SL-4 genome sequence

Gene locus of SCAP in SL-4 mutant. This sequence is related to Fig.

Forward Genetic Screening for Regulators Involved in Cholesterol Synthesis Using Validation-Based Insertional Mutagenesis

<div><p>Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s) due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM) system was used to isolate and characterize the 25-hydroxycholesterol (25-HC)-resistant and SR-12813-resisitant mutants. Here we report that five mutant cell lines were isolated. Among which, four sterol-resistant mutants either contain a truncated NH₂-terminal domain of sterol regulatory element-binding protein (SREBP)-2 terminating at amino acids (aa) 400, or harbor an overexpressed SREBP cleavage-activating protein (SCAP). Besides, one SR-12813 resistant mutant was identified to contain a truncated COOH-terminal catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). This study demonstrates that the VBIM system can be a powerful tool to screen novel regulatory genes in cholesterol biosynthesis.</p></div

The SR-12813-resistant mutant SL-5 produced a truncated COOH-terminal catalytic domain of HMG-CoA reductase.

<p><b>A.</b> Growth pattern of HeLa, SL-5 and its Cre recombinase treatment counterpart (SL-5+Cre). On day 0, the cells were set up in medium B with 10% FBS at 1×10⁴ per well in 6-well plate. On day 1, the cells were changed to medium B with 10% LPDS plus 12 µM SR-12813. The cells were refed every 3 days with fresh medium with SR-12813. On Day 14, the cells were washed, fixed and stained as descripted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112632#pone-0112632-g002" target="_blank">Fig. 2A</a>. <b>B.</b> The wild type HeLa cells and SR-12813-resistant cell line SL-5 were set up at 4×10⁵ per 60 mm dish in medium B supplemented with 10% FBS on day 0. Next day, the cells were changed to medium B containing 10% LPDS, 1 µM lovastatin and 10 µM mevalonate and incubated for 16 hr. On day 3, the cells were switched to medium B containing 10% LPDS, 1 µM lovastatin and 10 µM mevalonate in the absence (–) or presence of 25-HC or different concentration of SR-12813 plus 10 mM mevalonate as indicated for 5 hr, then the cells were harvested as described in “<b>Materials and methods</b>”. The aliquots were subjected to SDS-PAGE and immunoblot analysis. <b>C.</b> The total RNA was isolated and reverse-transcribed into cDNA as described in “<b>Materials and methods</b>” from the HeLa, SL-5 and SL-5+Cre cells. The aliquots of cDNA were subject to PCR analysis with the primers as indicated. The PCR products were analyzed by 1% agarose and purified for sequencing. <b>D.</b> Domain structure of <i>HMG-CoA reductase</i> gene and illustration of the insertion site of VBIM virus. The virus inserted into the 10^th intron of <i>HMG-CoA reductase</i> gene,and the CMV promoter drove transcription of downstream sequences which encoded a truncated COOH-terminal domain of HMG-CoA reductase.</p

Mutant cell lines with defects in SREBP processing and HMG-CoA reductase degradation^*.

<p>*This table is modified from TABLE II of reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112632#pone.0112632-Goldstein1" target="_blank">[15]</a>.</p><p>Mutant cell lines with defects in SREBP processing and HMG-CoA reductase degradation^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112632#nt101" target="_blank">*</a>}.</p

The growth patterns of CHO-7 cells in different selection regents and screen strategy.

<p><b>A.</b> On day 0, the CHO-7 cells or HeLa cells were set up at 1×10⁴ per 35 mm dish in medium A contains 5% FBS or medium B containing 10% FBS, respectively. On day 1, the cells were refed with medium A containing 5% LPDS (for CHO-7 cells) or medium B with 10% LPDS (for HeLa cells) with indicated concentration of 25-HC or SR-12813 respectively in the absence (–) or presence (+) of 5 µg/ml cholesterol. The medium were changed every 2 days. On day 14, the cells were washed once with PBS, fixed with 95% ethanol for 20 min and stained with 0.5% crystal violet for 1 hr at room temperature. <b>B.</b> The structure of provirus integrating into genome and the mutagenesis type of VBIM virus. LTR, long terminal repeats; CMV, cytomegalovirus promoter; GFP, green fluorescent protein; IRES, internal ribosome entry site; SD, splice donor site; LoxP, Cre-mediated recombination site. <b>C.</b> The forward genetic screen strategy. Cell pools were conducted with VBIM virus with MOI≈0.3. The mutagenized cells were selected with 25-HC or SR-12813. The survival clones were isolated, expanded and validated with Cre recombinase.</p

The 25-HC resistant cell line SL-4 contained overexpressed SCAP.

