Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor

Abstract

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

Figure 1:. Results from the genome-wide CRISPR interference screen.

A) Overall schematic of phenotypic selection. CRISPRi-ready cells are transduced with a genome-wide library of sgRNAs, surface labeled with antibody, sorted by flow cytometry, and deep sequenced to deconvolute putative gene functions. See text for details. B) Volcano plot showing the statistical significance (Mann-Whitney test) of the guides recovered for each gene against the mean ρ phenotype of the 3 guides with the strongest effect. ρ is defined as the log₂-fold enrichment for sgRNAs recovered from cells with high LDLR abundance cells to those recovered from cells with low LDLR abundance. Guides targeting known regulators of the LDLR are noted. C) Venn diagram showing the overlap between parallel LDLR and TFR screens. Six guides common to both had opposing abundance phenotypes in the respective screens and were included as specific hits. D) Venn diagram of hits between the LDLR genome-wide CRISPRi screen (GWCS) and putative genes correlated with serum LDL-C from GWAS. The dotted line indicates a relaxed threshold for hit selection from LDLR screen, with only an additional 3 genes in the overlap. Overlap genes shown at right.

Figure 2:. Validation of LDLR CRISPRi hits.

Heatmap showing receptor abundance (LDLR, TFR, and LDLR/TFR ratio) and function (LDL uptake) for dCas9-KRAB HepG2 cells transduced with sgRNA targeting the indicated gene, analyzed by flow cytometry. Hits are grouped according to directional effect on LDLR abundance, and then within groups, by effect on LDL uptake (with uptake from FOXL3-OT1, CIT, and DHX15 sgRNAs not significantly different, at P > 0.05, from negative control sgRNA). CSDE1 is highlighted in blue. Control sgRNAs are shown at right. Readouts show log₂ fold change compared to transduction with negative control sgRNA and represent the weighted average of the effects from both sgRNAs targeting each gene. Viability indicates the relative number of cells surviving to flow cytometry in the experiments. Functional classification of genes is shown in fig. S3. Note that LDLR/TFR is a separately ascertained value from individual cells, and not a derived parameter from aggregate data. Only the hits for which two separate sgRNAs independently validated for receptor expression are shown, defined as P < 0.05 via Holm-Sidak corrected t-test. Data represent summary information from 3 to 4 independent experiments.

Figure 3:. Synergy of CRISPRi knockdowns with simvastatin or PF-846.

Heatmap showing synergy score for knockdowns of indicated genes combined with simvastatin (top) or PF-846 (bottom). Separate LDLR abundance and function (LDL uptake) experiments are shown. Hits are grouped first according to overall effect on LDLR abundance, and secondarily by effect on LDL uptake, as in Fig. 2. CSDE1 is highlighted in blue. Data represent summary information from 4 independent experiments.

Figure 4:. CSDE1 mediates LDLR mRNA decay.

A) Relative LDLR abundance in CRISPRi HepG2 cells transduced with indicated sgRNAs and grown in the indicated media. B) Relative LDL uptake in dual-sgRNA CRISPRi HepG2 cells. The pie chart shows the relative contribution of LDLR-dependent (CSDE1^LDLR, blue with purple stripes) and LDLR-independent (CSDE1^non-LDLR, purple with magenta stripes) CSDE1-mediated mechanisms, as well as SREBP2-mediated mechanisms (SREBP2^LDLR, white with grey stripes) to LDL uptake. Data is normalized to the control cells in standard media (dashed line, data shown in fig. S10C). C) Relative LDLR abundance in HepG2 cells overexpressing indicated CSDE1 isoforms. D) Relative expression of indicated mRNA targets in CRISPRi cells under sterol-depleted conditions. E) Relative expression of LDLR mRNA in CRISPRi cells after arrest of transcription with actinomycin D. Data normalized at T=0 within the sgRNA evaluated to illustrate the change in time, and thus no comparison can be made at T=0. t_1/2 indicates data fit to a one-stage exponential decay equation and analyzed by extra sum-of-squares F test. E) Schematics (not to scale) of Luc2-Prom_LDLR reporter constructs, illustrating LDLR promoter, start site (arrowhead), P2A ribosomal skipping sequence, AREs in 3’ UTR, stop codon (red octagon), and indicated regions of the LDLR gene. G to I) Ratiometric luciferase outputs of CRISPRi cells transfected with indicated reporters. “Luc2 Only” used in G, all constructs used in H, and “3’UTR_LDLR” used in I. Outputs normalized to negative control in H and I. All panels) Data represent summary information from 3 to 4 independent experiments. n.s. = non-significant (P ≥ 0.05), * = P < 0.05, ** = P < 0.01,*** = P < 0.001, and **** = P < 0.0001.

Figure 5:. CSDE1 disruption upregulates Ldlr mRNA and LDLR function in mice.

A) Relative expression of hepatic eGFP, Csde1, and Ldlr transcripts in chow-fed mice transduced with indicated moderate-dose AAV8-shRNA. Matched two-way ANOVA with Holm-Sidak multiple comparisons test shown. B) Immunoblots of liver extracts from mice in A. Each lane represents an individual mouse. Quantification of protein, normalized to loading control, shown at right. Unpaired t-tests shown. C) Mean plasma cholesterol concentrations of Western diet-fed mice before and 2 weeks after transduction with moderate-dose AAV8-shRNA. Matched two-way ANOVA with Sidak multiple comparisons test shown. D) Mean LDL cholesterol concentrations, fractionated by gel filtration, from individual mice from C. E) Mean plasma cholesterol concentrations of Paigen diet-fed mice 2 weeks after transduction with low-dose AAV8-shRNA. F) Mean plasma cholesterol concentration of mice in E 2 weeks after transduction with a second low-dose AAV8-shRNA. G) top: Mean cholesterol concentrations of fractions collected from gel filtration of plasma from individual mice in F. bottom: Immunoblots of fractions from representative mice against ApoB are shown. Note that fractions shown begin with the elution front from the size-exclusion column. Two-way ANOVA with Sidak’s multiple comparisons test shown to illustrate comparison between treatment arm within a given fraction. Error bars indicate standard error of the mean. H) Mean plasma cholesterol concentrations of Pcsk9-D377Y overexpressing and Paigen diet-fed mice 2 weeks after transduction with a second dose of low-dose AAV8-shRNA (8 weeks after first dose, for singly dosed mice). Note that Pcsk9-targeted mice were only given the first dose of AAV8. I and J) Leading upregulated (I) and downregulated (J) biological process GO terms in the differentially expressed genes (adj. P < 0.05, log₂FC > |1|) in Csde1 knockdown mice on the Paigen diet. All panels) n.s. = non-significant (P ≥ 0.05), * = P < 0.05, ** = P < 0.01,*** = P < 0.001, and **** = P < 0.0001.