Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 2507–2512, March 1997
Immunology
Fig1, an interleukin 4-induced mouse B cell gene isolated by
cDNA representational difference analysis
CHARLES C. CHU* AND WILLIAM E. PAUL†
*North Shore University Hospital, Department of Medicine, Manhasset, NY 11030; and †Laboratory of Immunology, National Institute of Allergy and Infectious
Diseases, Bethesda, MD 20892
Contributed by William E. Paul, December 26, 1996
ABSTRACT
Interleukin 4 (IL-4) is a cytokine that regu-
lates growth and differentiation of lymphoid and nonlym-
phoid cells. To study the molecular basis of IL-4 function, we
used a cDNA subtraction approach based on the represen-
tational difference analysis method. This subtractive am-
plification technique allowed us to use small amounts of
RNA from lipopolysaccharide
IL-4-stimulated normal B
cells to obtain IL-4-induced genes from these cells. We
report here on one such gene, Fig1 (interleukin-four induced
gene 1), the first characterized immediate–early IL-4 in-
ducible gene from B cells. Fig1 expression is strikingly
limited to the lymphoid compartment. B cells, but not T cells
or mast cells, express Fig1 in response to IL-4 within 2 hr in
a cycloheximide resistant manner. IL-2, IL-5, and Il-6 do not
induce Fig1 in culture. Fig1 maps between Klk1 and Ldh3 on
mouse chromosome 7, near two loci involved with murine
lupus, Sle3 and Lbw5. The Fig1 cDNA sequence encodes a
predicted 70-kDa flavoprotein with best homology to the
monoamine oxidases, particularly in domains responsible
for FAD binding.
Interleukin 4 (IL-4) is a multifunctional cytokine that regulates
growth and differentiation among many cell types. It was first
recognized because of its actions as a comitogen of B cells and
as a determinant of immunoglobulin (Ig) class switching
specificity of B lymphocytes stimulated with lipopolysaccha-
ride (LPS). It is now known to promote B cell survival in
culture and to induce transcription and/or expression of a
series of genes including the germ-line and mature forms of the
Ig and 1 H chains, major histocompatibility complex class
II molecules, Thy-1, and CD23 (1). In general, the induction of
these genes by IL-4 is relatively slow (2 days), although
germ-line 1 H chain (I1) transcripts have been observed
within 4 hr of culture of normal B cells with IL-4 (2). Genes
induced earlier in the response to IL-4 have not been charac-
terized in this or any other cell type. To understand the
molecular basis of IL-4 function, an analysis of such ����early����
genes would be most valuable.
To identify IL-4-induced genes in normal B cell cultures, we
used a subtractive cDNA hybridization and amplification
method based on genomic representational difference analysis
(RDA) (3). We report here the isolation and extensive char-
acterization of one such gene,
Fig1, a putative flavoprotein
with homology to monoamine oxidase (MAO).
Fig1 is induced
in resting B cells within 2 hr in response to IL-4 alone; its
induction was not inhibited by cycloheximide. Thus, it qualifies
as an immediate–early IL-4-inducible gene.
MATERIALS AND METHODS
Mice. Female 6–16-week-old BALB/c mice, obtained from
the Frederick Cancer Research and Development Center,
National Cancer Institute (Frederick, MD), were maintained
and used in experiments under National Institutes of Health
guidelines that meet or exceed standards set forth by the
National Research Council (4).
Cell Culture. As described (5), resting B cells were prepared
from the 60–70% Percoll gradient fraction of dispersed
splenocytes treated with antibodies [monoclonal rat anti-
mouse anti-Thy1.2 (HO-13-4-9) (6), anti-CD5 (53-7.313) (7),
anti-CD8 (3.155) (8), and mouse anti-rat (MAR 18.5) (9)] and
complement. B cells were cultured in complete RPMI 1640
medium (5) supplemented, depending on the protocol of the
experiment, with 20
g/ml LPS (LPS W
Escherichia coli
055:B5, Difco), 20 g/ml cycloheximide (Sigma), 400 units/ml
recombinant human IL-2 (gift from Steven Rosenberg, Na-
tional Cancer Institute, Bethesda), and the following baculo-
virus-derived recombinant mouse cytokines: 100–10,000
units/ml IL-4 (10), 15 ng/ml (150 units/ml) IL-5 (R & D
Systems), and 1000 units/ml IL-6 (Genzyme).
