From the
The cholesterol analogue 25-hydroxycholesterol kills animal
cells by blocking the proteolytic activation of two sterol-regulated
transcription factors designated sterol regulatory element binding
protein-1 and -2 (SREBP-1 and SREBP-2). These proteins, each
Two transcription factors, designated sterol regulatory element
binding protein-1 (SREBP-1)
When sterols accumulate within
cells, the SREBPs are no longer proteolyzed. They remain bound to the
extranuclear membranes, and transcription of the sterol-regulated genes
declines
(3, 4) . This feedback mechanism appears
designed to supply cholesterol to cells in times of need and to prevent
overloading with cholesterol under conditions of reduced
demand
(5) . SREBP-1 and -2 are coordinately regulated in
cultured cells such as Chinese hamster ovary (CHO) cells, HeLa cells,
and human fibroblasts
(3, 4, 6) . Each protein is
capable of acting independently, and there is no evidence that
heterodimers are required
(2, 3) .
Functional analysis
of the SREBPs has been enhanced through study of mutant hamster cells
with defects in the regulation of cholesterol
metabolism
(6, 7, 8, 9, 10) . One
class of mutant cells exhibits constitutive overexpression of the low
density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A
synthase genes. These cells are unable to suppress transcription even
when cellular sterol levels are greatly elevated. Our laboratory has
described three independent cell lines of this class, designated
sterol-regulatory defective (SRD)-1, SRD-2, and SRD-3
cells
(7, 8) .
The SRD-1, -2, and -3 cells were each
produced after treatment of cultured hamster cells with a different
mutagen between 1975 and 1987. The cells were selected for a
sterol-resistant phenotype by growth in the presence of
25-hydroxycholesterol in a lipoprotein-free
medium
(7, 8) . 25-Hydroxycholesterol kills wild-type
hamster cells by blocking the proteolysis of the SREBPs, thereby
halting cholesterol biosynthesis. 25-Hydroxycholesterol cannot
substitute for cholesterol in cell membranes, and the cells die of
cholesterol deficiency. SRD-1, -2 and -3 cells survive in the presence
of 25-hydroxycholesterol because they have lost the ability to suppress
the sterol-regulated genes. When transfected with reporter genes
containing sterol regulatory elements (SREs), the cells show high level
transcription that is dependent on the presence of the SRE, and they
fail to repress this transcription in the presence of
25-hydroxycholesterol. We therefore postulated that the SRD-1, -2, and
-3 cells constitutively produce a transcription factor that acts upon
the SRE and is not inactivated in the presence of
sterols
(7, 8) .
This prediction was borne out by
molecular studies of the SRD-1 cells (6). These cells have undergone a
recombination between an intron in the gene for SREBP-2 and an intron
in the gene for a previously cloned protein designated Ku p70
autoantigen. The cells produce an abnormal mRNA that encodes the first
460 amino acids of SREBP-2 followed by 11 amino acids derived from an
out-of-frame reading of the Ku p70 mRNA. The SREBP-2 fragment does not
contain the transmembrane segments, and it enters the nucleus and
activates transcription without a requirement for
proteolysis
(6) . Its activity is therefore not reduced by
25-hydroxycholesterol.
In addition to the recombined gene, the SRD-1
cells retain a copy of the normal SREBP-2 gene, which gives rise to a
normal full-length SREBP-2 protein. Surprisingly, this protein was not
processed to the mature form
(6) . The SRD-1 cells also failed to
process SREBP-1. We concluded that the constitutively active
NH
In the current study we have extended these
investigations to SRD-2 and SRD-3 cells. We have made the surprising
observation that both of these cell lines also produce truncated forms
of SREBP-2 that end precisely at amino acid position 460, just as in
the SRD-1 cells. The mechanism for the truncation differs in the two
cases. The SRD-2 cells have undergone a recombination between an intron
in SREBP-2 and an intron in a previously unidentified gene that we have
designated Gene X. The SRD-3 cells have undergone a selective
amplification of the exons encoding the protein sequence of SREBP-2 up
to residue 460. Based on these findings, we conclude that
25-hydroxycholesterol selected for hamster cells that produce truncated
forms of SREBP-2, thereby further implicating this protein as a central
regulator of sterol metabolism. The results also dramatically
illustrate the central role of introns in evolution.
The SRD-3 cDNA
continues for only 247 nucleotides past codon 460
(Fig. 1C). This 247-nucleotide sequence is identical to
that of the intron following codon 460 in the wild-type ham-ster gene.
It terminates in a string of 16 adenosine residues. These residues are
not added posttranscriptionally. Rather, they are present in genomic
DNA where they number 15 rather than 16. The 16th adenosine in the
SRD-3 mRNA is likely to have been added during the oligo(dT)-primed
cDNA synthesis. The open reading frame of the SRD-3 mRNA extends for
only one novel residue, which is followed by a termination codon
(Fig. 1C).
Fig. 2
shows the sizes of the mRNAs
for SREBP-2 and the Gene X product in SRD-2 cells as compared with the
parental CHO-7 cells as determined by Northern blotting. In CHO-7 cells
the SREBP-2 probe bound to a single species of mRNA of
The
selective advantage of the truncation at position 460 is attributable
to the ability of the soluble truncated fragment to enter the cell
nucleus directly without a requirement for proteolysis, thereby
bypassing the sterol-suppressible step. Theoretically, a similar
advantage could have been obtained with a truncation at any position
between the end of the bHLH-Zip domain at residue 400 and the beginning
of the membrane attachment domain at residue 480
(4) . A similar
result would also be expected if SREBP-1 were truncated between its
corresponding residues (amino acids 389 and 477). The fact that the
SREBP-2 sequence of all three mutant proteins terminates precisely at
residue 460 is clearly attributable to the plasticity inherent in the
presence of an intron at this site. Why did the cells use only the
intron following codon 460 of SREBP-2, and why was SREBP-1 not used?