<p><b>A.</b> The growth patterns of CHO-7, SL-4 mutant and its Cre recombinase treatment counterpart (SL-4+Cre) in the presence of 25-HC. The cells were set up at 1×10⁴ per well for 6-well plate in medium A containing 5% FBS on day 0. Next day, the cells were changed into the medium containing 5% LPDS plus 0.1 µg/ml 25-HC. Fresh medium was changed every 2 days. On day 14, the cells were washed, fixed with 95% ethanol and stained with crystal violet as descripted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112632#pone-0112632-g002" target="_blank">Fig. 2A</a>. <b>B.</b> On day 0, the wild type CHO-7, SL-4 and SL-4+Cre cells were set up at 4×10⁵ cells per 60 mm dish in medium A containing 5% FBS. On day 1 the cells were changed to medium A containing 5% LPDS, 1 µM lovastatin and 10 µM mevalonate and incubated at 37°C for 16 hr. Then the cells were switched to medium A containing 5% LPDS, 1 µM lovastatin and 10 µM mevalonate in the absence (–) or presence of different concentration of 25-HC plus 10 mM mevalonate as indicated. 5 hr after treatment, the cells were harvested as described in “<b>Materials and methods</b>”. The aliquots were subjected to SDS-PAGE and immunoblot analysis with IgG A9 antibody against HMGCR, IgG-7D4 antibody against SREBP-2, IgG-9D5 against SCAP and anti-actin monomial antibody against Actin. P and N denote the precursor and NH₂-teriminal mature forms of SREBP-2, respectively. <b>C.</b> The neutral lipids of CHO-7, SL-4 and SL-4+Cre cells were visualized by Oil Red O staining. On day 0, the cells were set up at 5×10⁴ cells per well for 12-well plate with coverslips in medium A supplemented with 5% FBS. On day 2, the cells were fixed with 4% PFA, and stained with Oil Red O for lipid droplets (red) and hematoxylin for nuclei (blue). <b>D.</b> Genomic PCR analysis for CHO-7, SL-4 and SL-4+Cre cells. Genomic DNA from CHO-7, SL-4 and SL-4+Cre cells were subjected to PCR with primers as indicated. The PCR products were analyzed by 1% agarose and purified for sequencing.</p

Three 25-HC resistant clones SL-1, SL-2 and SL-3 were identified to contain a truncated SREBP-2.

<p><b>A.</b> On day 0, the CHO-7, SL-1, SL-2 and SL-3 and their Cre recombinase processed counterpart (labeling with + CRE) were set up at 1×10⁴ per well for 6-well plate in medium A supplemented with 5% FBS. On day 1, the cells were switched into medium A containing 5% LPDS and 0.1 µg/ml 25-HC. Cells were refed per 2 days. On day 14, the cells were washed once with PBS, fixed with 95% ethanol and stained with 0.5% crystal violet. <b>B.</b> CHO-7, SL-1, SL-2 and SL-3 mutants and their Cre recombinase processed counterparts were set up at 4×10⁵ cells per 60 mm dish on day 0 in medium A containing 5% FBS. On day 1 the cells were refed with medium A containing 5% LPDS, 1 µM lovastatin and 10 µM mevalonate. After 16 hr cells were changed to medium A containing 5% LPDS, 1 µM lovastatin and 10 µM mevalonate in the absence (–) or presence of different concentration of 25-HC plus 10 mM mevalonate as indicated. 5 hr post incubation, the cells were harvested as described in “materials and methods”. The aliquots were subjected to SDS-PAGE and immunoblot analysis with IgG-7D4 antibody against SREBP-2, IgG A9 antibody against HMG-CoA reductase and IgG-9D5 against SCAP. P and N denote the precursor and NH2-teriminal mature forms of SREBP-2, respectively. <b>C.</b> RT-PCR analysis of three 25-HC resistant clones. The total RNA was isolated and reverse-transcribed into cDNA as described in “materials and methods”. The aliquots of cDNA were subject to PCR analysis with the primers as indicated. The PCR products were analyzed by 1% agarose and purified for sequencing. <b>D.</b> Invert-PCR analysis one of three mutants SL-1. The genomic DNA were isolated and carried out invert-PCR analysis as described in “materials and methods”. The PCR products were purified and cloned into pGEM-T easy vector for sequencing. <b>E.</b> The localization and regulation of SREBP-2 in WT and SL-1 cells. The fixed cells were stained with polyclonal anti-calnexin antibody and monoclonal anti-SREBP-2 antibody IgG-7D4. <b>F.</b> Domain structure of the SREBP-2 of wild type and three sterols-resistance clones. The numbers correspond to the amino acid sequence.</p