RNA Preparation. Total RNA was prepared using a guani-
dine thiocyanate protocol (11) or a similar RNAzol (Biotecx
Laboratories, Houston) protocol. Briefly, 2
107 to 1
108
cultured cells or homogenized tissue from 1–2 mice were lysed
in guanidine thiocyanate solution, extracted with a phenol/
chloroform solution, and then precipitated with isopropanol.
The resulting RNA was resuspended in diethyl-pyrocarbonate-
treated H2O and quantitated by UV absorption measurements
at 260 nm.
cDNA RDA Subtractive Amplification. Amplicons were
prepared from poly(A) mRNA isolated from B cells cultured
for 12 and 36 hr with LPS (driver) or LPS and IL-4 (tester) by
converting to cDNA, digesting with
Sau3AI restriction en-
zyme, ligating adaptor sequences to ends of
Sau3AI fragments,
filling in ends, and amplifying by PCR using a primer with
sequence complementary to the adaptor (unpublished work).
Driver amplicons were modified by PCR amplification with
biotinylated primer. Tester amplicons were modified by re-
placing adaptor with a different adaptor sequence.
For the first round of subtraction, a 20:1 ratio of driver to
tester amplicon (combined 12 and 36 hr) was denatured and
hybridized together. After complete hybridization, driver-
containing hybrids were removed by streptavidin magnetic
beads and tester hybrids were RDA amplified by PCR with
primers specific for the tester adaptor, resulting in RDA
amplicon 1. RDA amplicon 1 was modified by replacing the
adaptors with yet another adaptor sequence. To this, a 100-fold
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ����
advertisement���� in
accordance with 18 U.S.C. ��1734 solely to indicate this fact.
Copyright
1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA
0027-8424/97/942507-6$2.00/0
PNAS is available online at
http://
www.pnas.org.
Abbreviations: IL, interleukin; RDA, representational difference
analysis; LPS, lipopolysaccharide; EF, elongation factor; MAO, mono-
amine oxidase; TMO, tryptophan 2-monooxygenase; PDS, phytoene
desaturase; FRD/SDH, fumarate reductase/succinate dehydroge-
nase; RACE, rapid amplification of cDNA ends.
Data deposition: The sequences reported in this paper have been
deposited in the GenBank database (accession nos. U70429, U70430,
and U80211).
2507
excess of biotinylated driver amplicon was added for the
second round of subtraction. Driver-containing hybrids were
depleted and tester hybrids were RDA amplified as above,
resulting in RDA amplicon 2. After cloning this material, the
sequence of 154 individual clones revealed 37 different genes,
including
Fig1. Two different cDNA RDA subtractive ampli-
fication clones, 20-8T#17 and 20-8T#22, cover overlapping
parts of
Fig1ps and
Fig1, respectively.
cDNA Library Clones. A gt11 cDNA library made from
random-primed cDNA prepared from CBA/J resting splenic
B cells stimulated with 300 units/ml of IL-4 for 18 hr (CLON-
TECH) was used to isolate nearly full-length cDNA clones of
Fig1. PCR product (50 ng), containing the insert of subtraction
clone 20-8T#22 (505 bp), was labeled with [-32P]dCTP (3000
Ci/mmol, 1 Ci
37 GBq; Amersham) using an oligolabeling
kit (Pharmacia) and used to probe for
Fig1 cDNA clones. After
three rounds of plaque hybridization according to the manu-
facturer��s directions, four clones were isolated: 20-11T1, 20-
11T2, 20-11T3, and 20-11T4. Phage DNA was prepared from
each clone according to manufacturer��s instructions and insert
cDNA was subcloned into pBluescript SK (Stratagene). A
3.5-kb
BsiWI–
PvuI fragment from 20-11T1 was ligated to
Asp718I-digested pBluescript SK resulting in a linear frag-
ment. This fragment was blunt-ended with T4 DNA polymer-
ase (Boehringer Mannheim) according to manufacturer��s pro-
tocol and then circularized by ligation, resulting in plasmid
20-12R1-9. A 0.6-kb
BsiWI fragment from 20-11T2 was ligated
into the
Asp718I site of pBluescript SK, resulting in plasmid
20-12C2-1. A 2.0-kb
BsiWI fragment from 20-11T3 was ligated
into the
Asp718I site of pBluescript SK, resulting in plasmid
20-12C3-5. A 4.8-kb
BsiWI–
HindIII fragment from 20-11T4
was ligated into the
Asp718I–
HindIII sites of pBluescript SK,
resulting in plasmid 20-12T4-2.