The answer may relate to the size of the intron. The complete structure
of the human SREBP-1 gene has been published
(17) , and partial
structures of the hamster SREBP-1 and -2 genes have been elucidated by
PCR methods.
In the SRD-1 and SRD-2 cells the intron
following codon 460 of the SREBP-2 gene recombined with an intron from
another gene, i.e. the Ku P70 autoantigen gene and the
previously unknown Gene X, respectively. The Ku P70 gene and Gene X
both contribute splice acceptor sites that allow the recombinant mRNA
to splice between codon 460 of the SREBP-2 gene and the newly adjacent
exon from the respective gene. In the case of Ku P70 the open reading
frame is interrupted, and the protein terminates after only 11
additional amino acids. The Gene X splicing event maintains the open
reading frame so that the protein continues to the normal terminus of
the Gene X protein. In both cases the recombinant gene uses the normal
transcription termination and polyadenylation signals from the
downstream gene.
The SRD-3 cells have used partial gene
amplification instead of recombination to produce an mRNA encoding the
truncated form of SREBP-2. Genomic Southern blots revealed a relative
increase in the amount of DNA from the 5`-end of the gene up to codon
460, but not beyond (Fig. 7). This relative increase was
reproducible on all genomic Southern blots that were performed. Based
on densitometric scans of multiple blots, we concluded that the SRD-3
cells had approximately 3.5 times as much DNA from this region of the
gene as they did from the 3`-end. Assuming that the SRD-3 cells have
two chromosomes containing SREBP-2 genes and assuming that the
amplification occurred on one chromosome, the data suggest that this
segment of the gene has been reiterated six times on the rearranged
chromosome.
cDNA cloning from SRD-3 cells revealed a mutant RNA in
which the intron following codon 460 was not removed by splicing
(Fig. 1). The open reading frame continues for only one residue
past codon 460. Reverse transcriptase PCR confirmed the presence of
mRNAs with this unspliced intron in SRD-3 cells (Fig. 6).
Immunoblot analysis confirmed that these cells produce a shortened form
of SREBP-2 whose size corresponds to that expected for the truncated
protein (Fig. 8), suggesting that this RNA is functional. The
cloned transcript terminates with a sequence of 16 adenosine residues
that begins at nucleotide position 232 of the intron
(Fig. 1C). Fifteen of these 16 adenosines are present in
the intron sequence itself. Therefore, this polyadenosine sequence does
not represent posttranscriptional polyadenylation. The polyadenosine
sequence served as the site of priming by the oligo(dT) that was used
to construct the cDNA library. Whether this represents the true
termination of the mRNA is unknown. The length of the cloned transcript
was 1.7 kb. In Northern blots with isolated poly(A)
Based on all of the above data, we conclude that one
or more of the copies of the partially amplified gene in the SRD-3
cells give rise, albeit inefficiently, to a transcript that terminates
after the polyadenosine sequence in the intron following codon 460. The
polyadenosine sequence may serve the same function as a
posttranscriptionally-added polyadenosine sequence in mediating nuclear
export of the transcript and stabilizing it in the cytoplasm. Although
the process is inefficient, it results in sufficient amounts of mRNA to
allow the cells to escape the 25-hydroxycholesterol selection.
The
large intron after codon 460 allows the 5`-end of the SREBP-2 gene to
be amplified or to undergo recombination as a unit, thereby markedly
increasing the chance of a rearrangement that allows cells to survive
an environment that suddenly challenges them with a new agent, namely,
25-hydroxycholesterol. The likelihood of a productive recombination
appears to be greater than the likelihood of a point mutation or a
frameshift that would terminate SREBP-2 (or SREBP-1) at a position
between residues 399 and 480. These findings are in concert with the
postulation originally made by Gilbert
(20) in 1978.
Soon
after introns were discovered, Gilbert
(20) wrote a prescient
article in which he suggested that introns are spacers that facilitate
evolution by permitting the shuffling of exons encoding modular units
of proteins. The concept of exon shuffling was illustrated vividly by
the observation that the low density lipoprotein receptor and the
epidermal growth factor precursor contain the same repeated
cysteine-rich elements that were derived from duplication and shuffling
of exons, most of which encode single elements
(21, 22) .
The literature is now replete with examples of mosaic proteins created
by exon shuffling
(23) . These events occurred in distant
evolutionary time, and it is not possible to trace the recombination
steps that facilitated them
(23) . In the case of the SREBP-2
gene recombinations, the selective advantage is sufficient to allow
multiple different recombination events to be visualized in the same
intron in a short period of time. The selection system that revealed
the SRD mutants can now be used to expand the spectrum of mutational
events that can lead to rearrangements within a single normal intron of
an animal cell gene.
We thank our colleagues Xiaodong Wang, Xianxin Hua,
and Rob Rawson for helpful suggestions; Charles Nguyen for excellent
technical assistance; Lisa Beatty for invaluable help with tissue
culture experiments; and Jeffrey Cormier and Michelle Laremore for DNA
sequencing.
1150
amino acids in length, are embedded in the membranes of the nucleus and
endoplasmic reticulum by virtue of hydrophobic COOH-terminal segments.
In cholesterol-depleted cells the proteins are cleaved to release
soluble NH
-terminal fragments of
480 amino acids that
enter the nucleus and activate genes encoding the low density
lipoprotein receptor and enzymes of cholesterol synthesis.
