CSDE1 is a Post-Transcriptional Regulator of the LDL Receptor

Abstract

In humans, clearance of LDL cholesterol, which causes atherosclerotic heart disease, is mediated by the hepatic LDL receptor (LDLR). As a result, therapies that upregulate the LDLR are highly effective treatments for atherosclerosis. Since cardiovascular disease remains the leading cause of death in Western countries, we sought to identify regulators of the LDLR beyond the known genetic causes of familial hypercholesterolemia. Here we show that CSDE1, an RNA-binding protein involved in mRNA stability, enhances LDLR mRNA degradation to modulate LDLR expression and function. Using parallel phenotypic genome-wide screens, based on the CRISPR interference platform, we identified over 100 specific regulators of surface LDLR expression in HepG2 cells, characterized their effects on LDLR function, and leveraged pharmacologic strategies to probe their mechanistic pathways. Among our hits, we found that CSDE1 participates in post-translational control of the LDLR independent from well-established, and clinically exploited, transcriptional and lysosomal regulatory mechanisms. Overall, our results reveal a network of novel LDLR modulators left undiscovered by human genetics, many of which have phenotypic strengths similar to bona fide targets in the clinic, offering hope for new therapeutic strategies against atherosclerosis. We anticipate that our approach of modelling a clinically relevant phenotype in an in vitro experimental system amenable to a forward genetic screen, followed by high throughput validation and mechanistic pharmacologic dissection, will serve as a template for the identification of novel therapeutic targets for other disease states.

Systematic elucidation of genetic mechanisms underlying cholesterol uptake

“… 22 Altogether, the four screens identify a total of 490 genes whose knockout alters LDL-C uptake under either SER or SS conditions (299 decrease uptake, 180 increase uptake, 11 do both at FDR <0.1; Figure 1 F; Table S2 ), increasing the number of such genes compared with previously published screens. 21 , 22 , 33 , 34 We observed a positive correlation in the gene-level phenotype across our screens (R = 0.36–0.53 for 229 union gene set; Figure S1 F). We find robust but incomplete correlation in the gene-level log2 fold change in screens of SER vs. SS conditions (R = 0.43 for 1,800 genes in KO-Library 3; Figure S1 G) and in our HepG2 SS screens vs. previous screens in SS HuH7 cells (R = 0.50 for 203 genes in KO-Library 4; Figure S1 H), suggesting that environmental conditions and cell line influence the genetic control of LDL-C uptake.…”

Systematic elucidation of genetic mechanisms underlying cholesterol uptake

“…The final library (KO-Library 4) contains four gRNAs targeting 522 genes with significant or near-significant effects on LDL-C uptake from the three previous screens as well as from a recently published genome-wide LDL-C uptake CRISPR-Cas9 screen performed in Huh7 cells 23 . Altogether, the four screens identify a total of 490 genes whose knockout significantly alters LDL-C uptake in either SER or SS conditions (299 decrease uptake, 180 increase uptake, 11 do both at FDR <0.1, Figure 1F, Supplemental Table 2), substantially increasing the number of such genes as compared to previous published screens 22,23,34,35 . We observe a positive correlation in the gene-level phenotype across our screens (R=0.36-0.53 for 229 union gene set, Figure S1F).…”

Regulation of cellular LDL uptake by PROX1 and CHD7

“…Additional transcription factors have been identified that modify LDLR expression through sterol-independent mechanisms [28][29][30]. Unbiased genome-wide CRISPR screens by our group and others have identified other transcriptional regulators whose perturbation influences LDL uptake or LDLR abundance [7,31].…”