RACE (Rapid Amplification of cDNA Ends). To obtain the
5 end of the
Fig1 cDNAs, we performed 5 RACE using the
5-AmpliFINDER RACE kit (CLONTECH). First-strand
cDNA was prepared from 1 g poly(A) RNA from LPSIL-
4-stimulated B cells (12
36 hr). The AmpliFINDER anchor
oligonucleotide was ligated to the 3 end of the cDNA using
RNA ligase in a 10-l reaction. This and other oligonucleotides
used in this work are listed in Table 1. An aliquot (1 l of 1:10
dilution) of this material was amplified by PCR in a 50-l
reaction containing 50 mM KCl, 22.5 mM Tris (pH 9.0), 0.2
mM dNTP, 2.0 mM MgCl2, 1 M CCC53 or CCC54 primer,
1 M anchor primer, 2.5 units
Taq DNA polymerase, and
0.00625 unit
Pfu DNA polymerase (Stratagene) in a GeneAmp
PCR system 9600 thermal cycler under the following condi-
tions: 94C for 30 sec, 35 cycles of [94C for 5 sec, 55C for 15
sec, 72C for 1 min], 72C for 2 min, and a final soak at 4C.
This material was diluted 106-fold and 1 l reamplified with a
nested primer CCC55, CHU44, or CCC58 and anchor primer
or CCC56 as above, except with a higher annealing tempera-
ture (63C or 65C). The obtained PCR products were ana-
lyzed by 1% agarose gel electrophoresis and products greater
than 200 bp were cut out from the gel and purified (12).
Purified PCR product was ligated into TA cloning vector pCR3
(Invitrogen); the resulting RACE clones obtained are shown
in Table 2.
Sequencing from Plasmid or PCR Product. Plasmid DNA
was prepared from 4.5-ml cultures of individual clones by a
modified mini-alkaline lysis/polyethylene glycol precipitation
procedure recommended by Applied Biosystems (Perkin–
Elmer). Alternatively, plasmid DNA was prepared by a Qiagen
Plasmid Midi kit (Qiagen, Chatsworth, CA) involving a similar
alkaline lysis procedure followed by binding to and elution
from a plasmid DNA binding column.
Plasmid DNA was initially sequenced using United States
Biochemical (Amersham) Sequenase Version 2.0 DNA Se-
quencing kit with [-33P]dATP (Amersham) and appropriate
oligonucleotide primer. Primers CCC32, CCC34, CCC52,
NEB#1218, and NEB#1222 were used in the preliminary
sequencing of cDNA library subclones. Sequence reactions
were electrophoresed through 6% or 8% polyacrylamide se-
quencing gels, which were then dried down, autoradiographed,
and read.
Plasmid DNA of cDNA library subclones, 20-12R#1-9 and
20-12C#3-5 was sequenced initially with CCC34 and CCC35
primers or NEB#1218 and NEB#1222 primers, respectively,
by Paragon Biotechnology on a 373 Stretch Sequencer (Ap-
plied Biosystems) using an ABI PRISM Dye Terminator Cycle
Sequencing Ready Reaction kit with Ampli
Taq DNA poly-
merase FS (Applied Biosystems Part Number 402122). The
sequence of these cDNA library subclones was completed by
primer walking (sequences available on request).
RACE clones were sequenced from purified PCR product.
