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The Journal of Neuroscience, December 1, 2002, 22(23):10217-10231
Identification of Genes That Are Downregulated in the Absence of
the POU Domain Transcription Factor pou3f1 (Oct-6, Tst-1, SCIP)
in Sciatic Nerve
John R.
Bermingham Jr1,
Susan
Shumas2,
Tom
Whisenhunt3,
Erich E.
Sirkowski2,
Shawn
O'Connell3,
Steven S.
Scherer2, and
Michael G.
Rosenfeld3
1 McLaughlin Research Institute, Great Falls, Montana
59405, 2 Department of Neurology, University of
Pennsylvania, Philadelphia Pennsylvania 19104-6077, and
3 Howard Hughes Medical Institute, Department of Medicine,
University of California, San Diego, La Jolla, California 92093-0648
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ABSTRACT |
Despite the importance of myelinating Schwann cells in health and
disease, little is known about the genetic mechanisms underlying their
development. The POU domain transcription factor pou3f1 (Tst-1,
SCIP, Oct-6) is required for the normal differentiation of myelinating
Schwann cells, but its precise role requires identification of the
genes that it regulates. Here we report the isolation of six genes
whose expression is reduced in the absence of pou3f1. Only one of these
genes, the fatty acid transport protein P2, was
known previously to be expressed in Schwann cells. The LIM domain proteins cysteine-rich protein-1 (CRP1) and CRP2 are
expressed in sciatic nerve and induced by forskolin in cultured Schwann cells, but only CRP2 requires pou3f1 for normal expression. pou3f1 appears to require the claw paw gene product for
activation of at least some of its downstream effector genes.
Expression of the novel Schwann cell genes after nerve injury suggests
that they are myelin related. One of the genes,
tramdorin1, encodes a novel amino acid transport protein
that is localized to paranodes and incisures. Our results suggest that
pou3f1 functions to activate gene expression in the differentiation of
myelinating Schwann cells.
Key words:
myelin; pou3f1; Oct-6; Tst-1; SCIP; claw paw; Schwann cells; tramdorin; dendrin; CRP1; CRP2; P2; representational difference analysis
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INTRODUCTION |
Schwann cells generate myelin in the
peripheral nervous system. One myelinating Schwann cell forms a single
myelin sheath around a single axon, whereas nonmyelinating Schwann
cells typically ensheathe several unmyelinated axons without
myelinating any of them. Axons are thought to regulate the
differentiation of Schwann cells; presumptive myelinated axons signal
Schwann cells into the myelinating lineage, whereas the axons lacking
this signal remain associated with nonmyelinating Schwann cells (Mirsky
and Jessen, 1996 , 1999; Taylor and Suter, 1997; Garbay et al.,
2000).
pou3f1 [also known as octomer-6 (Oct-6), Testes-1 (Tst-1), suppressed
cAMP-inducible POU (SCIP), Octamer transcription factor-6 (Otf6)] is a
member of the POU domain family of transcription factors (for
review, see Ryan and Rosenfeld, 1997). pou3f1 is expressed transiently
during Schwann cell development (Monuki et al., 1989), specifically in
promyelinating Schwann cells, which have formed a 1:1 relationship with
axons but have not yet formed a myelin sheath (Zorick et al., 1996;
Arroyo et al., 1998). In the absence of pou3f1, the differentiation of
myelinating Schwann cells is delayed, transiently arrested at the
promyelinating stage (Bermingham et al., 1996; Jaegle et al.,
1996).
The role of pou3f1 in Schwann cell differentiation is unknown. It has
often been considered to be a repressor of Schwann cell gene expression
and/or differentiation. Cotransfection experiments suggest that pou3f1
inhibits the expression of the P0
(Mpz), myelin basic protein (Mbp), and p75
(low-affinity neurotrophin receptor) genes (Monuki et al., 1990, 1993;
He et al., 1991). Furthermore, an N-terminal deletion of pou3f1
( SCIP) acts as a dominant-negative inhibitor of pou3f1 repression of
a 1.1 kb rat Mpz (P0) promoter in
cotransfection experiments. In transgenic mice, SCIP causes precocious PNS myelination, taken as evidence that pou3f1 represses the
progression from promyelinating Schwann cells to myelinating Schwann
cells (Weinstein et al., 1995). However, Schwann cells in
pou3f1-null mice show delayed differentiation, and their
expression of myelin-related mRNAs, including those of Mpz
and Mbp, was not higher than in wild-type mice (Bermingham
et al., 1996; Jaegle et al., 1996). These results contradict the idea
that pou3f1 represses the expression of these genes in Schwann cells.
To reconcile these observations, pou3f1 has been hypothesized to
activate genes required for Schwann cell differentiation but to repress
terminal differentiation (Mirsky and Jessen, 1996; Zorick and Lemke,
1996; Jaegle and Meijer, 1998). In this scheme, pou3f1-null
mice lack both functions, whereas SCIP inhibits only the repression function.
Our approach sought to identify target genes of pou3f1 in Schwann cells
to better understand the mechanisms by which it controls their
differentiation. Representational difference analysis (RDA), a
PCR-based technique that permits the isolation of DNA fragments that
are present in one DNA sample but absent in another (Lisitsyn and
Wigler, 1993; Lisitsyn, 1995), was used with a carrier RNA facilitating
the isolation of differentially expressed genes (Erkman et al.,
2000; Bermingham et al., 2001). We found six genes that are
misexpressed in the sciatic nerves of pou3f1-null mice, and the expression of five of these genes correlates with the expression of
other myelin-related genes after nerve injury. Because all of these
mRNAs are expressed at lower levels in the sciatic nerves of
pou3f1-null mice, pou3f1 appears to be an activator of gene expression in Schwann cells. The identification of these genes indicates that pou3f1 functions to induce membrane biogenesis, cytoskeletal rearrangement, and amino acid transport during the differentiation of myelinating Schwann cells.
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MATERIALS AND METHODS |
Isolation of RNA from cultured Schwann cells. Schwann
cells were isolated from 3-d-old rat pups (Brockes et al., 1979) and expanded on 10 cm plates coated with
poly-L-lysine in DMEM supplemented with 10% FCS,
2 µM forskolin (Porter et al., 1987), and 10 ng/ml recombinant human secreted  isoform of neuregulin (glial
growth factor-2). The cells were passaged six times, grown to
confluence, and then maintained for 3 d in DMEM and 10% FCS. The
cells were maintained for an additional 3 d in DMEM and 10% FCS
alone or supplemented with 20 µM forskolin. All
of the cultures used in these experiments were essentially free of
fibroblasts. RNA was isolated from rat sciatic nerves and Schwann cells
by CsCl2 gradient centrifugation (Chirgwin et
al., 1979).
Isolation of RNA from sciatic nerves from lesioned rat sciatic
nerves. Adult Sprague Dawley rats were anesthetized with 50 mg/kg
pentobarbital intraperitoneally, and the sciatic nerves were exposed at
the obturator tendon. To prevent axonal regeneration, nerves were
doubly ligated and transected between the ligatures. Nerves were
crushed by compression with flattened forceps twice, each time for 10 sec. Animals were allowed to survive for various periods of time before
being killed by CO2 inhalation. For RNA extraction, several millimeters of nerve adjacent to the lesion site
were trimmed off, and the distal nerve stumps were frozen in liquid
nitrogen. Where indicated, the distal stumps of crushed nerves were
subdivided into proximal and distal segments of equal lengths.
Unlesioned nerves were taken from animals of various ages. Total RNA
was isolated by CsCl2 gradient centrifugation (Chirgwin et al., 1979).