25-Hydroxycholesterol blocks this cleavage, and cells die of
cholesterol deprivation. We previously described a mutant
25-hydroxycholesterol-resistant hamster cell line (SRD-1 cells) in
which the SREBP-2 gene had undergone a recombination between the intron
following codon 460 and an intron in an unrelated gene. The SREBP-2
sequence terminated at residue 460, eliminating the membrane attachment
domain and producing a constitutively active factor that no longer
required proteolysis and thus was not inhibited by
25-hydroxycholesterol. Here, we report that two additional
sterol-resistant cell lines (SRD-2 and SRD-3) have also undergone
genomic rearrangements in the intron following codon 460 of the SREBP-2
gene. Although the molecular rearrangements differ in the three mutant
lines, each leads to the production of a constitutively active
transcription factor whose SREBP-2 sequence terminates at residue 460.
These findings provide a dramatic illustration of the advantage that
introns provide in allowing proteins to gain new functions in response
to new environmental challenges.
(
)
and SREBP-2,
regulate cholesterol metabolism in animal cells. These proteins, each
greater than 1100 amino acids in length, are attached to membranes of
the endoplasmic reticulum and nuclear envelope by means of hydrophobic
sequences located in the middle of each
protein
(1, 2, 3, 4) . In sterol-depleted
cells a protease cleaves each SREBP, releasing an
NH
-terminal fragment of
480 amino acids that contains
a basic helix-loop-helix-leucine zipper (bHLH-Zip) motif and a
transcriptional activating domain. This fragment translocates to the
nucleus where it binds to a sterol regulatory element in the promoter
of the gene encoding the low density lipoprotein receptor, thereby
increasing transcription. The SREBPs also stimulate transcription of
the gene for 3-hydroxy-3-methylglutaryl coenzyme A synthase and
possibly other enzymes of cholesterol biosynthesis. The combined effect
is to increase sterol input from exogenous lipoproteins and from
endogenous synthesis
(5) .
-terminal SREBP-2 fragment suppresses the proteolysis of
full-length SREBP-1 and SREBP-2 through some type of feedback
mechanism
(6) .
General Methods and Materials
Standard molecular
biology techniques were used
(11) . DNA sequencing was performed
with the dideoxy chain termination method
(12) on an Applied
Biosystems model 373A DNA sequencer. Newborn calf lipoprotein-deficient
serum (d >1.215 g/ml) was prepared by
ultracentrifugation
(13) . Sterols, ALLN, and other chemicals
were obtained from sources described previously
(3) . Protein was
measured with the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories).
cDNA probes were P-labeled with a Random Primed DNA
Labeling Kit (Boehringer Mannheim). Hamster SREBP-2 probes
corresponding to nucleotides 1-1594 and 1595-5003 were
generated by EcoRI digestion of p02JY9
(6) . A probe for
Gene X corresponding to nucleotides 1865-4271 was generated by
XcmI digestion of p2D17, the plasmid containing the mutant
SREBP-2/Gene X fusion cDNA. Restriction enzymes were obtained from New
England Biolabs. Blot hybridization of genomic DNA and
poly(A)
RNA was performed as described previously
(6) except that the hybridization was carried out at 65 °C
for 2 h in Rapid-hyb Buffer (Amersham Corp.). All blots were exposed to
DuPont NEF-496 film at -80 °C with two intensifying screens.
Cell Culture
SRD-2 and SRD-3 cells, two mutant
lines of 25-hydroxycholesterol-resistant hamster cells, were isolated
as previously reported
(7, 8) . All cells were grown in
monolayer at 37 °C in an atmosphere of 9% CO. CHO-7 and
V-79 cells were maintained in medium A (a 1:1 mixture of Ham's
F-12 medium and Dulbecco's modified Eagle's minimum
essential medium containing 100 units/ml penicillin, 100 µg/ml
streptomycin sulfate, 2 mM glutamine, and 5% (v/v) newborn
calf lipoprotein-deficient serum). SRD-2 and SRD-3 cells were
maintained in medium A containing 1 µg/ml 25-hydroxycholesterol.
cDNA Cloning of Mutant SREBP-2 from SRD-2 and SRD-3
Cells
A cDNA library from SRD-2 cells in gt22A was
described previously
(4) . Poly(A)
RNA from
SRD-3 cells was purified with oligo(dT) affinity columns (Life
Technologies, Inc.) and used to construct a cDNA library with a
Superscript
Lambda System (Life Technologies, Inc.). Both
libraries were screened with the 5` 1594-bp
P-labeled
fragment of p02JY9, a cDNA clone of hamster SREBP-2
(6) at 65
°C for 2 h in Rapid-hyb Buffer (Amersham Corp.). Among 10
plaques from the SRD-2 library, 15 positive clones were isolated.
Five of them (p2D5, p2D7, p2D15, p2D17, and p2D21) encoded a truncated
form of SREBP-2 with an identical added sequence of 2911 bp at their
3`-ends. Among 10
plaques from the SRD-3 library, 10
positive clones were isolated. Three of them (p3D6, p3D9, and p3D12)
encoded a truncated form of SREBP-2 with an identical added sequence of
247 bp at their 3`-ends. p2D17 and p3D9, which contained 4419 and 1758
bp, respectively, were sequenced on both strands.
PCR Analysis of Genomic DNA and Poly(A)
Genomic DNA and poly(A)RNA
RNA were
prepared as described previously
(6) . Poly(A)
RNA was reverse-transcribed with a Stratascript
kit
(Stratagene) according to the provided instructions. Classic PCR
(14) was carried out with Taq polymerase (Stratagene)
in the manufacturer's buffer for 30 cycles of 1 min at 94 °C,
1 min at 55 °C, and 3 min at 72 °C. Long and accurate PCR (LA
PCR)
(15) was carried out with a mixture of µl of Pfu DNA polymerase (Stratagene) and µl of Klentaq1 (AP
Peptides, Inc.) in the manufacturer's buffer for 30 cycles of 30
s at 99 °C, 30 s at 67 °C, and 8 min at 68 °C. PCR products
were cloned into TA vectors (Invitrogen).