PCR product was prepared from boiled bacteria (unpublished
work) using CCC35 and CCC57 primers and purified through
Chromaspin-100 columns (CLONTECH). Purified PCR prod-
Table 1. Oligonucleotides
Name*
Sequence (5 3 3)†
Source‡
AmplifFINDER anchor
cacgaattcaCTATCGATTCTGGAACCTTCAGAGG
CLONTECH
Anchor primer
ctggttcggcccaCCTCTGAAGGTTCCAGAATCGATAG
CLONTECH
CCC32 (SK primer)
TCTAGAACTAGTGGATC
LI
CCC34 (T3 primer)
AATAACCCTCACTAAAG
LI
CCC35 (T7 primer)
AATACGACTCACTATAG
LI
CCC52 (
Fig1 524–542)
cggaattcggtaccGGAGAAGATGCCAGAAAAG
Paragon
CCC53 (
Fig1ps 1165–1149)
cggaattcggtacCACAAGACTCTTGGGGG
Paragon
CCC54 (
Fig1 790–773)
cggaattcggtacCGTAAGGCTTCTGCAAAG
Paragon
CCC55 (
Fig1 610–588)
cgtgatcaagcTTGAGTGCCATCTGGTAGATGTC
Bio-Synthesis
CCC56
CCTCTGAAGGTTCCAGAATCGATAG
Bio-Synthesis
CCC57 (SP6 primer)
ATTTAGGTGACACTATAG
Bio-Synthesis
CCC58 (
Fig1 410–393)
GTGAGAGCTGGGCATTCG
Bio-Synthesis
CHU3 (
Fig1ps 2213–2194)
CCTCGGACATCACATCTCCC
Paragon
CHU39 (
Fig1ps 1711–1730)
GGGCCAGTAGAGGTGGGACT
Paragon
CHU44 (
Fig1ps 811–789)
AGGAGTCGGGGTGGGGATAGGGG
Paragon
gt11 forward primer
GGTGGCGACGACTCCTGGAGCCCG
NEB#1218
gt11 reverse primer
TTGACACCAGACCAACTGGTAATG
NEB#1222
*Oligonucleotides complementary to
Fig1 or partially spliced
Fig1 (
Fig1ps) have sequence coordinates shown.
†Lowercase sequences indicate added restriction site tails for CCC52, CCC53, CCC54, and CCC55. AmpliFINDER anchor
and anchor primer lowercase letters indicate sequences not complementary to each other.
‡Oligonucleotides were synthesized in the Laboratory of Immunology (LI) as described (5) or purchased from CLONTECH,
Paragon Biotechnology, Bio-Synthesis (Lewisville, TX), or New England Biolabs.
2508
Immunology: Chu and Paul
Proc. Natl. Acad. Sci. USA 94 (1997)
uct (100 ng/l) was sequenced as before by Paragon Biotech-
nology with the same primers used to generate the PCR
product.
Sequence Analysis. With the aid of the VAXcluster at the
Frederick Biomedical Supercomputing Center (National Can-
cer Institute, Frederick, MD) and the GCG computer package
(Genetics Computer Group, Madison, WI), the full-length
Fig1 cDNA sequence was assembled. Coding sequence was
predicted by CODONUSE program (Conrad Halling, personal
communication). Regions of
Fig1 protein structure and ho-
mology were determined using the GCG package including
PROFILESCAN (13), supported BLAST (14) service provided by
the National Center for Biotechnology Information (Be-
thesda), and FASTA program (15) on the VAXcluster, and
BLOCKS on the internet (http://www.blocks.fhcrc.org) (16).
Comparisons were made to GenBank (release 97.0), EMBL
(release 47.0), and Swiss-Prot (release 34.0) databases.
Northern Blot Analysis. Denatured RNA samples were
electrophoresed in 1% agarose/formaldehyde gels and North-
ern blotted to supported nitrocellulose membrane (Optitran,
Schleicher & Schuell) by downward capillary method (12).