Isolation of RNA from sciatic nerves from newborn mice.
pou3f1TM1Rsd mutant mice (Bermingham et
al., 1996) were maintained either on a pure 129Sv genetic background or
on a mixed 129/Sv-C57BL/6 genetic background. Newborn mice on the
129/Sv genetic background were euthanized by decapitation. Sciatic
nerves were dissected from hindquarters and immediately frozen in
liquid nitrogen. Additional tissue was taken for genotyping by PCR
(Bermingham et al., 1996). Homozygous mutant and homozygous wild-type
nerves were pooled separately in Trizol reagent (Invitrogen,
Carlsbad, CA), and RNA and DNA were purified according to the
manufacturer's instructions. The DNA was analyzed by PCR to confirm
the purity of the samples.
Carrier RNA. The template that lacks sites for
DpnII and NlaIII was chosen and generated as
follows. A 584 bp BstXI-XbaI fragment of
Drosophila genomic DNA, located 1310 bp 5' to the
transcription initiation site of the Antennapedia
(Antp) P1 promoter (Laughon et al.,
1986), was cloned into the XbaI and SmaI sites of
pBKSII+ (Stratagene, La Jolla, CA). Oligonucleotides
encoding an artificial poly(A) tract were introduced adjacent to the
Antp sequences. The carrier itself was synthesized in
vitro using T7 polymerase (Promega, Madison, WI).
cDNA synthesis. Carrier RNA (150 ng) was added to 1 µg of
total RNA; the amount of carrier added was designed to bring the total
amount of poly(A+) RNA to 200 ng.
Poly(A+) RNA was selected using Oligotex
beads (Qiagen, Hilden, CA), and cDNA was synthesized using the
Superscript system (Invitrogen); both oligo(dT) and random
oligonucleotides were used to prime the first-strand synthesis.
Adaptors/primers. The J, N, and R oligonucleotides were
designed by Lisitsyn and Wigler (1995) for use on genomic RDA with BglII and are used here with DpnII. The K, O, and
S primers are the corresponding adaptors/primers designed to be used
with NlaIII: K Sph 24 (RDA with NlaIII),
5'-GAC AAC CGA CGT CGA CTA TGC ATG-3'; K Sph 12 (RDA with
NlaIII), 5'-CAT AGT CGA CGT-3'; O Sph 24 (RDA with
NlaIII), 5'-AGG CAA CTG TGC TAT CCG AGC ATG-3'; O Sph 12 (RDA with NlaIII), 5'-CTC GGA TAG CAC-3'; S Sph 24 (RDA with
NlaIII), 5'-GGC ACT CTC CAG CCT CTC AGC ATG-3' and S Sph 12 (RDA with NlaIII), 5' CTG AGA GGC TGG 3'.
Representational difference analysis. RDA was performed as
described previously (Hubank and Schatz, 1994; Lisitsyn and Wigler, 1995; Edman et al., 1997) with the following modifications. For most
experiments, carrier mRNA was added to total RNA before the isolation
of poly(A+) RNA. cDNAs that were derived
from mouse tissue were digested separately with DpnII or
NlaIII. DpnII cleaves at the recognition site
GATC, and because the four base 5' overhang that results from its
cleavage is identical to that generated by BglII, RDA primers designed previously for experiments that use BglII
were used to amplify DpnII-generated fragments. N adaptors
(Lisitsyn and Wigler, 1995) were ligated to the DpnII ends,
and O primers were ligated to NlaIII ends. Two rounds of PCR
were performed with Invitrogen Taq polymerase and buffer, 2 mM MgCl2, and 0.2 mM deoxyNTPs. The first round of PCR consisted of
four separate 50 µl reactions run in parallel for five cycles (95°C
for 1 min; 72°C for 3 min) and then pooled. The second round of PCR
consisted of 16 separate 100 µl reactions run in parallel for 12 cycles and subsequently pooled. Only fragments with two appropriately spaced adaptors (typically 50-600 bp apart) are amplified efficiently. It was determined empirically that amplification remained exponential under these conditions. Excess primers were removed from the PCR products using Qiaquick columns (Qiagen). A small proportion of each of
the drivers was digested with DpnII to remove the N adaptors or with NlaIII to remove the O adaptors, after which they
were replaced with R or S adaptors, respectively, yielding "tester" DNA. Tester DNA was mixed with an excess of driver that was derived from the other source, denatured, and permitted to anneal in a 5 µl
volume at 67°C, under oil, for 24-36 hr, which corresponds to a
Cot of ~300-400. Because single-copy genomic DNA reassociates between Cot 100 and 104, and because
~3% of the nonrepetitive genome is transcribed (Lewin, 1994), a Cot
of 3-300 should be sufficient for cDNA. The reannealed DNA was
amplified in four parallel 100 µl reactions for 10 cycles of PCR
using R or S primers. This material was phenol extracted, ethanol
precipitated, resuspended in 0.2 × echo time, and digested with mung bean nuclease (New England Biolabs, Beverly, MA) to remove
the single strands that result from priming at only one end of a
template. The resulting material was subjected to a second round of
amplification in four parallel 100 µl PCRs for 16 cycles with R or S
primers. Difference product 1 (DP1) cDNA was digested with
DpnII or NlaIII to remove the R or S adaptors,
after which they were replaced with J or K adaptors. This material was
used as the tester for a second round of subtraction, after mixing with
an excess of driver. The products of the second round of subtraction
were subjected to two rounds of PCR amplification to produce the second
difference product (DP2). The linkers were removed from the DP2 DNA,
after which it was cloned. DpnII fragments were ligated into
the BamHI site of pBKSII+
(Stratagene), whereas the NlaIII fragments were ligated into the Sph site of pGEM7Zf+ (Promega).
Ligations were transfected into DH5 (Invitrogen).
Sequence analysis and rapid amplification of cDNA ends.
Twenty-four clones were sequenced for each experiment (96 total). Some
clones contained multiple inserts. The sequences were compared with the
GenBank nonredundant and expressed sequence tag (EST) databases
using the National Center for Biotechnology Information (NCBI) basic
local alignment search tool (BLAST) (blastn) program (www.ncbi.nlm. nih.gov/blast) (Altschul et al., 1997). Similar queries were performed using the Celera mouse genome assembly (www.celera.com). The sequence indicated which cDNAs were derived from
the same gene and which were duplicates. Based on sequence, some
inserts were not analyzed further, such as those corresponding to
ribosomal RNAs (rRNAs) that were found in both +/+ and  / samples. Clones 18 and 138 contained overlapping cDNAs of a novel gene,
18-138. 5' and 3' rapid amplification of cDNA ends (RACE) was performed with the SmartRACE kit (Clontech, Cambridge, UK), using
cDNA that had been synthesized using Superscript II polymerase (Invitrogen) and P0-P2
Sprague Dawley rat sciatic nerve RNA.
"Snorthern" blots. cDNA that was amplified by PCR for
use as driver in the RDA experiments was used also for Snorthern blots. As described above, the PCR conditions were optimized to ensure that
amplification was exponential, and parallel PCRs were pooled to
minimize variation from individual samples and obtain sufficient material. Driver DNA (0.5 or 1 µg) was electrophoresed through 3%
NuSieve 3:1 agarose (FMC Bioproducts, Rockland, ME) or 4% acrylamide gels and then transferred by conventional Southern blotting or electroblotting onto Hybond N+ membranes
(Amersham Biosciences, Arlington Heights, IL).
