
(2001)
Science
293,
1068-1070 |
38. | Paulsen, M., and Ferguson-Smith, A. C. (2001) J. Pathol. 195, 97-110[CrossRef][Medline] [Order article via Infotrieve] |
39. | Pfeifer, K. (2000) Am. J. Hum. Genet. 67, 777-787[CrossRef][Medline] [Order article via Infotrieve] |
40. | Siegfried, Z., Eden, S., Mendelsohn, M., Feng, X., Tsuber, B.-Z., and Cedar, H. (1999) Nat. Genet. 22, 203-206[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Beohar, N.,
and Kawamoto, S.
(1998)
J. Biol. Chem.
273,
9168-9178 |
42. | Cao, Y.-X., Jean, J.-C., and Williams, M. C. (2000) Biochem. J. 350, 883-890[CrossRef][Medline] [Order article via Infotrieve] |
43. | Lübbert, M., Tobler, A., and Daskalakis, M. (1999) Leukemia (Baltimore) 13, 1420-1427[CrossRef] |
44. |
Newell-Price, J.,
King, P.,
and Clark, A. J.
(2001)
Mol. Endocrinol.
15,
338-348 |
45. | Persengiev, S. P., and Kilpatrick, D. L. (1996) Neuroreport 8, 227-231[Medline] [Order article via Infotrieve] |
46. |
Schwab, J.,
and Illges, H.
(2001)
Int. Immunol.
13,
705-711 |
47. | Takizawa, T., Nakashima, K., Namihira, M., Ochiai, W., Uemura, A., Yanagisawa, M., Fujita, N., Nakao, M., and Taga, T. (2001) Dev. Cell 1, 749-758[Medline] [Order article via Infotrieve] |
48. | Ross, J. (1995) Microbiol. Rev. 59, 423-450[Abstract] |
49. | Kozak, M. (1991) J. Cell Biol. 115, 887-903[Abstract] |
50. | Dibb, N. J. (1993) FEBS Lett. 325, 135-139[CrossRef][Medline] [Order article via Infotrieve] |
51. | Duret, L. (2001) Trends Genet. 17, 172-175[CrossRef][Medline] [Order article via Infotrieve] |
52. | Hurst, L. D., Brunton, C. F. A., and Smith, N. G. C. (1999) Trends Genet. 15, 437-439[CrossRef][Medline] [Order article via Infotrieve] |
53. | Hanke, J., Brett, D., Zastro, I., Aydin, A., Delbrück, S., Lehmann, G., Luft, F., Reich, J., and Bork, P. (1999) Trends Genet. 15, 389-390[CrossRef][Medline] [Order article via Infotrieve] |
54. | Carvalho, A. B., and Clark, A. G. (1999) Nature 401, 344[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Comeron, J. M.,
and Kreitman, M.
(2000)
Genetics
156,
1175-1190 |
56. | Davies, J. L., Kawaguchi, Y., Bennett, S. T., Copeman, J. B., Cordell, H. J., Pritchard, L. E., Reed, P. W., Gough, S. C., Jenkins, S. C., Palmer, S. M., Balfour, K. M., Rowe, B. R., Farrall, M., Barnett, A. H., Bain, S. C., and Todd, J. A. (1994) Nature 371, 130-136[CrossRef][Medline] [Order article via Infotrieve] |
57. | German, M. (2000) in Diabetes Mellitus: A Fundamental and Clinical Text (LeRoith, D. , Taylor, S. I. , and Olefsky, J. M., eds), 2nd Ed. , pp. 11-19, Lippincott Williams & Wilkins, Philadelphia |
58. | Lucassen, A. M., Screaton, G. R., Julier, C., Elliott, T. J., Lathrop, M., and Bell, J. I. (1994) Hum. Mol. Genet. 4, 501-506[Abstract] |
59. | Kennedy, G. C., German, M. S., and Rutter, W. J. (1995) Nat. Genet. 9, 292-298 |
60. | Pugliese, A., Zeller, M., Fernandez, A., Jr., Zalcberg, L. J., Bartlett, R. J., Ricordi, C., Pietropaolo, M., Eisenbarth, G. S., Bennett, S. T., and Patel, D. D. (1997) Nat. Genet. 15, 293-297[Medline] [Order article via Infotrieve] |
61. | Egwuagu, C. E., Charukamnoetkanok, P., and Gery, I. (1997) J. Immunol. 159, 3109-3112[Abstract] |
62. |
Sospedra, M.,
Ferrer-Francesch, X.,
Dominguez, O.,
Juan, M.,
Foz-Sala, M.,
and Pujol-Borrell, R.
(1998)
J. Immunol.
161,
5918-5929 |
63. | Smith, K. M., Olson, D. C., Hirose, R., and Hanahan, D. (1997) Int. Immunol. 9, 1355-1365[Abstract] |
64. | Vafiadis, P., Bennett, S. T., Todd, J. A., Nadeau, J., Grabs, R., Goodyer, C. G., Wickramasinghe, S., Colle, E., and Polychronakos, C. (1997) Nat. Genet. 15, 289-292[Medline] [Order article via Infotrieve] |
65. | Werdelin, O., Cordes, U., and Jensen, T. (1998) Scand. J. Immunol. 47, 95-100[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Pugliese, A.,
Brown, D.,
Garza, D.,
Murchison, D.,
Zeller, M.,
Redondo, M.,
Diez, J.,
Eisenbarth, G. S.,
Patel, D. D.,
and Ricordi, C.
(2001)
J. Clin. Invest.
107,
555-564 |
67. | Pietropaolo, M., Giannoukakis, N., and Trucco, M. (2002) Nat. Immunol. 3, 335 |