After baking for 1 hr at 80C in a vacuum oven, membranes
were prehybridized at 42C in prehyb buffer [40% deionized
formamide (Fluka)/4 SSC/10 mM Tris (pH 7.5)/2 Den-
hardt��s solution/0.1 mg/ml denatured salmon sperm DNA] for
1 hr and then hybridized overnight at 42C to radioactive-
labeled probe in hyb buffer (same as prehyb buffer except it
includes 10% dextran sulfate). Probes used were either from
cDNA RDA subtraction clone 20-8T#22 (Chromaspin-100-
purified PCR product of the
Fig1 insert) or clone 20-8T#40
[gel-purified 400-bp
Sau3AI-digested PCR product containing
the elongation factor-2 (EF-2) insert] labeled with
[-32P]dCTP as above (
cDNA Library Clones). Membranes
were washed two times in 2 SSC/0.1% SDS at 42C for 15
min and two times in 0.1 SSC/0.1% SDS at 65C for 30 min.
After air drying, membranes were autoradiographed by expo-
sure to x-ray film (Kodak XAR-2) with intensifying screens at
70C from 19 hr to 11 days.
Genetic Mapping. To map the genetic location of
Fig1, we
used the BSS [(C57BL/6JEi
Mus spretus SPRET/Ei)
SPRET/Ei] Backcross DNA Panel Map Resource (The Jack-
son Laboratory) (17). We identified a polymorphism between
C57BL/6 and
M. spretus by sequencing a 607-bp PCR product
that spans an intron–exon border. PCR was performed in two
20-l reactions containing 50 ng
M. spretus genomic DNA, 50
mM KCl, 22.5 mM Tris (pH 9.0), 0.2 mM dNTP, 1.5 mM
MgCl2, 1 M CHU3 primer, 1 M CHU39 primer, 2.5 units
Taq DNA polymerase, and 0.00625 unit
Pfu DNA polymerase
in a GeneAmp PCR system 9600 thermal cycler under the
following conditions: 94C for 30 sec, 35 cycles of [94C for 5
sec, 64C for 15 sec, 72C for 30 sec], 72C for 2 min, and a final
soak at 4C. The PCR reactions were combined with 10 l of
10 mM Tris/1 mM EDTA (pH 8.0), purified through Chro-
maspin-100 columns, and sequenced by Paragon Biotechnol-
ogy using CHU3 and CHU39 primers. Comparison of
M.
spretus sequence (GenBank accession no. U80211) to
Fig1ps
sequence revealed
HphI and
BfuI restriction enzyme site
polymorphisms in this fragment. This polymorphism was con-
firmed to exist between
M. spretus and C57BL/6 by restriction
enzyme digestion of PCR product generated from genomic
DNA using CHU3 and CHU39 primers. The
HphI polymor-
phism was used to type PCR product generated from each
sample in the BSS Backcross DNA Panel using CHU3 and
CHU39 primers. The typing results were then sent to The
Jackson Laboratory for determination of mapping position.
RESULTS
Subtraction. To isolate IL-4-induced genes from a subtrac-
tion between small populations of normal mouse B lympho-
cytes with very slight differences, we developed a PCR method
of subtraction based on genomic RDA (3), which we combined
with a physical separation method using magnetic beads
(unpublished work). This cDNA RDA technique was used to
isolate genes expressed in BALB/c splenic B cells cultured in
LPS and IL-4 after subtraction from cDNA obtained from B
cells cultured with LPS only. Of the 37 genes obtained, three
were strongly induced by IL-4: the previously characterized
immunoglobulin C1 gene fragment, the mouse homolog of
human transcription factor E4BP4, and an unknown gene with
no sequence homology to the public databases.
Fig1 Gene. We have further characterized this unknown
gene originally called clone 20-8T#17/22, which we have
renamed
Fig1 (interleukin-
four
induced
gene
1). Northern blot
analysis of RNA from B cells stimulated with LPS
IL-4
confirmed that
Fig1 is induced by IL-4 (data not shown).
Fig1
probes hybridize to mRNAs of 2.0 and 3.5 kb.