In situ hybridization. Mice were anesthetized and perfused
with formalin. Hindquarters were dissected to expose the sciatic nerve
and then stored at 4°C in formalin. Tissues were frozen in a 1:1
mixture of optimal cutting temperature compound (O.C.T.) and Aquamount
and sectioned at 20 µm. In situ hybridization was performed as described by Simmons et al. (1989). Single-stranded antisense transcripts were prepared from the following clones: pou3f1,
AP700 (Bermingham et al., 1996); LacZ, JBSN3, which consists of
423 bp of sequence starting at the DpnII site at 2344 in
V00296; P0, rat cDNA as described by Bermingham
et al. (1996); cysteine-rich protein-2 (CRP2), JBSN61B, which
contains 231 bp starting at the DpnII site at 171 in D88792;
Dendrin, JBSN64, which consists of 361 bp between nucleotides 2856 and
3203 of X96589; tramdorin1 (tramd1), 343 bp
between nucleotides 1549 and 1892 of AF512429; and 21-70 gene, a 673 bp fragment of a mouse RACE clone that contains the 3' 222 nt of JBSN70, all of JBSN21, and 114 nt of additional 3' untranslated
region sequence. For 18-138, a 185 bp DpnII RDA fragment
(clone JBSN18) and an overlapping 245 bp NlaIII fragment (JBSN 138) were fused at a common BglI site to generate a
333 bp DpnII-NlaIII fragment, 18-138, which was
used for in situ hybridization. Emulsion-dipped sections
were exposed for 7-17 d, counterstained in 0.001% bisbenzimide
(Sigma, St. Louis, MO), and photographed using dark-field optics and
either Kodak (Rochester, NY) Ectachrome 400 or 160T film.
RNase protection assays. Single-stranded antisense
transcripts were generated with the appropriate RNA polymerase. Equal
amounts (10 µg) of total RNA from mouse tissues and rat Schwann cells were incubated with 100,000 cpm of riboprobes. The RNA was denatured at
85°C for 10 min, hybridized overnight at 48°C, and then digested with RNase T1 and RNase A (final concentrations: 1 µg/µl and 40 µg/ml, respectively) for 1 hr at 30°C. The reaction was stopped by
adding proteinase K and SDS (final concentrations, 0.28 µg/µl and
0.56%, respectively) for 30 min at 37°C. The RNA was purified by
phenol-chloroform extraction and precipitation with LiCl, yeast tRNA,
and 100% ethanol. The RNA pellet was resuspended in loading dye and
counted, and the protected fragments were separated on a sequencing gel.
Northern blot analysis. Equal amounts (10 µg) of total RNA
were electrophoresed in 1% agarose and 2.2 M
formaldehyde gels, transferred to nylon membranes (Duralon; Stratagene)
in 6× SSC, and ultraviolet cross-linked (0.12 J). Blots were
prehybridized, hybridized, and washed using standard techniques; the
final stringency of the wash was 0.2× SSC at 65°C for 30 min
(Sambrook et al., 1989). The following cDNAs were used as probes:
JBSN64, a 361 bp RDA fragment of mouse dendrin for the Northern blot
tissue; a 2 kb fragment of mouse dendrin cDNA (kindly provided by
Torsten Shultz and Peter Seeburg, Max-Planck Institute,
Heidelberg, Germany) for the injury Northern blots; a 1 kb cDNA
that corresponds to the 18-138 gene; a 590 bp fragment of
mouse CRP2 and a 0.6 kb fragment of mouse CRP1, both kindly provided by
S.-F. Yet (Harvard University, Boston, MA), and a 2 kb fragment of
mouse tramdorin1 cDNA 1920302. Plasmid inserts were isolated after
restriction endonuclease digestion by agarose gel electrophoresis and
purified by electroelution. 32P-labeled
cDNA probes with specific activities of 2-5 × 109 cpm/µg were prepared by primer
extension with random hexamers using the Prim-a-gene kit (Promega)
according to the manufacturer's instructions.
Preparation of tramdorin1 antiserum. Peptide
ESAKKLQSQDPSPANGTSC, containing amino acids 22-39 near the N terminus
of tramdorin1, was coupled to KLH and injected into two rabbits, one of
which generated useful antiserum.
Transfections. A 1566 bp BglII-XmnI
fragment of EST cDNA 1920302, containing the entire coding region, was
cloned between the BamHI and EcoRV sites in the
expression plasmid pcDNA3.1 (Invitrogen) and transiently transfected
into Cos, HeLa, and 293 cells. The cells were grown in low-glucose DMEM
supplemented by 10% fetal bovine serum and antibiotics (100 µg/ml
penicillin-streptomycin) in a humidified atmosphere containing 5%
CO2 at 37°C. Both Lipofectin (Invitrogen) and
plasmid DNA were incubated in Optimen for 30 min at room temperature
and then combined for another 15 min. The cells (~80% confluent)
were washed with Optimen and then incubated with the combined
Lipofectin/DNA solution for 6 hr at 37°C. After 6 hr, the cells were
washed once with HBSS (calcium or magnesium free),
incubated for 3 d in DMEM at 37°C, and then replated for immunoblotting or immunostaining.
Immunoblotting. Plates of confluent cells in 100 mm plates
were harvested in cold Dulbecco's PBS lacking calcium and magnesium (Invitrogen). The cell pellet was lysed in ice-cold 50 mM Tris, pH 7.0, 1% SDS, and 0.017 mg/ml
phenylmethylsulfonyl fluoride (Sigma), followed by a brief sonication
on ice with a dismembrator (Fisher Scientific, Houston, TX). Protein
concentration was determined using the Bio-Rad (Richmond, CA) kit
according to the manufacturer's instructions. For each sample, after a
5-15 min incubation in loading buffer at room temperature, 100 µg of
protein lysate was loaded onto a 12% SDS-polyacrylamide gel,
electrophoresed, and transferred to an Immobilon-polyvinylidene
fluoride membrane (Millipore) over a period of 1 hr, using a semidry
transfer unit (Fisher Scientific). The blots were blocked (5% powdered
skim milk and 0.5% Tween 20 in Tris-buffered saline) overnight at
4°C and incubated for 24 hr at 4°C in a rabbit antiserum against
tramdorin1 (diluted 1:1000). After washing in blocking solution and
Tris-buffered saline containing 0.5% Tween 20, blots were visualized
by enhanced chemiluminescence (Amersham Biosciences) according to the
manufacturer's protocols.
Immunostaining. Transfected cells were plated onto four
chamber glass slides (Nalge; Nunc, Roskilde, Denmark) and incubated for
2-3 d to ~60% confluency. The cells were washed in PBS, fixed in
acetone at 20°C for 10 min, and then blocked with 5% fish-skin gelatin in PBS containing 0.1% Triton X-100 for 1 hr at room
temperature. Cells were labeled with rabbit anti-tramdorin1 (1:500) and
processed as described below. Unfixed rat sciatic nerves were embedded
in O.C.T. and immediately frozen in a dry ice-acetone bath.
Five-micrometer-thick cryostat sections were thaw mounted on SuperFrost
Plus glass slides (Fisher Scientific) and stored at 20°C. Teased
nerve fibers were prepared from adult rat sciatic nerves, dried on
SuperFrost Plus glass slides overnight at room temperature, and stored
at 20°C. Sections and teased fibers were postfixed and
permeabilized by immersion in 20°C acetone for 10 min, blocked at
room temperature for  1 hr in 5% fish-skin gelatin containing 0.5%
Triton X-100 in PBS, and incubated 16-48 hr at 4°C with various
combinations of primary antibodies: rabbit anti-tramdorin1 (1:500),
mouse anti-rat myelin-associated glycoprotein (MAG)
(clone 513, 1:100; Boehringer Mannheim, Indianapolis, IN), and mouse
anti-lysosome-associated membrane protein (LAMP)1 (1:10;
Developmental Hydridoma Bank). After incubating with the primary
antibodies, the slides were washed and incubated with the appropriate
fluorescein- and rhodamine-conjugated donkey
cross-affinity-purified secondary antibodies (diluted
1:100; Jackson ImmunoResearch, West Grove, PA). Slides were mounted
with Vectashield (Vector Laboratories, Burlingame, CA), examined
by epifluorescence with tetramethylrhodamine isothiocyanate (TRITC) and
FITC optics on a Leica (Nussloch, Germany) DMR light
microscope, and photographed with a cooled Hamamatsu (Tokyo, Japan)
camera or followed by image manipulation with Adobe Systems (San Jose, CA) PhotoShop.