Expression Plasmids
Expression plasmids encoding
mutant SREBP-2 from SRD-2 cells and SRD-3 cells were constructed by
cloning the 4419-bp insert from p2D17 and the 1758-bp insert from p3D9,
respectively, into the SalI-NotI sites of pRc/CMV7SB,
a neo-containing expression vector driven by the
cytomegalovirus (CMV) enhancer-promoter
(4) . A similar
neo-containing plasmid encoding mutant SREBP-2 from SRD-1
cells was described previously
(6) .
Antibodies and Immunoblot Analysis
A monoclonal
antibody IgG-7D4 (IgG subclass) was produced by immunizing
mice (16) with a bacterially produced fusion protein encoding 6
consecutive histidines followed by amino acids 32-250 of hamster
SREBP-2
(6) . Monoclonal antibody IgG-2A4 against human SREBP-1
was described previously
(4) . For immunoblot analysis, a 100,000
g membrane fraction and a high salt nuclear extract
fraction of cultured cells were prepared as described by Wang et
al.(3) . The enhanced chemiluminescence (ECL) Western
blotting detection system kit (Amersham Corp.) was used according to
the manufacturer's instructions with some
modifications
(3) . The blots were exposed to DuPont NEN 496 film
at room temperature.
RESULTS
Fig. 1A shows the domain structure of
wild-type hamster SREBP-2 and the truncated forms found in SRD-1,
SRD-2, and SRD-3 cells. All of the DNA binding and transcriptional
activation functions of SREBP-2 are contained in the
NH-terminal portion, which terminates at residue 399. The
first transmembrane domain begins at residue 480. cDNAs encoding
truncated forms of SREBP-2 were isolated from libraries prepared from
SRD-1, SRD-2, and SRD-3 cells using radiolabeled SREBP-2 cDNA probes as
described under ``Experimental Procedures.'' The sequence of
the truncated cDNA from SRD-1 cells was reported previously
(6) .
The sequences of the cDNAs from SRD-2 and SRD-3 cells are shown in
Fig. 1
, B and C.
Figure 1:
Truncated forms of SREBP-2 produced in
SRD-1, -2, and -3 cells. A, domain structures of wild-type
and mutant SREBP-2. Numbers refer to the amino acid sequence.
The bHLH-Zip region is denoted by the hatchedbox and
the COOH-terminal domain by the shadedbox, and the
two putative transmembrane domains are indicated. All three mutant
proteins contain the wild-type sequence up to amino acid 460, after
which they diverge. The novel protein sequences are denoted by the
blackboxes. B, nucleotide sequence of the
aberrant cDNA from SRD-2 cells in the region where the coding sequence
of SREBP-2 (boxed) is fused to a fragment of an unknown gene,
designated Gene X. The underline denotes the putative signal
for polyadenylation. C, nucleotide sequence of the aberrant
cDNA from SRD-3 cells in the region where the coding sequence of
SREBP-2 (boxed) continues into an unspliced intron. The open
reading frame continues for one more amino acid. In panels B and C, the stop codons are indicated by
asterisks. The full-length nucleotide sequences of aberrant
cDNAs from SRD-2 and SRD-3 cells have been deposited in GenBank
(accession number U22818 and U22819).
All three truncated
proteins contain wild-type sequences up to residue 460, which lies
between the bHLH-Zip domain and the first membrane domain
(Fig. 1A). Thereafter, the three sequences diverge. The
cDNA from SRD-1 cells continues into sequences derived from the Ku p70
gene
(6) . The junction is out of frame from that of the Ku p70
protein, so the protein contains 11 novel amino acids followed by a
premature termination codon. The 3`-end of the cDNA from SRD-2 cells
consists of 2911 nucleotides derived from an unknown gene, which we
designate Gene X (Fig. 1B). The junction is in the
correct reading frame for Gene X, and the open reading frame extends
for 379 amino acids. This is followed by a 3`-untranslated region of
1774 nucleotides, which includes a polyadenylation signal and a
poly(A) tract. Searches of nucleic acid and protein
data banks failed to reveal a protein with significant identity to the
379-amino acid fragment of the Gene X protein.
5 kb in
length (lane 1). The SRD-2 cells exhibited this normal band
plus an additional band immediately below it at
4.4 kb (lane
2). In CHO-7 cells the Gene X probe revealed a single mRNA species
of
4 kb (lane 3). The SRD-2 cells exhibited this band
plus an additional band of
4.4 kb, which was the same size as the
abnormal band revealed with the SREBP-2 probe in these cells (lane
4). We believe that the abnormal band represents the mRNA that
encodes the SREBP-2/Gene X fusion protein.
Figure 2:
Northern blot analysis of
poly(A) RNA from CHO-7 and SRD-2 cells probed for
SREBP-2 and Gene X. An aliquot of poly(A)
RNA (5
µg/lane) from the indicated cell line was subjected to
agarose gel electrophoresis, transferred to nylon membranes, and
hybridized with the indicated
P-labeled probe (2
10
cpm/ml) at 65 °C for 2 h. The following probes were
used: lanes 1 and 2, nucleotides 1-1594 of
wild-type SREBP-2 cDNA; lanes 3 and 4, nucleotides
1865-4271 of p2D17, which represent the portion of the mutant
cDNA that was derived from Gene X. The filters were exposed at
-80 °C for 24 h. The same filters were stripped and reprobed
with a
P-labeled cDNA probe for rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (10
cpm/ml) as described (6) and were exposed at -80 °C for
16 h.
To demonstrate the gene
rearrangement in SRD-2 cells directly, we used the ``long and
accurate'' modification
(15) of the PCR technique (LA PCR).