The full-length cDNA sequences of both
Fig1 mRNAs were
obtained and shown schematically in Fig. 1. The sequence of
the larger mRNA reveals that it is a partially spliced pre-
mRNA (
Fig1ps) that still contains two introns. The first exon
Table 2. RACE clones
First round
primers
Second round
primers
RACE
clones
Insert, bp
CCC53/anchor
CHU44/anchor
20-13#T20
722
20-13#T49
588
CCC54/anchor
CCC55/anchor
20-13#M38
313
20-13#M45
294
CCC53/anchor
CCC58/CCC56
20-13#V5
443
20-13#V8
435
CCC54/anchor
CCC58/CCC56
20-13#U20
234
20-13#U28
269
20-13#U29
445
FIG. 1. Schematic of both 2.0- and 3.5-kb
Fig1 mRNA sequences.
The sequences of two distinct
Fig1 mRNAs were largely obtained from
cDNA library clones and the remaining 5 end sequences were
obtained by RACE. Both mRNA forms are shown schematically with
coding regions shown in large solid rectangles and 5 and 3 untrans-
lated regions in large shaded rectangles; areas corresponding to
introns and to the poly(A) tail are shown in thin rectangles. Exons and
introns of
Fig1ps are numbered. Locations of original subtraction
library clones 20-8T#17 and 20-8T#22 are indicated. Clone 20-8T#17
contains exon 2 and part of intron 2 from
Fig1ps. Clone 20-8T#22
contains exon 2 and part of exon 3 from
Fig1. , splice sites, with one
indicating a splice site found in the 607-bp genomic sequence obtained
for genetic mapping. ,
Sau3AI restriction enzyme sites. Lengths in
bp are shown in parentheses.
Fig1 (GenBank accession no. U70429)
and
Fig1ps (GenBank accession no. U70430) had slight sequence
differences (7 bp) in the untranslated area close to the poly(A) tail.
Immunology: Chu and Paul
Proc. Natl. Acad. Sci. USA 94 (1997)
2509
could possibly encode a short protein of 13 kDa, which would
have no additional coding sequence contributed by the first
intron due to an immediate translational stop codon found in
the intron.
Fig1ps does not represent genomic DNA since some
introns have been spliced out. A 607-bp genomic sequence
revealed that a short intron in exon 3 has been spliced out (Fig.
1 and genetic mapping). Homology to MAO (see below)
suggests many more introns have been removed. Thus,
Fig1ps
is unlikely to produce a functional protein and most likely
represents a partially spliced pre-mRNA.
The sequence of the shorter mRNA (
Fig1) reveals a single
large open reading frame with good codon usage that encodes
a 630 amino acid protein with a predicted mass of 70 kDa. The
full-length sequence contains some amino acid homology to
many proteins. These homologies were not found in the
sequence of the two original cDNA RDA subtraction library
clones 20-8T#17 and 20-8T#22.
The predicted
Fig1 protein contains a putative signal peptide
sequence for translation into the endoplasmic reticulum and
hence may possibly be secreted (Fig. 2). It contains three
possible
N-glycosylation sites and two possible tyrosine phos-
phorylation sites. The protein is mostly hydrophilic with five
pockets of hydrophobic nature (including the signal peptide
sequence). The strongest homology is found with proteins that
bind FAD or NAD as a cofactor. Five areas (1–5), which may
be involved in FAD binding are illustrated in Fig. 2: (
i) binds
the ADP moiety of FAD based on homology to a host of FAD
and NAD binding proteins that contain the consensus se-
quence for the dinucleotide binding fold (18); (
ii) binds the
ribityl moiety of FAD based on homology to MAO (19); (
iii)
may bind to an unknown portion of FAD based on its
identification by the PROFILESCAN program and its homology
to MAO; (
iv) may bind to another unknown region of FAD
based on its identification by PROFILESCAN and its homology to
phytoene desaturase (PDS); and (
v) probably binds the flavin
component of FAD based on homology to MAO (20). Of these
five areas, the homologies in
iii and
iv have not been clearly
defined as FAD binding regions. The best homologies were
found with MAO (23% identical over 460 amino acids),
tryptophan 2-monooxygenase (42% identical over 92 amino
acids), and PDS (38% identical over 80 amino acids). Another
weak area of homology to the active site of the fumarate
reductase/succinate dehydrogenase (FRD/SDH) family of
proteins (26% over 47 amino acids) (21) was uncovered by the
BLOCKS program. Although most similar to MAO,
Fig1 is not
MAO, because it does not contain the MAO conserved
cysteine residue used to covalently bind FAD, its splice sites
are in different locations, and despite five large areas of
homology (Fig. 2), there are large stretches of nonhomologous
amino acids in contrast to rat and human MAO, which are 88%
identical over 519 amino acids. Based on these homologies,
Fig1 appears to be an enzymatic flavoprotein with a possible
active site similar to FRD/SDH.