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RESULTS |
RDA using pou3f1 +/+ and / sciatic nerve
To identify target genes of pou3f1 in peripheral nerves, sciatic
nerves were isolated from newborn [postnatal day 0 (P0)] pups. Not
only do most pou3f1-null mice die at this time (Bermingham et al., 1996; Jaegle et al., 1996), but P0 is near the peak of pou3f1
mRNA expression (Monuki et al., 1989), so the expression of pou3f1
target genes should be maximally affected at this time. The confounding
effects of myelin gene expression per se should be minimized at this
time, because myelination has not commenced in mice at P0 (Webster,
1993), and the expression of genes whose function or expression
correlates with myelination increases primarily after birth (Stahl et
al., 1990; Baron et al., 1994). Furthermore, because the maturation of
myelinating Schwann cells is delayed in pou3f1-null mice
(Bermingham et al., 1996; Jaegle et al., 1996), comparing gene
expression after the onset of myelin gene expression would be
complicated by the expression of myelin-related genes (i.e., genes
whose function or expression correlates with myelination) in the
wild-type nerves.
RDA on pou3f1 / and +/+ sciatic nerves was performed as
described previously (Erkman et al., 2000; Bermingham et al.,
2001). Nerves were dissected from newborn pups of heterozygous pou3f1 intercrosses and saved separately for each animal. After the pups were
genotyped, the nerves from 7 to 10 pou3f1 +/+ and / pups were pooled separately, and total RNA was isolated from each pool. DNA
was also isolated from the pooled nerves and used to confirm their
genotypes (data not shown). Poly(A+) RNA
was isolated from a mixture of 1 µg of total RNA and 150 ng of
carrier RNA and used to generate cDNA. cDNAs from +/+ and / nerves
were digested separately with DpnII or NlaIII,
ligated to adaptors, and amplified by PCR to generate driver DNAs (Fig. 1A). From each driver
DNA, tester DNAs were derived by replacing adaptor sequences, after
which four RDA experiments were performed. For each enzyme, / cDNA
served as driver and +/+ cDNA as tester, to isolate putative
pou3f1-activated genes. Separately, +/+ cDNA was used as driver and
/ cDNA as tester to isolate putative pou3f1-repressed genes. Figure
1A shows +/+ and / drivers, as well as the first
and second difference products, DP1 and DP2, generated from one and two
rounds of the RDA procedure, respectively. Amplified DP2 DNA was cloned
into plasmid vectors and, after transformation, 24 random colonies from
each experiment (96 total) were sequenced.
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Figure 1.
Isolation of genes that are misexpressed in
sciatic nerve in the absence of pou3f1. A, Photograph of
an ethidium bromide-stained acrylamide gel containing driver, DP1, and
DP2 cDNA from P0 pou3f1 +/+ and / sciatic nerves.
The sizes of the cDNAs can be estimated from the  X174
HaeIII digest (lane M). RDA was
performed in parallel using cDNAs that were digested with either
DpnII or NlaIII. Faint bands, presumably attributable to ribosomal RNA or other highly
repetitive RNAs, can be seen in the lanes containing the
driver cDNA. Note the absence of any band derived from carrier RNA (584 bp). After one round of RDA (DP1 lanes), specific bands,
many of similar sizes, begin to appear in both the +/+ and /
lanes, with either DpnII- or
NlaIII-digested cDNA. After two rounds of RDA, the +/+
and / lanes contain distinct banding patterns
(DP2 lanes). DP2 DNAs were cloned into
pBKSII+ for analysis. In DP3 cDNA, only a few bands
that correspond to Neo and LacZ increase in intensity relative to DP2
cDNA (data not shown). B, Southern blots of
PCR-amplified cDNA (Snorthern blots) from pou3f1 +/+ and
/ sciatic nerves after hybridization to different radiolabeled cDNA
fragments generated by RDA. The sizes of bands are shown to the
right of each panel and include the 24 bp
linkers on the ends of the fragments. All of the misexpressed genes
that were confirmed by this analysis are shown, except for Neo. The
lacZ probe was made from JBSN11, a plasmid with a double insert. In
addition to lacZ, it contains an unknown sequence, which is not
differentially expressed, and thus serves as an internal control.
JBSN18, JBSN21, JBSN61B, JBSN64, JBSN57, JBSN70, and JBSN125 are
activated by pou3f1, because they are expressed at lower levels in cDNA
derived from pou3f1 / nerves. The pou3f1 probe was derived from a
separate RDA experiment and is used here as a control to confirm its
absence in / cDNA. The names of the genes containing these
fragments are given below each pair of
lanes.
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The sequences were compared to the NCBI nonredundant and EST databases.
This analysis revealed, consistent with our previous RDA results
(Bermingham et al., 2001), that some clones contained multiple cDNAs,
some cDNAs were isolated multiple times, some cDNAs represented
different parts of the same gene, and some RDA fragments were
homologous to EST clones that correspond to two different genes. A
total of 114 RDA cDNA fragments were sequenced. Of these, 23 RDA
fragments corresponded to 18S and 28S ribosomal RNA genes; these were
found in both the pou3f1 / and +/+ samples and were not studied
further. Similarly, fragments that correspond to presumed
"housekeeping" genes and to known muscle-specific genes such as
perinatal myosin heavy chain and troponin C, probably introduced by
contaminating muscle tissue in the / nerves, also were eliminated.
We were left with 78 RDA fragments that corresponded to 51 different
candidate pou3f1 target genes. To determine whether these candidate
genes were differentially expressed, we performed Southern blot
hybridization of 44 of these genes to blots of PCR-amplified cDNA from
wild-type/pou3f1 +/+ and mutant/pou3f1 / sciatic nerves (Table
1). In our hands, these Snorthern blots
have proved to be reliable indicators of differential expression
(Bermingham et al., 2001) (our unpublished observations).
As summarized in Figure 1B, 11 RDA fragments from
eight genes were confirmed to be differentially expressed by Snorthern
blots. Hybridization to JBSN18 was seen only in +/+ cDNA; an identical result was seen with JBSN138 (data not shown), which is a different fragment of the same gene (see below). JBSN21 hybridized to a differentially expressed cDNA and, to a lesser degree, with two cDNAs
that are not differentially expressed. JBSN61B (CRP2), JBSN64 (dendrin), and JBSN67 (myelin P2) are all
differentially expressed, as is JBSN70. Clone JBSN125 contains two
inserts, a 215 bp unknown sequence, which is not differentially
expressed and serves as an internal control, and a differentially
expressed cDNA fragment that is a fragment of tramdorin1
(see below). A subclone (125-10) that contains only the smaller cDNA
hybridizes only to the differentially expressed fragment (data not
shown). pou3f1 itself was not identified in the experiments
presented here, presumably because of an insufficient number of
clones that were analyzed. However, hybridization of a Snorthern blot
to a mouse pou3f1 RDA fragment from brain demonstrates that pou3f1 is
present in +/+ but not in / cDNA, providing further confirmation of
the genotype of the tissue from which the cDNA is derived.
