©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Three Different Rearrangements in a Single Intron Truncate Sterol Regulatory Element Binding Protein-2 and Produce Sterol-resistant Phenotype in Three Cell Lines
ROLE OF INTRONS IN PROTEIN EVOLUTION (*)

Jianxin Yang , Michael S. Brown , Y. K. Ho , Joseph L. Goldstein

From the (1) Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Two transcription factors, designated sterol regulatory element binding protein-1 (SREBP-1)() 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) .

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-terminal SREBP-2 fragment suppresses the proteolysis of full-length SREBP-1 and SREBP-2 through some type of feedback mechanism (6) .

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.


EXPERIMENTAL PROCEDURES

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)RNA

Genomic DNA and poly(A) 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.

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 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 NaCO (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 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.() 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.

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) 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.

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.


FOOTNOTES

*
This work was supported by research grants from the National Institutes of Health (Grant HL20948) and the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: SREBP, sterol regulatory element binding protein; ALLN, N-acetyl-leucyl-leucyl-norleucinal; bHLH-Zip, basic helix-loop-helix-leucine zipper; bp, base pair(s); CHO cells, Chinese hamster ovary cells; CMV, cytomegalovirus; PCR, polymerase chain reaction; LA PCR, long and accurate PCR; SRD cells, mutant CHO cells with a sterol regulatory defective phenotype; kb, kilobases; PAGE, polyacrylamide gel electrophoresis.

J. Yang, J. L. Goldstein, and M. S. Brown, unpublished observations.


ACKNOWLEDGEMENTS

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.


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