Fig1 Genetic Mapping.
Fig1 was genetically mapped using
The Jackson Laboratory BSS Backcross DNA Panel Mapping
Service to the proximal portion of mouse chromosome 7
between the
Klk1 and
Ldh3 markers (Fig. 3). Human
Fig1 is
deduced to reside on chromosome 19q13.3-q13.4 because
mouse
Fig1 is located between genetic markers that are
syntenic with this portion of human chromosome 19 (see The
Jackson Laboratory BSS backcross mapping data at the World
Wide Web address http://www/jax/org/resources/documents/
cmdata). Interestingly, several genetic traits that affect anti-
body responses map to this region. In the SAM-P/1 mouse,
one of two genes responsible for the
Lar (low antibody
response) trait maps to the proximal portion of chromosome
7 (22). In the mouse (NZW
NZB)F1 hybrid lupus model, two
susceptibility genes,
Lbw5 and
Sle3, map to this region (23, 24).
FIG. 2. Schematic of predicted
Fig1 protein. The results of com-
puter program-aided sequence analysis of
Fig1 is summarized in
schematic form. Possible N-linked glycosylation sites (N-Gly) and
tyrosine phosphorylation sites (Y) are indicated. Locations of se-
quence inferred from original subtraction library clones 20-8T#17 and
20-8T#22 are indicated. Predicted hydrophobic regions and FAD
cofactor-binding regions (numbered 1–5) are shown. In addition,
regions homologous to many known FAD binding and NAD binding
proteins (MANY) and to specific FAD binding proteins (MAO, PDS,
TMO, and FRD/SDH) are shown. The predicted signal peptide
domain is also indicated (SIGNAL). , Location of the homologous
FRD/SDH active site. The consensus sequence for the dinucleotide
binding fold (FAD-binding region 1) is -#-X-#-G-X-G-X-X-G-X-
X-X-#-X-X-#-X-X-X-X-X-X-#-X-#-X-.
, a hydrophilic, posi-
tively charged residue; #, a hydrophobic residue; X, any amino acid;
G, glycine; , an acidic residue.
FIG. 3. Genetic mapping. Haplotype figure from The Jackson
Laboratory BSS backcross showing part of chromosome 7 with loci
linked to
Fig1. Loci are listed in order, with the most proximal at the
top. Solid boxes represent the C57BL6/JEi allele and open boxes the
SPRET/Ei allele. The number of animals with each haplotype is given
at the bottom of each column of boxes. The percent recombination (R)
between adjacent loci, equivalent to centimorgans, is given to the right
of the figure, with the standard error (SE) for each R.
FIG. 4. Tissue expression of
Fig1. Total RNA (10 g) prepared
from the indicated mouse tissues was electrophoresed, Northern
blotted, and probed with
Fig1 or EF-2 (internal mRNA ����housekeep-
ing���� standard). Ethidium bromide stained 28S and 18S rRNA bands
are shown for comparison.
2510
Immunology: Chu and Paul
Proc. Natl. Acad. Sci. USA 94 (1997)
Fig1 Expression. Expression of
Fig1 is strikingly limited to
lymphoid tissues (Fig. 4), with highest expression in lymph
node and spleen. A trace amount of
Fig1 is detectable in
thymus upon longer exposure.
In B cells,
Fig1 is induced specifically by IL-4 and not by
several other type I cytokines (IL-2, IL-4, IL-5, and IL-6). This
induction does not require LPS (Fig. 5).