P0 and peripheral myelin protein 22 kDa (PMP-22)
cDNA fragments were isolated but were not differentially expressed on
Snorthern blots, consistent with their apparent normal mRNA expression
in pou3f1-null mice (Bermingham et al., 1996). In summary, six genes,
representing nine RDA fragments, were expressed at lower levels in the
absence of pou3f1 and thus are putatively activated by pou3f1. Two
genes, LacZ (Fig. 1B) and Neo
(data not shown), were expressed at higher levels in
pou3f1-null sciatic nerve. These genes replace
pou3f1 in the knock-out mutation and demonstrate that we
could have detected repressed genes; the absence of other such genes
suggests that pou3f1 is not a repressor in Schwann cells. Finally,
these results demonstrate that misexpressed genes can be isolated from
small amounts of mutant tissue using an artificial mRNA as a carrier.
Of the six activated genes, only one was known previously to be
expressed by Schwann cells: the fatty acid transport protein myelin
P2 (Narayanan et al., 1991). Two genes had been
identified previously but were not known to be expressed by
Schwann cells: the LIM-domain protein CRP2 [smooth muscle
LIM (smLIM)] (Weiskirchen et al., 1995; Jain et al., 1996),
which binds to components of the actin cytoskeleton (Louis et al.,
1997), and dendrin, a protein of unknown function found in dendrites of
CNS neurons (Neuner-Jehle et al., 1996; Herb et al., 1997). The
subclone 125-10 (from JBSN125) corresponds to numerous EST clones,
including AI786604 (I.M.A.G.E. clone 1920302), AI035402 (1380757), and
AI005767 (1363993). As described below, we have named this gene
tramd1. Gene 18-138 is represented by clone JBSN18
(DpnII-derived) and clone JBSN138 (NlaIII-derived) fragments that overlap; the full
characterization of this gene will be published elsewhere. RDA clones
JBSN21 and JBSN70, which show limited or no homology to sequences
currently in the public databases, have been shown by RACE experiments
to correspond to adjacent DpnII fragments of a novel
putative extracellular matrix gene, 21-70 (data not shown).
Schwann cells express putative pou3f1
target genes
To determine whether the cDNAs that appeared to be differentially
expressed on Snorthern blots are in fact misexpressed in vivo, in situ hybridization was performed with
antisense cRNA probes. Figure
2A shows adjacent
sections of P0 sciatic nerves from
pou3f1 +/+ and / mice. As expected, pou3f1
mRNA was expressed in +/+ but not in / nerves, LacZ mRNA
was expressed in the opposite pattern, and
P0 mRNA was abundantly expressed in both
genotypes (Bermingham et al., 1996). In contrast to
P0 mRNA, P2 mRNA was more
abundant in +/+ nerves than in / nerves. Dendrin, 18-138, and CRP2
mRNA were all present in +/+ nerve, but the signals in / nerve were
not above background levels. Minimal expression was detected for 21-70, and no signal was observed using a sense cRNA probe from clone 18-138 or for tramdorin1 (data not shown).
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Figure 2.
Expression of novel Schwann cell genes
in pou3f1 +/+ and / sciatic nerves at P0 and P9.
Sections of sciatic nerves are shown after in situ
hybridization with antisense probes for pou3f1, LacZ, P0
(Mpz), P2 (P2), dendrin,
18-138, CRP2, and 21-70 mRNA. A, P0 sciatic nerves. The
left panels show wild-type nerves (+/+); the
right panels show knock-out / nerves. pou3f1
hybridizes to +/+ but not to / nerves, whereas LacZ is the
opposite; these results confirm the genotypes of the tissues.
Hybridization to mRNA for P0, whose expression is
independent of pou3f1, is observed in both +/+ and / sciatic nerves
and serves to demarcate them. Myelin P2 and CRP2 are
expressed in +/+ sciatic nerve, but in pou3f1 mutant nerve, little or
no signal is observed. Dendrin and 18-138 hybridization produce faint
signals in +/+ and no signals in /. At this age, 21-70 mRNAs were
not detected. These results demonstrate that differences in expression
can be detected by in situ hybridization, and that the
genes isolated by RDA are misexpressed in pou3f1 mutant nerves
in vivo. B, P9 sciatic nerves. The same
probes that were hybridized to P0 sciatic nerves were used to assay
expression in P9 sciatic nerves. pou3f1 and LacZ expression confirm the
genotypes of the tissues, although by P9, pou3f1 expression is
declining, whereas LacZ expression in mutants remains high, as has been
observed previously (Jaegle and Meijer, 1998). Myelin P0
mRNA expression confirms no significant differences in P0
expression in +/+ and pou3f1 / mice. At P9, myelin P2
and 18-138 expression in mutant sciatic nerve is much stronger than at
P0 but remains reduced relative to wild type. Dendrin expression is
present in +/+ nerves, but there is little or no signal from mutant
nerves. CRP2 expression appears equivalent in +/+ and /; at this
time, expression is declining in +/+ nerves but is probably
increasing in / nerves. Faint 21-70 expression can be observed in
+/+ nerves but not in / nerves. Scale bar, 0.5 mm.
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To determine whether these changes persisted in older nerves, we also
performed in situ hybridization on sciatic nerves from P9
pou3f1 +/+ and / mice (Fig. 2B). As
expected (Monuki et al., 1989), pou3f1 mRNA expression in P9 +/+
sciatic nerves was reduced from that in P0 +/+ nerves. No
pou3f1 mRNA expression was observed in P9 / sciatic nerves, whereas
LacZ mRNA expression was observed in / nerves but not in +/+
nerves, confirming the genotypes of these tissues. The
P0 mRNA signal increased from P0 to P9 but was
comparable in +/+ and / nerves, as has been described previously (Bermingham et al., 1996). Compared with +/+ nerves, the
P2 signal was still relatively reduced in P9
/ nerves but increased compared with P0. Dendrin mRNA was detected
in P9 +/+ nerves but not in / nerves. The mRNA level of 18-138 was
increased in both +/+ and / nerves between P0 and P9, but the
levels were lower in P9 / nerves. Expression of CRP2 mRNA was
comparable in P9 +/+ and / nerves, but compared with its expression
at P0, its expression had decreased in P9 +/+ nerve and increased in P9
/ nerve (see below). Expression of 21-70 mRNA was faint but greater
in P9 +/+ nerve than in P0 +/+ nerve and greater in +/+ than in /
nerve. Tramdorin1 expression was too faint to be reliably detected
(data not shown). These results demonstrate that the genes isolated by
RDA are not only expressed in sciatic nerve, but also that their
relative expression is altered in the predicted direction in the
pou3f1 / nerves. However, the pattern of expression
between P0 and P9 differed; the expression of P2,
CRP2, and 18-138 mRNA in pou3f1 / nerves appeared to
partially recover, whereas dendrin expression did not.
To evaluate whether the genes that we have identified are important for
timely peripheral myelination, we examined their expression in
claw paw (clp) mutant mice (Fig. 3). Like
pou3f1-null mice, homozygous clp mice display
delayed peripheral myelination, whereas central myelination is
unaffected (Henry et al., 1991). Myelin P2 and 18-138 expression is reduced in P1.5
clp / sciatic nerves but recovered somewhat by P13.
Dendrin expression also is reduced in P1.5 clp / nerves, but the
recovery of its expression by P13 is less obvious. In wild-type nerves,
CRP2 expression is robust at P1.5 but is greatly reduced at P13. In
clp mutant nerves, CRP2 activation is reduced, but its
expression at P13 is elevated, consistent with the delay in
myelination. P0 mRNA expression appears unaffected, although the nerves are thinner in clp mutant
mice. These results demonstrate that delayed expression of the genes that we have identified correlates with delayed peripheral myelination in clp as well as pou3f1 mutant mice. In
contrast, pou3f1 expression appears normal in P1.5 clp /
nerves but fails to be downregulated at P13. Therefore, expression of
pou3f1 is insufficient for activation of its putative target genes,
indicating either that pou3f1 regulates these genes indirectly or that
it requires a cofactor to activate them.