Genomic DNA from CHO-7 and SRD-2 cells was subjected to PCR with an
upstream primer (P1) that hybridizes to a sequence in the exon that
encodes residue 460 of SREBP-2 (Fig. 3A). The downstream
primer was either P2, which hybridizes to a sequence in the SREBP-2
exon that begins with codon 461, or P3, which hybridizes to the exon of
Gene X that is immediately downstream of residue 460 in the aberrant
cDNA from SRD-2 cells. In CHO-7 cells and SRD-2 cells, the P1/P2 primer
pair give a band of 3.1 kb, which represents the intron separating
the codon sequences for residues 460 and 461 (lanes 1 and
3). As expected, the P1/P3 primer pair gave no amplified band
in the CHO-7 cells (lane 4). In SRD-2 cells the P1/P3 primers
produced a novel band of
4.5 kb. These data are consistent with
the Northern blot of Fig. 2and indicate that SRD-2 cells have a
copy of the normal SREBP-2 gene and a copy of a rearranged SREBP-2 gene
in which a coding exon from Gene X has recombined into a position
downstream of the SREBP-2 exon that encodes residue 460.
Figure 3:
Comparison of genomic DNA from CHO-7 and
SRD-2 cells using LA PCR. Panel A shows structures of the
cDNAs encoding wild-type and SRD-2 mutant forms of SREBP-2. The novel
sequence in the mutant SREBP-2 cDNA is denoted by the hatchedbox. The intron that is the site of the gene
rearrangement is indicated by the arrow. A pair of primers P1
and P2 (5`-CTTCTCTCCCTATTCTATTGACTCTGAGCC-3` and
5`-GAGGACACACAGCAGAATTCGAGACGGTC-3`) corresponding to nucleotides
1483-1512 and 1586-1615 of the wild-type SREBP-2 cDNA,
respectively, was designed to amplify the intron of the wild-type
SREBP-2 gene. Primers P1 and P3 (5`-ATGAAACACTTGCTGGTTGCCAGGAGACG-3`)
corresponding to the novel nucleotides 1572-1601 of the mutant
SREBP-2 (p2D17) were used to amplify the intron of the mutant SREBP-2
gene. B, LA PCR was carried out as described under
``Experimental Procedures'' with aliquots of genomic DNA (1
µg) from CHO-7 and SRD-2 cells. Lanes 1 and 3,
intron of the wild-type SREBP-2 gene amplified with primers P1 and P2.
Lanes 2 and 4, intron of the mutant SREBP-2 gene
amplified with primers P1 and P3. An aliquot (10 µl) of each
50-µl reaction was subjected to electrophoresis on a 1% agarose gel
and stained with ethidium bromide.
Fig. 4
shows an immunoblot analysis of SREBP-2 in CHO-7 and SRD-2
cells. In nuclear extracts of CHO-7 cells, we observed the mature,
processed 68-kDa form of SREBP-2 (bottom panel, lane
1). This fragment was no longer detectable when cells were
incubated with sterols (lane 2), and it was increased in
amount when the cells were incubated with compactin, an inhibitor of
cholesterol synthesis that lowers cellular sterol levels (lane
3). The mature form was also increased in the presence of ALLN, a
protease inhibitor that blocks the degradation of the mature form of
SREBP-2 (lane 4)
(3) . The wild-type 125-kDa precursor
form of SREBP-2 in cell membranes was not affected by any of these
treatments (upper panel, lanes 1-4). Nuclear
extracts and membranes from SRD-2 cells contained an abnormal band of
116 kDa, which is somewhat larger than the predicted size of the
SREBP-2/Gene X fusion protein (92 kDa). We have shown previously that
the SREBPs migrate more slowly than predicted for their calculated
molecular mass
(4) . This aberrant migration is attributable to
the acidic NH
terminus, since it is normalized when this
domain is deleted
(4) . The amount of the SREBP-2/Gene X fusion
protein in the membrane or nuclear fraction was not affected by
sterols, compactin, or ALLN (lanes 5-8). The membrane
fraction of the SRD-2 cells also contained a band corresponding to the
wild-type precursor of SREBP-2. Only trace amounts of wild-type mature
SREBP-2 protein were seen in the nuclear extracts and only when the
cells were treated with compactin or ALLN (bottom panel,
lanes 7 and 8). We showed previously that
constitutive expression of a nuclear form of SREBP-2 down-regulates the
processing of wild-type SREBP-2 in SRD-1 cells
(6) . The data of
Fig. 4
suggest that the same phenomenon occurs in SRD-2 cells.
Figure 4:
Immunoblot analysis of SREBP-2 in CHO-7
and SRD-2 cells. On day 0, the indicated cells were set up at 5
10
cells/100-mm dish in medium A. On day 2, cells in
lanes 3 and 7 were refed with fresh medium A
containing 50 µM compactin and 50 µM sodium
mevalonate. Cells in all other lanes received only medium A.
On day 3, the following additions were made directly to the dish:
lanes 2 and 6, 1 µg/ml 25-hydroxycholesterol plus
10 µg/ml cholesterol; lanes 4 and 8, 100
µg/ml ALLN. The medium in all dishes was adjusted to contain 0.2%
(v/v) ethanol and 0.2% (v/v) dimethyl sulfoxide, the solvents for
sterols and ALLN, respectively. After incubation for 4 h at 37 °C,
the cells were harvested, and the 100,000
g membrane
fractions (top panel) and high salt nuclear extracts
(bottom panel) were prepared as described under
``Experimental Procedures.'' An aliquot of each fraction (50
µg of protein) was subjected to SDS-PAGE and immunoblot analysis
with 4 µg/ml of IgG-7D4, a monoclonal antibody directed against
SREBP-2. The gels were calibrated with prestained SDS-PAGE standards,
high range (Bio-Rad). The filters were exposed to films for 15
s.
Inasmuch as the SREBP-2/Gene X fusion protein in SRD-2 cells lacks
the putative transmembrane domains, it should not be attached
intrinsically to membranes in the same fashion as is wild-type SREBP-2.