Fig1 is induced within
the first 2 hr and remains elevated for at least 36 hr (Fig. 5).
Fig1 induction by IL-4 is cycloheximide resistant implying that
new protein synthesis is not required for its induction (Fig. 6).
Thus,
Fig1 is the first immediate–early IL-4-inducible gene
characterized in B cells.
Fig1 is
not expressed in naive T cells; there may be some very
weak
Fig1 expression in response to IL-4 in primed TH1 and
TH2 cells (data not shown). Mast cells treated with or without
IL-4 also do not express
Fig1 (data not shown). Thus,
Fig1
appears to be a largely B cell-specific IL-4-inducible gene.
DISCUSSION
We have isolated
Fig1 from mouse B cells by the cDNA RDA
method, a PCR-based subtraction technique.
Fig1 expression
has thus far been observed only in lymphoid tissues and
appears to be largely limited to expression to B cells. It is
induced within 2 hr in normal mouse B cells and its induction
is cycloheximide resistant. Thus, it is the first characterized
IL-4-dependent immediate–early gene.
Initial studies using a mouse B cell line transfected with
various mutants of the human IL-4 receptor indicate that the
region of the receptor required for STAT6 activation is
required for upregulating
Fig1 expression (C.C.C., J. J. Ryan,
and W.E.P., unpublished results). This is consistent with
requirements for induction of other IL-4-dependent genes but
differs from requirements for IL-4-mediated cell growth (25).
Is
Fig1 only inducible by IL-4? IL-2, IL-5, and IL-6 did not
induce
Fig1 in culture. However, we have not tested a variety
of other factor combinations
in vitro. Indeed,
Fig1 mRNA
expression in lymph nodes of IL-4 knockout mice was similar
to that of the wild type (unpublished results), indicating the
existence of an IL-4-independent mechanism for induction of
Fig1. This is probably not via cytokines that signal through
receptors that contain the c chain (IL-2, IL-7, IL-9, IL-15),
because the IL-2 receptor includes the c chain in common
with the IL-4 receptor. Whether such
Fig1 expression requires
the IL-4 receptor chain, possibly activated by IL-13, remains
to be established. Other receptor systems that signal via
STAT6 (platelet-derived growth factor, leptin, B cell antigen
receptor) may be involved (25). However, the possibility of a
totally different signaling system for
Fig1 induction must be
considered.
Genetic mapping located
Fig1 to a region of mouse chro-
mosome 7 that contains
Lbw5 and
Sle3, which are involved in
the lupus-like disease found in (NZB
NZW)F1 hybrid mice.
Because two different groups (23, 24) identified this mutation
in the same region,
Lbw5 and
Sle3 are likely to be the same
gene. The idea that this lupus-associated locus is the same as
Fig1 is attractive, because
Fig1 is expressed in antibody-
producing B cells induced with IL-4 (a regulator of the
humoral immune response). Preliminary analysis of the induc-
tion by IL-4 and size of
Fig1 transcripts in B cells of (NZB
NZW)F1 hybrid and
Sle3 affected mice (24) show no notice-
able alterations (L. M. Morel, C.C.C., W.E.P., and E. K.
Wakeland, unpublished results). However, the possibility still
exists that
Fig1 may be mutated at the protein level in these
mice.
The function of
Fig1 remains to be solved. Its homologies
suggest that it is a secreted FAD binding protein. From its
similarity to MAO, it is tempting to speculate that
Fig1 may
also inactivate monoamine transmitters in a similar manner to
the MAO inactivation of serotonin. This is particularly pro-
vocative in view of the role IL-4 plays in allergic inflammatory
responses.
We thank Hangjiong Chen, Cyndy Watson, Carol Kinzer, S. Z.
Ben-Sasson, Jane Hu-Li, and Hua Huang for help in preparing cell
cultures and RNA, Lucy Rowe for the mapping figure, Michail
Sitkovsky for cycloheximide, Shirley Starnes for preparation of this
manuscript, and Keats Nelms and Ronald Germain for helpful dis-
cussion.
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