Axotomy results in a prompt decline in the levels of myelin-related
mRNAs distal to the lesion, including those of
P0, MBP, P2, connexin32,
PMP-22, myelin-associated glycoprotein, and periaxin (Poduslo, 1993;
Scherer et al., 2001). To determine whether CRP2, tramd1, 18-138, and
dendrin are expressed in parallel with other myelin-related genes, we
performed Northern blot analysis on rat sciatic nerves collected at
various times after crush or transection (Fig.
4). For comparison, the blot was
also hybridized for CRP1, pou3f1, P0, p75, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The transections were
performed to prevent axonal regeneration, whereas nerve crush allows
axonal regeneration, and many of the regenerating axons are
remyelinated in a proximal-to-distal manner. To better illustrate these
points, the distal stumps of crush-injured nerves were divided into
proximal (D1) and distal (D2) segments. Tramd1, 18-138, and dendrin
mRNA levels paralleled that of P0; their mRNA
levels fell after transection and did not increase thereafter, whereas
their mRNA levels returned after crush, first in the D1 segment (at
24 d) and then in the D2 segment (at 58 d). The expression of
CRP2 mRNA, in contrast, was remarkably similar to that of pou3f1, being
low in unlesioned adult nerves and increasing after nerve crush but not
transection. These data indicate that tramd1, 18-138, and dendrin are
expressed in myelinating Schwann cells and are expressed as part of the
program of myelin-related gene expression.
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Figure 4.
Expression of novel Schwann cell genes after nerve
injury. Each lane contains an equal amount (10 µg) of
total RNA isolated from the distal stumps of rat sciatic nerves after
transection or crush injury. The number of days after these lesions is
indicated; the 0 time point is from unlesioned nerves. For the crushed
nerves, RNA was made separately from two distal segments, one
immediately distal to the injury (D1) and one more
distal to the injury (D2). The blots were successively
hybridized with radiolabeled cDNA probes for tramd1,
18-138, dendrin, CRP2,
CRP1, pou3f1, NGF receptor
(NGFR), GAPDH, and P0
and exposed to film for 14, 5, 14, 14, 7, 14, 14, 4, and 0.15 d,
respectively. The steady-state levels of tramd1, 18-138, and dendrin
mRNA parallel that of P0, whereas CRP2 mRNA levels
parallel that of pou3f1.
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Expression of CRP genes in Schwann cells
CRP2 mRNA expression parallels that of pou3f1 both during
development as well as after nerve injury. In situ
hybridization of CRP2 mRNA at P0, P1.5, P9, and P13 (Figs. 2,
3) indicates that CRP2 expression is
transient, like that of pou3f1 (Monuki et al., 1989). To evaluate the
relationship between CRP2 and pou3f1 further, we examined CRP2 mRNA
expression in cultured Schwann cells and observed that CRP2 is induced
by forskolin (Fig. 5A), as is
pou3f1 (Monuki et al., 1989). These results further indicate that CRP2 expression closely mimics that of pou3f1 and that CRP2, like pou3f1, may be expressed in promyelinating Schwann cells (Arroyo et al., 1998).
To determine whether CRP2 performs an essential role in Schwann cell
myelination, we examined sciatic nerves from P4 and P5 mice that lack
the CRP2 gene (kindly provided by S.-F. Yet and M.-E. Lee,
Harvard Medical School). We found no differences in myelin from these
mice and their wild-type littermates in epoxy sections (data not
shown), suggesting that CRP2 is not essential or that other CRP genes
may compensate for the absence of CRP2. To test the latter possibility,
we examined the expression of CRP1, which is closely related (Liebhaber
et al., 1990; Crawford et al., 1994). CRP1 is also induced by
forskolin, but unlike CRP2, its expression is not regulated by pou3f1
(Fig. 5B) or nerve injury (Fig. 4). As potential regulators
of the actin cytoskeleton, the CRP proteins may act in the changes in
Schwann cell morphology that accompany myelination.
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Figure 3.
Expression of novel Schwann cell genes in
claw paw mutant sciatic nerves. Adjacent sections of sciatic
nerves are shown, after in situ hybridization with
antisense probes for pou3f1, P0,
P2, CRP2, 18-138, and dendrin mRNA. For each probe,
the sections were processed in parallel. A, Sciatic
nerves from day P1.5 pups: left, wild type
(WT); right,
clp/clp. Although nerves are thinner in claw
paw mutant mice, pou3f1 and P0 expression is readily
apparent. At this stage, expression of myelin P2,
18-138, and CRP2 is robust, whereas dendrin expression is faint. In
contrast to pou3f1, all four genes are not significantly expressed in
P1.5 clp/clp mutant mice. B, P13 sciatic
nerves: pou3f1 expression is minimal in wild-type nerve but remains
robust in clp/clp nerve. P13 wild-type expression for
P2, dendrin, and 18-138 is comparable with that of
P1.5, and expression of these genes appears to recover in P13
clp mutant nerves, consistent with the delay in peripheral
myelination observed in these mice. CRP2 expression is greater in
clp/clp mutant nerve than in wild-type nerve at P13.
Note that in wild-type nerves, both CRP2 and pou3f1 are transiently
expressed, high at P1.5 and low at P13.
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Figure 5.
Comparison of CRP1 and CRP2 Schwann cell
expression. A, RNase protection demonstrates that
forskolin (F) increases the steady-state level of
CRP2 mRNA in cultured rat Schwann cells. B, Snorthern
blots demonstrate that CRP1 is also induced by forskolin in cultured
Schwann cells, but unlike CRP2, it is not misexpressed in
P0 nerves in the absence of pou3f1 (compare +/+ with
/).
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Tissue-specific expression of putative pou3f1 target genes
The expression of dendrin, 18-138, and tramdorin1 mRNAs was
analyzed on a Northern blot of adult mouse tissues to determine their
tissue distribution and the sizes of their transcripts. A dendrin probe
hybridized to a 4 kb mRNA in adult cerebrum, as expected (Neuner-Jehle
et al., 1996; Herb et al., 1997), and more weakly to a similarly sized
mRNA from sciatic nerve (Fig.
6A). The 18-138 probe
generated a strong hybridization signal with a 1.9 kb mRNA and a weaker
signal with a 4.7 kb transcript (Fig. 6B); of the
tissues examined, 18-138 mRNA was detected only in sciatic nerve.
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Figure 6.
Tissue-specific expression of dendrin, 18-138, and
tramdorin1 genes. Each lane contains an equal amount (10 µg) of total RNA isolated from the indicated mouse tissues. The blot
was successively hybridized to a radiolabeled probe corresponding to
dendrin (A), 18-138 (B),
and tramdorin1 (C) and exposed to film for 8, 7, and 14 d, respectively, after each hybridization. The sizes of the
transcripts are estimated according to the sizes of 28S and 18S rRNAs
(4712 and 1869 nt, respectively) (Hassouna et al., 1984; Raynal et al.,
1984). Sciatic N., Sciatic nerve.
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The major tramdorin1 transcripts in sciatic nerve were ~2 and 2.7 kb;
the 2.7 kb species is the most prominent tramdorin1 mRNA in
lung, adrenal gland, and thymus (Fig. 6C). No expression was
observed in the spleen, but a faint band (>6.5 kb) was observed in
cerebrum. These results indicate that EST cDNA 1920302, as well as the
RACE cDNAs, define a nearly full-length tramd1 mRNA that likely
corresponds to the larger 2.7 kb transcript, because they are ~2.45
kb without their poly(A) tails. Therefore, tramdorin1 expression is
neither ubiquitous nor restricted to sciatic nerve. A similar Northern
blot analysis of rat tissue showed expression in sciatic nerve but not
in lung and thymus (data not shown), suggesting either variation in
tramdorin1 expression among species or cross-hybridization of the
tramd1 probe to related sequences.