To test this hypothesis, we washed the membranes of CHO-7 and SRD-2
cells with sodium carbonate, a treatment that is known to remove
extrinsic, but not intrinsic, membrane proteins
(3) . The mixture
was subjected to centrifugation, and the supernatant and pellet
fractions were subjected to SDS gel electrophoresis and immunoblotting.
As reported previously for SREBP-1
(3) , wild-type SREBP-2 was
resistant to removal from membranes by sodium carbonate (Fig. 5,
lanes 3 and 4). In contrast, nearly all of the
SREBP-2/Gene X fusion protein was removed from the membrane by this
treatment (lanes 7 and 8). These data indicate that
the SREBP-2/Gene X fusion protein is associated weakly with membranes
and that it is free to enter the nucleus without a requirement for
proteolysis.
Figure 5:
Immunoblot
analysis of SREBP-2 in membranes from CHO and SRD-2 cells. On day 0,
the indicated cells were set up at 5 10
cells/100-mm dish in medium A. The cells were refed with the same
medium on day 2 and harvested on day 3. The 100,000
g membrane fraction was prepared as described under
``Experimental Procedures.'' The membrane pellets were
suspended in buffer containing 10 mM Tris-HCl (pH 6.8) and 100
mM NaCl (lanes 1, 2, 5, and
6) or in 0.1 M Na
CO
(lanes 3, 4, 7, and 8) and
spun at 100,000
g for 30 min. All pellets were
resuspended in 10 mM Tris-HCl (pH 6.8), 0.1 M NaCl,
and 1% (v/v) SDS. Equal volumes of supernatant (S) and pellet
(P) fractions were subjected to SDS-PAGE and immunoblot
analysis with 4 µg/ml of IgG-7D4. The filters were exposed to film
for 15 s.
We next turned our attention to the mutant protein
produced in SRD-3 cells. cDNA cloning suggested that these cells
produce an mRNA with an unspliced intron following the sequence
encoding amino acid 460 in SREBP-2 (Fig. 1). To confirm the
presence of this abnormal mRNA, we performed a reverse transcriptase
PCR analysis with SRD-3 mRNA. The upstream primer was P4, which
hybridizes to a sequence near the 5`-end of the SREBP-2 mRNA
(Fig. 6A). The downstream primer was either P2, which
corresponds to a sequence on the 3`-side of residue 461, or P5, which
corresponds to a sequence in the intron that follows codon 460 in the
SREBP-2 gene. As a template we used mRNA from SRD-3 cells and from
Chinese hamster V-79 cells, which are the parental cells from which the
SRD-3 cells were derived
(8) . The P4/P2 primer pair gave a band
of the expected size (1.4 kb) in V-79 cells (Fig. 6B,
lane 3) and in SRD-3 cells (lane 1). The P4/P2 primer
pair gave no amplified band in the V-79 cells (lane 4), but it
produced an abnormal band of 1.5 kb in the SRD-3 cells (lane
2). These data indicate that the SRD-3 cells, but not the V-79,
produce a mutant messenger RNA that retains the intron following codon
460.
Figure 6:
Reverse transcriptase/PCR analysis of
poly(A) RNA from V-79 and SRD-3 cells. Panel A shows the structures of the cDNAs encoding wild-type and SRD-3
mutant forms of SREBP-2. A pair of primers P2 (described in the legend
to Fig. 3) and P4 (5`-GGACATCGATGAAATGCTACAGTTCGTCAGC-3`) was designed
to amplify nucleotides 235-1675 of the wild-type SREBP-2 cDNA. Primer
P4 and P5 (5`-GAGATGGTCTTGGTACCATCTCAAGATCTTC-3`) were used to amplify
nucleotides 208-1735 of the mutant SREBP-2 (p3D9). The novel sequence
of the mutant SREBP-2 is denoted by the blackbox.
B, an aliquot (1 µg) of poly(A)
RNA from
V-79 and SRD-3 cells was reverse-transcribed with oligo(dT) primers. An
aliquot (5 µl) of each 50-µl reaction was subjected to PCR
amplification as described under ``Experimental Procedures.''
Lanes 1 and 3, wild-type SREBP-2 cDNA amplified with
P4 and P2. Lanes 2 and 4, mutant SREBP-2 cDNA amplified with
P4 and P5. An aliquot (10 µl/lane) of each reaction (50
µl) was run on 1% agarose gel and stained with ethidium
bromide.
To gain insight into the reason for the production of the
abnormal mRNA in the SRD-3 cells, we performed Southern blotting
analysis (Fig. 7). These blots revealed gross differences between
the organization of the SREBP-2 gene in parental V-79 and mutant SRD-3
cells. Four restriction enzymes cut genomic DNA in the intron between
codon 460 and codon 461 (panel B). When genomic DNA was
digested with these enzymes and probed with the 5`-end of the cDNA
(lanes 1-8), all of the visualized bands were
3.5-fold more intense in the SRD-3 DNA than they were in the V-79
DNA (indicated by closedcircles in panel A and quantified by Fuji phosphorimaging). Two abnormal bands were
also detected (denoted by asterisks in lanes 6 and
8). When a probe from the 3`-end of the cDNA was used
(lanes 9-16), all of the bands showed equal intensity in
the V-79 and SRD-3 DNA (<1.2-fold difference by quantification). The
simplest interpretation of these data is that the mutant gene in the
SRD-3 cells has undergone selective amplification of the exons encoding
amino acids 1-460 together with at least part of the intron
following codon 460.
