Leix, K. | Search bioRxiv

bioRxiv2025

Functional interrogation of candidate cis-regulatory elements at the LDLR locus

Regulation of LDLR gene expression plays an important role in the development of atherosclerotic diseases including heart attack and stroke. Although LDLR regulation by sterol response elements has been well characterized, the functional significance of other noncoding regions at the LDLR locus remains poorly defined. We developed and applied a high throughput CRISPR screen to test the functional significance of candidate LDLR cis-regulatory elements in their native genomic context. Analysis of our screen results revealed a discrete region in the first intron of LDLR with a significant impact on cellular LDL uptake. We validated the presence of enhancer activity in this region by confirming that its disruption reduced endogenous LDLR expression while its insertion upstream of a minimal promoter augmented reporter gene expression. We then applied a massively parallel reporter assay to fine map enhancer activity in this region to a 129 bp interval that is highly conserved among vertebrates, exhibits biochemical hallmarks of enhancer activity, is enriched for transcription factor binding motifs, and contains a common genetic variant (rs57217136) that has been associated with human LDL cholesterol levels by genome-wide association studies. Overall, these findings demonstrate the power of CRISPR screening to interrogate candidate CREs and support the functional significance of an enhancer in the first intron of LDLR.

genomics↗

bioRxiv2023

Type I interferon signaling induces a delayed antiproliferative response in Calu-3 cells during SARS-CoV-2 infection

Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain poorly understood. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top 4 genes identified in our screen encode components of the same type I interferon signaling complex - IFNAR1, IFNAR2, JAK1, and TYK2. The 5th gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

microbiology↗