Chromosomal localization of putative pou3f1 target genes
The chromosomal locations of all six putative pou3f1 target genes
were examined to ascertain any linkage to known human or mouse
peripheral neuropathy loci. Dendrin has been mapped to distal mouse
chromosome 15 (Brady et al., 1997), and myelin P2
(Pmp2) has been mapped to proximal mouse chromosome 3 (Dietrich et al., 1996). The mouse chromosomal locations of the CRP2
(Csrp2), 18-138, 21-70, and tramd1 genes were
determined by interspecific backcross analysis using progeny derived
from matings of [(C57BL/6J × Mus spretus)F1 × C57BL/6J] mice (N. A. Jenkins, D. Gilbert, and N. Copeland, unpublished observations). The
location of Csrp2 in mice near Myf6 on chromosome
10 confirms its previous mapping to human to 12q21.1 (Weiskirchen et
al., 1997) (data not shown). 18-138 resides near the Lag
locus on distal mouse chromosome 4, in a region that corresponds to
human 1q36. 21-70 is closely linked to the Atm and
Csk loci on mouse chromosome 9; the corresponding human
locus is on 15q21. The chromosomal locations of these genes were
confirmed using Celera and public mouse and human genome databases.
Currently, there are no peripheral neuropathy loci for which dendrin,
myelin P2, CRP2, 18-138, or 21-70 are candidate loci.
The results of backcross mapping of the tramd1 locus
confirm its localization to 11 exons residing between nucleotides
56,775,171 and 56,748,143 on mouse chromosome 11 in the Celera mouse
chromosome database (May 5, 2002 update; our unpublished
observations; data not shown). The proximal region of mouse chromosome
11 is syntenic with human chromosome 5q31-33, and the homology of
mouse tramdorin1 coding exons to 5q sequences in the Celera and public
human genome assemblies (Lander et al., 2001; Venter et al., 2001)
confirms the mapping in mouse. Although no candidate mouse mutations
reside near the tramd1 locus, kindreds with an autosomal
recessive form of inherited demyelinating neuropathy have been mapped
to this region (LeGuern et al., 1996; Gabreels-Festen et al., 1999;
Guilbot et al., 1999a,b), prompting us to characterize this gene further.
cDNAs homologous to clone 125-10 encode a novel gene
product, tramdorin1
Rat and mouse cDNAs that correspond to clone 125-10 were isolated
by 5' and 3' RACE (Frohman, 1993, 1994) and by obtaining and sequencing
the homologous EST clone AI786604 (I.M.A.G.E. clone 1920302). As shown
in Figure 7A, the combined 5'
and 3' rat RACE fragments define a 2.5 kb cDNA, as does mouse EST
1920302. The putative full-length rat and mouse cDNAs contain a 1.4 kb open reading frame that contains an ATG codon that closely matches the
Kozak consensus for initiator ATGs (Fig. 7B), predicted to encode a protein of 52 kDa. The rat and mouse cDNAs and the
hypothetical proteins they encode do not match any genes in the GenBank
nonredundant database or the SwissProt database (release 39), although
while this manuscript was in preparation, a homologous but not
identical gene was cloned (Sagne et al., 2001; see below). Therefore,
the cDNAs that we have cloned are derived from a novel gene.
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Figure 7.
RDA clone 125-10 identifies the
gene for a novel protein, tramdorin1. A, cDNAs that
correspond to RDA clone 125-10. Five cDNA fragments are shown; they
include the RDA fragment 125-10, two independent, nonidentical 5' RACE
rat cDNAs, a 3' RACE rat cDNA, and a mouse EST cDNA. Of these, the 2.45 kb EST cDNA 1920302 appears to include the entire open reading frame,
which is depicted in black. This cDNA appears to be
nearly full length, based on the calculated size of tramd
mRNA (Figs. 3, 7). The organism and tissue of origin of each cDNA
fragment are listed. The open reading frame commences with an ATG
initiation codon (in bold) that closely matches the
Kozak consensus (Kozak, 1987); nucleotides that are an exact match are
underlined. 14dpc, 14 d postcoitum. B,
Amino acid sequences encoded by mouse tramdorin EST cDNA 1920302 and a
hypothetical rat tramdorin1 cDNA, derived from 5' and 3' RACE
sequences, are shown. These sequences have been assigned GenBank
accession numbers AF512429 and AF512430, respectively. The mouse cDNA
encodes a 478 aa protein, whereas the corresponding rat protein is 481 aa. The amino acid sequence of mouse EST cDNA 1920302 was analyzed
using the transmembrane domain prediction programs Memsat2 (McGuffin et
al., 2000) and TMHMM (Sonnhammer et al., 1998). Eleven
putative transmembrane domains are numbered. Transmembrane domains
predicted by TMHMM are marked with capital Ts. For
Memsat2, transmembrane domains are marked as follows: O,
Outside transmembrane helix cap; X, central
transmembrane helix segment; I, inside transmembrane
helix cap. For both Memsat2 and TMHMM, predicted cytoplasmic domains
are marked by +, whereas noncytoplasmic domains are marked by . Five
consensus glycosylation sites within the protein sequence are shown in
bold and are boxed. C,
Diagram depicting the predicted topology of tramdorin1 with 11 transmembrane domains, as predicted by Memsat2 and TMHMM. The
three extracellular/lumenal glycosylation sites are marked with
branched structures. However, the electrophoretic mobility of tramdorin
suggests that it is not glycosylated extensively (Fig. 8).
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The protein encoded by mouse cDNA 1920302 was predicted by the Memsat2
(PSIPRED) (McGuffin et al., 2000) and transmembrane hidden
Markov model (TMHMM) (Sonnhammer et al., 1998) protein structure prediction programs to consist of 11 transmembrane helices, with the N terminus facing the cytoplasm (Figs.
7B,C). Although other programs
produced differing predictions (data not shown), the following
considerations support this topology. First, those programs that
predicted 11 transmembrane domain helices more accurately predicted the
number and topology of the transmembrane helices of a set of
transmembrane proteins of known structure (Tusnady and Simon, 2001).
Second, a signal sequence prediction program (Nielsen et al., 1997)
indicates that tramdorin1 does not appear to contain a cleaved
N-terminal signal sequence that would be expected if the N terminus was
lumenal/extracellular. Third, membrane proteins are glycosylated only
on their extracytoplasmic faces, and the distribution of glycosylation
consensus sites
(NXS/T, where X is
any amino acid except proline) within the protein supports the proposed
topology. Five putative glycosylation sites were found, of which three
are predicted to be extracytoplasmic in the structures predicted by
Memsat2 and TMHMM. A fourth is not conserved in humans (our
unpublished observations), and a fifth is buried in predicted
transmembrane helix 5. Fourth, the protein displays homology to the
amino acid and anxin permease superfamily of amino acid
transport proteins, which are predicted to possess 11 transmembrane
domains (Young et al., 1999). However, the related vesicular GABA
transporters are thought to consist of 10 transmembrane domains that
correspond to predicted transmembrane domains 2-11 (McIntire et al.,
1997; Sagne et al., 1997). Regardless of their differing topological
predictions, all of the programs indicated that cDNA 1920302 encodes a
polytopic transmembrane protein. Therefore, we have named the putative
gene product tramdorin, for transmembrane domain rich protein. The
tramdorin1 gene has been assigned the human and mouse gene
symbol tramd1. The known proteins that are most closely
related to tramdorin1 are two additional tramdorins: tramdorin2 and
tramdorin3 (our unpublished observations). While this
manuscript was in preparation, tramdorin1 was isolated independently
and shown to encode a proton-dependent amino acid transporter (Boll et
al., 2002). A recently cloned rat lysosomal amino acid transporter
(LYAAT), LYAAT-1 (Sagne et al., 2001), is the rat homolog of
tramdorin3; rat tramdorin1 is 67% identical to this protein.