Figure 7:
Southern analysis of SREBP-2 gene in V-79
and SRD-3 cells. Aliquots of genomic DNA (10 µg/lane) from
the indicated cell line were digested with the indicated restriction
enzyme, subjected to electrophoresis on 0.8% agarose gels, transferred
to nylon membranes, and hybridized with the indicated
P-labeled wild-type SREBP probes. A,
hybridization was carried out with
P-labeled cDNA probes
(2
10
cpm/ml) corresponding to nucleotides
1-1594 (lanes 1-8) or nucleotides 1595-5003
(lanes 9-16) of the SREBP-2 cDNA (see diagram in
panel B) at 65 °C for 2 h as described under
``Experimental Procedures.'' The filters were exposed at
-80 °C for 48 h. Amplified bands are indicated by
dots, and abnormal bands in lanes 6 and 8 are indicated by asterisks. B, location of cDNA
probes used for Southern blots. Numbers refer to the amino
acid sequence of SREBP-2. The coding sequence of the SREBP-2 cDNA is
denoted by solidboxes, and the 5`- and
3`-untranslated regions by solidlines. The intron
between the codons for amino acids 460 and 461 is denoted by a
brokenline. The sites of the restriction enzymes
within the intron sequence are indicated (B for
BamHI, R for EcoRI, H for
HindIII, and P for PstI). The 5`-probe
corresponds to nucleotides 1-1594 of wild-type SREBP-2 cDNA. The
3`-probe corresponds to nucleotides
1595-5003.
Fig. 8
shows immunoblots designed to
detect the nuclear and membrane forms of SREBP-2 in SRD-3 cells. In the
parental V-79 cells, as in CHO-7 cells, the wild-type mature nuclear
form of SREBP-2 was suppressed by sterols and increased in the presence
of compactin and ALLN (lower panel, lanes 1-4).
The SRD-3 cells produced a mutant nuclear form of SREBP-2 that was
smaller than the wild-type mature form (lane 5). Its apparent
size of 62 kDa was in line with predictions based on the sequence
of the cDNA, taking into account the aberrant electrophoretic behavior
created by the acidic NH
-terminal domain. The amount of
this nuclear protein was not suppressed significantly by sterols
(lane 6), nor was it induced by compactin (lane 7).
ALLN increased the amount of this protein by severalfold (lane
8). The SRD-3 cells produced normal amounts of the wild-type
precursor form of SREBP-2, which was presumably transcribed from the
normal copy of the SREBP-2 gene that is present in the SRD-3 cells
(top panel, lanes 5-8). Only trace amounts of
this precursor were converted to the mature form (lower panel,
lanes 5-8).
Figure 8:
Immunoblot analysis of SREBP-2 in V-79 and
SRD-3 cells. The design of this experiment is identical to that
described in Fig. 4 except that V-79 and SRD-3 cells were
studied.
The SRD-1 and SRD-2 mutations both
involve apparent recombinations within the intron that follows codon
460 in the SREBP-2 gene. To determine whether both recombination events
occurred at the same position in the intron, we used the LA PCR
technique to amplify the intron from genomic DNA (Fig. 9). In
CHO-7 cells the intron was approximately 3100 bp long, as revealed with
primers P1 and P2 from the flanking exons (panel B, lane
1). In the SRD-1 and SRD-2 cells, we amplified the intron
containing the recombination point by using an upstream primer derived
from the SREBP-2 gene and a downstream primer derived from the Ku p70
gene and Gene X, respectively (panel A). The PCR analysis
revealed that the size of the intron was reduced to 1 kb in the
SRD-1 cells (panel B, lane 2), and it was increased
to
4.5 kb in the SRD-2 cells (panel B, lane 3).
The PCR products were cloned into plasmid vectors and sequenced. These
data revealed that the break point in the SRD-1 DNA occurred at
nucleotide position 626 of the intron sequence, whereas in the SRD-2
cells the break point occurred at nucleotide position 1171 (panels
C and D). There were no identities between the sequences
on either side of the break points. A computer search failed to reveal
any known repetitive hamster genomic elements at the sites of either
break point. Thus, these data provided no evidence for a recombination
hot spot.
Figure 9:
Sequences at the recombination break
points within the intron of SREBP-2 in SRD-1 and SRD-2 cells.
A, locations of primers derived from SREBP-2 cDNAs isolated
from CHO-7, SRD-1, and SRD-2 cells. Primers P1, P2, and P3 were
described in the legend to Fig. 3. Primer P6
(5`-CAGCTCTTCTTCTGATAACTCTACCTTGGG-3`) corresponds to nucleotides
1561-1590 of p02JY7, a cDNA clone of the mutant SREBP-2 from
SRD-1 cells (6). The novel sequences following the break point in the
SRD-1 and SRD-2 cDNAs are denoted by the black and hatchedboxes, respectively. The site of the intron harboring the
gene rearrangement is indicated by the arrow. B, an
aliquot of genomic DNA (1 µg) from the indicated cell line was
subjected to LA PCR as described under ``Experimental
Procedures.'' Lane 1, CHO-7 DNA amplified with P1 and P2;
lane 2, SRD-1 DNA amplified with P1 and P6; lane 3,
SRD-2 DNA amplified with P1 and P3. C, the nucleotide
sequences at the exon (capital letters)/intron (lowercase
letters) junctions are presented. Codons for amino acids 460 and
461 are underlined. The locations of the break points where
the SREBP-2 intron is fused to the introns of the other genes are
indicated. The numbers above the lines refer to the
sizes of the respective intron segments. D, comparison of the
intron sequences in the regions of the break points between the
wild-type SREBP-2 gene in CHO-7 cells and the mutant SREBP-2 gene in
SRD-1 and SRD-2 cells. Identical sequences are boxed. The
break points are denoted by the arrows. Numbers refer
to nucleotide sequence of the wild-type intron. Position 1 denotes the
first bp at the 5`-end of the intron.
