Biology subjects

Khan, T. G.

Publications and source records attributed to Khan, T. G..

3 recordsLinked to original sources

bioRxiv2024

Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening

An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial. In this study, we performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Strikingly, the top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort. Together, our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.

genetics↗

bioRxiv2022

Regulation of cellular LDL uptake by PROX1 and CHD7

An elevated level of low-density lipoprotein (LDL) in the bloodstream is a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). The low-density lipoprotein receptor (LDLR) is a critical regulator of circulating LDL, and increasing LDLR activity is an effective therapeutic approach to reduce circulating LDL cholesterol levels. In this study, we characterize PROX1 and CHD7, two genes we previously identified in a genome-scale CRISPR screen as positive regulators of LDL uptake in HuH7 cells. We found that although disruption of either PROX1 or CHD7 significantly reduced LDL uptake, only PROX1 disruption significantly reduced the cellular levels of LDLR mRNA and surface-displayed LDLR protein. Consistent with a direct role for PROX1 in LDLR gene regulation, we also observed in publicly available data sets the presence of two liver-specific PROX1 binding sites near the LDLR locus, one of which colocalized with biochemical hallmarks of enhancer activity in hepatic tissue. Both PROX1 LDLR binding sites contained predicted PROX1 transcription factor binding motifs and colocalized with binding sites for HNF4, a known interactor for PROX1 and regulator of hepatic lipid metabolism and LDL uptake. In contrast to PROX1, no CHD7 binding sites were detected near the LDLR locus. Together, our results support a model in which both PROX1 and CHD7 promote cellular LDL uptake through distinct mechanisms, with PROX1 directly promoting LDLR gene expression and CHD7 functioning through an LDLR-independent pathway.

molecular biology↗

bioRxiv2021

Identification of ACE2 modifiers by CRISPR screening

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. We identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2 in HuH7 cells. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry and suggest potential targets for therapeutic development.

genomics↗