To characterize tramdorin1, an antiserum was raised against a sequence
near the N terminus that was divergent among the tramdorin gene family
(see Materials and Methods). In immunoblots of adult rat sciatic nerve,
the antiserum binds to a single band close to the predicted molecular
weight of unglycosylated tramdorin1 (Fig.
8A). A band of similar
molecular weight is observed on immunoblots of cells transfected with
full-length mouse tramdorin1 cDNA (Fig. 8B).
Transfected cells were stained with the anti-tramdorin1 antiserum, whereas parental cells were unstained (Fig.
8C,D). To localize tramdorin1, we labeled unfixed
myelinated fibers from rat sciatic nerves with this antiserum.
Tramdorin1 immunoreactivity was observed in the paranodes and in most
incisures, as shown by colabeling with a monoclonal antibody against
MAG (Fig. 9A-C). To determine whether tramdorin1, like tramdorin3/LYAAT-1, is associated with lysosomes, teased fibers were double labeled for tramdorin1 and LAMP1,
a lysosomal marker. Figure 9D-F shows that tramdorin1 and LAMP1 are found in paranodes but do not colocalize. These observations indicate that tramdorin1 is localized to noncompact myelin but is not a
component of lysosomes.
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Figure 8.
Immunoblot analysis of tramdorin1.
A, B, Western blot analysis of
anti-tramdorin1 antiserum. Lysates (100 µg) from adult rat sciatic
nerve (sciatic N; A) and 293 cells
(B) transfected with tramdorin1 expression vector
(+) and untransfected 293 cells () were used for Western blot
analysis of the tramdorin1 antiserum (diluted 1:1000); the blots were
developed with chemiluminescence. Arrowheads mark the
location of immunoreactive tramdorin protein. C,
D, Micrographs showing transfected
(C) or untransfected (D)
293 cells viewed by immunofluorescence using the anti-tramdorin1
antiserum. Nuclei were visualized using
4',6'-diamidino-2-phenylindole.
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Figure 9.
Localization of tramdorin1 in myelinating Schwann
cells. Images of unfixed teased fibers from adult rat sciatic nerve,
double labeled with a rabbit tramdorin1 antiserum (A,
D) (TRITC) and a mouse monoclonal antibody against MAG
(B) (FITC) or LAMP1 (E)
(FITC) are shown. Tramdorin immunoreactivity is found at paranodes
(arrows) and most incisures (arrowheads),
colocalizing with MAG (A-C), and in puncta along
the outer surface. The failure to detect tramdorin in all incisures is
more likely the result of technical problems of antibody penetration
than of heterogeneity of expression. At paranodes
(D-F), tramdorin1 immunostaining does not appear
to colocalize with that of LAMP1, a lysosomal marker. Both markers
possess cone-like expression patterns in the paranode, in which the
tramdorin expression domain is nested within the LAMP1 expression
domain.
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DISCUSSION |
To identify candidate target genes of pou3f1, we used RDA in
conjunction with an artificial carrier mRNA to isolate genes that are
differentially expressed in sciatic nerves from newborn pou3f1 / pups compared with their +/+ littermates. The
sequences of the RDA fragments led to the discovery of six candidate
genes, each of which was verified to be misexpressed by Snorthern blots and in situ hybridization. Furthermore, the mRNA levels of
five of six of these genes paralleled other myelin-related genes in nerve-injury paradigms. These genes are good candidates to fulfill important functions in generating or stabilizing the myelin sheath or
in the metabolism of Schwann cell proteins.
Misexpressed genes in pou3f1 mutant mice
CRP2 is the only putative gene whose mRNA levels parallel those of
pou3f1 in developing as well as lesioned adult peripheral nerves and in
cultured Schwann cells treated with forskolin. Their parallel
expression patterns strongly support the idea that CRP2 is a direct
target of pou3f1. In keeping with this suggestion, the CRP2 promoter
(Yet et al., 1998) has seven consensus pou3f1-binding sites within 4.8 kb of the transcription start site (data not shown). However, pou3f1
expression is insufficient to activate CRP2 in clp mutant
mice, suggesting that if CRP2 is directly activated by pou3f1, this
activation also requires the clp gene product. Although
absence of CRP2 does not appear to affect Schwann cell myelination, it
is a second gene whose expression marks the promyelinating stage of
Schwann cell differentiation. The related protein CRP1 is induced in
Schwann cells by forskolin treatment, also expressed in adult sciatic
nerve, but its expression is not modulated by nerve injury or dependent
on pou3f1.
CRP1 and CRP2 (smLIM) are members of the cysteine-rich protein subclass
of LIM-only proteins, which consist of two LIM domains, each followed
by a glycine-rich region (Liebhaber et al., 1990; Arber et al., 1994;
Crawford et al., 1994; Jain et al., 1996). All three members of this
subclass are associated with actin fibers, bind to the actin
cytoskeletal proteins -actinin and zyxin (Louis et al., 1997), and
are thought to function in the differentiation of the cells that
express them. CRP1 and CRP2 are downregulated in transformed cells
(Weiskirchen and Bister, 1993; Weiskirchen et al., 1995), and mice that
lack the third member of the family, CRP3/MLP, display a
disruption of the cytoarchitecture of differentiated muscle cells
(Arber et al., 1997). Thus, if CRP2 and CRP1 perform similar functions
in Schwann cell differentiation, the loss of CRP2 may be compensated
for by CRP1. These proteins may participate in cytoskeletal changes
that accompany the transition from a promyelinating Schwann cell to a
myelinating Schwann cell (Kidd et al., 1996; Fernandez-Valle et al.,
1997; Bernier et al., 1998).
Myelin P2 is a member of the fatty acid binding
protein (FABP) family of cytosolic lipid binding proteins
(Narayanan et al., 1988), which are thought to function by binding and
transporting fatty acids and/or by modulating fatty acid-mediated
signal transduction (Glatz et al., 1995; Coe and Bernlohr, 1998).
Adipocyte P2 (aP2), the
most closely related FABP to myelin P2, activates
the nuclear receptor transcription factor peroxisome proliferator
activated receptor (PPAR) , which activates the
aP2 gene in a positive feedback loop that
controls lipid metabolism in adipocytes (Coe and Bernlohr, 1998;
Hertzel and Bernlohr, 1998). If myelin P2
performs an analogous function to a P2 in Schwann
cells, then lipid metabolism and perhaps PPAR-mediated gene regulation
may be compromised in pou3f1 / Schwann cells. In
addition, myelin P2 has been shown to bind
retinoic acid and retinol (Uyemura et al., 1984), suggesting that it
could modulate the activities of retinoid nuclear receptors. The myelin P2 promoter (Narayanan et al., 1991; Bharucha et
al., 1993) contains four consensus pou3f1-binding sites (data not
shown), located in sequences that are required for forskolin induction
of the myelin P2 gene and high-level
expression transient transfection assays. Together, these observations
suggest that myelin P2 is a target of pou3f1, and
that forskolin induction of myelin P2 (Monuki et
al., 1989) may be mediated by pou3f1.
As its name suggests, dendrin was originally found in dendrites
(Neuner-Jehle et al., 1996; Herb et al., 1997). It is a highly < |
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ABSTRACT
INTRODUCTION