To determine whether the production of a truncated form of
SREBP-2 blocked processing of wild-type SREBP-1 in SRD-2 and SRD-3
cells as in SRD-1 cells
(6) , we performed an immunoblot analysis
with an antibody against SREBP-1 (Fig. 10). In wild-type CHO-7
and V-79 cells, the antibody revealed a 125-kDa precursor form of
SREBP-1 and a 68-kDa mature form in the nucleus (lane 1). The
nuclear form was suppressed by sterols (lane 2) and induced by
compactin (lane 3) or ALLN (lane 4). SRD-2 and SRD-3
cells produced abundant amounts of the SREBP-1 precursor, but they
produced only trace amounts of the mature nuclear form (lanes
5-8). These data are consistent with previous findings in
SRD-1 cells.
Figure 10:
Suppression of proteolytic processing of
wild-type SREBP-1 in mutant SRD-2 (panel A) and SRD-3
(panel B) cells. Aliquots (50 µg of protein) of the same
membrane (top panels) and high nuclear salt extracts
(bottom panels) that were used in Figs. 4A and 8B were subjected to SDS-PAGE and immunoblot analysis with 4
µg/ml of IgG-2A4, a monoclonal antibody directed against SREBP-1.
P, precursor; M, mature form of
SREBP-1.
To confirm directly that the truncated forms of SREBP-2
rendered cells resistant to 25-hydroxycholesterol, we performed a
transfection experiment (Fig. 11). Wild-type CHO-7 cells were
transfected with plasmids encoding each of the truncated forms of
SREBP-2 under control of the CMV promoter plus a G-418 resistance
marker (neo). Transfected cells were selected initially for
G-418 resistance and then subjected to selection with
25-hydroxycholesterol. CHO-7 cells transfected with a control vector
survived the G-418 selection but were killed by 25-hydroxycholesterol.
Cells transfected with vectors encoding truncated SREBP-2 from SRD-1,
SRD-2, or SRD-3 cells survived both selections.
Figure 11:
cDNAs encoding mutant SREBP-2 from SRD-1,
-2, and -3 cells render transfected CHO-7 cells resistant to killing by
25-hydroxycholesterol. On day 0, 5 10
CHO-7 cells
were set up in 60-mm dishes containing medium A. On day 2, the cells
were transfected with the Transfectam reagent (Promega) containing 2
µg of the control vector pRc/CMV7SB or 2 µg of plasmid encoding
one of the mutant forms of truncated SREBP-2 under the control of the
CMV promoter. All vectors also contained a G-418 resistance gene
(neo). On day 4, each dish of transfected cells was split into
two replicate dishes and refed with medium A containing 500 µg/ml
G418 to select for stable transfectants. On day 9, one of the dishes
from each group was refed with the same medium (top row), and
the other dish was refed with medium A containing 500 µg/ml G418
plus 0.1 µg/ml of 25-hydroxycholesterol (bottom row). The
concentration of 25-hydroxycholesterol in the selection medium was
increased to 0.3 µg/ml on day 12 and to 1 µg/ml on day 15. The
colonies were stained with crystal blue dye on day 12 (G418 group) or
day 18 (G418 plus 25-hydroxycholesterol
group).
DISCUSSION
The current data dramatically illustrate two features of
animal cells: 1) the central role of SREBP-2 in regulating cholesterol
metabolism; and 2) the functional advantage of introns in gene
evolution. When mutagenized Chinese hamster fibroblasts were placed
under stringent selection for resistance to 25-hydroxycholesterol, in
all three instances examined to date they escaped by producing a
truncated form of SREBP-2. Even more remarkably, in all three instances
the SREBP-2 fragment terminated at the same amino acid position,
i.e. residue 460. This uniformity was seen with three
different mutagens (nitroethyl urea for SRD-1 cells, -irradiation
for SRD-2 cells, and ethylmethane sulfonate for SRD-3 cells) in two
different cell lines (Chinese hamster ovary cells for SRD-1 and SRD-2
cells and hamster V-79 lung fibroblasts for SRD-3 cells). In all three
mutant cell lines the truncation was made possible by a rearrangement
within the intron following codon 460 in the SREBP-2 gene.
(
)
The hamster SREBP-1 and SREBP-2
genes have two introns in the region between the end of the bHLH-Zip
domain and the beginning of the membrane attachment domain. The
locations and approximate lengths of these two introns in the two genes
are shown in Fig. 12. Only one of these four introns is greater
than 600 bp in length, and that is the intron following codon 460 in
the SREBP-2 gene. This intron is
3100 bp long, which is about 3.5
times as long as the total of the other three introns. The
corresponding intron in the SREBP-1 gene is only 116 bp. It is likely,
therefore, that the intron rearrangements occur at random and that the
enrichment for recombinations at the SREBP-2 intron following codon 460
is simply a reflection of the longer size of this intron as a target.
Figure 12:
Introns in the region between the end of
the bHLH-Zip domain and the beginning of the membrane attachment domain
in hamster SREBP-1 and SREBP-2 genes. The introns are denoted by
triangles. Numbersabovethe
triangles denote the length. Numbersbelowthe proteindomainstructure refer to the amino acid sequence.
In humans certain repetitive sequences, notably Alu elements, have been found at the sites of many germ line
recombination events
(18, 19) , suggesting that the
sequences are hot spots for recombination. There is no evidence that
the intron following codon 460 in the SREBP-2 gene contains a
recombination hot spot. In the SRD-1 and SRD-2 cells, in which the
break point could be localized precisely, the recombination occurred at
two different positions within the intron (nucleotides 626 and 1171,
respectively) (Fig. 9). There was no recognized repeated sequence
at either location. We believe, therefore, that these recombinations
occurred as random events and that they were observed only because of
the growth advantage that they imparted during selection with
25-hydroxycholesterol.
mRNA, we were able to visualize a truncated mRNA of 1.7 kb on
only one occasion (data not shown). Subsequently, repeated attempts to
visualize this mutant mRNA on Northern blots using either total RNA or
poly(A)
RNA failed. We also failed to visualize this
shortened mRNA in extracts of wild-type hamster cells, suggesting that
this intron does not contain a transcription termination signal that is
normally used.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.