ACE2 protein expression within isogenic cell lines is heterogeneous and associated with distinct transcriptomes

Abstract

The membrane protein angiotensin-converting enzyme 2 (ACE2) is a physiologic regulator of the renin-angiotensin system and the cellular receptor for the SARS-CoV-2 virus. Prior studies of ACE2 expression have primarily focused on mRNA abundance, with investigation at the protein level limited by uncertain specificity of commercial ACE2 antibodies. Here, we report our development of a sensitive and specific flow cytometry-based assay for cellular ACE2 protein abundance. Application of this approach to multiple cell lines revealed an unexpected degree of cellular heterogeneity, with detectable ACE2 protein in only a subset of cells in each isogenic population. This heterogeneity was mediated at the mRNA level by transcripts predominantly initiated from the ACE2 proximal promoter. ACE2 expression was heritable but not fixed over multiple generations of daughter cells, with gradual drift toward the original heterogeneous background. RNA-seq profiling identified distinct transcriptomes of ACE2-expressing relative cells to non-expressing cells, with enrichment in functionally related genes and transcription factor target sets. Our findings provide a validated approach for the specific detection of ACE2 protein at the surface of single cells, support an epigenetic mechanism ACE2 gene regulation, and identify specific pathways associated with ACE2 expression in HuH7 cells.

Identification of ACE2 modifiers by CRISPR screening

“…Given the sensitivity of our screens to detect subtle influences on ACE2 abundance, a number of negative findings are noteworthy. Cholesterol regulatory genes in the SREBP pathway have been implicated in SARS-CoV-2 infection, but we did not detect an association with ACE2 abundance either by focused analysis of canonical SREBP regulators or by systematic comparison to all HuH7 LDL uptake regulators identified in our previously reported genome-wide CRISPR screen (Emmer et al, 2021). These findings are consistent with the association of SREBP regulators with ACE2-independent coronaviruses and the lack of association with ACE2-dependent HCoV-NL63.…”

“…Consistently, these screens have lent further support to the central importance of ACE2 in SARS-CoV-2 infection, with guide RNAs targeting ACE2 among those conferring the greatest resistance to cytopathic effect. Our current study, enabled by our recently reported identification of a sensitive and specific platform for ACE2 flow cytometry (Sherman and Emmer, 2021), complements the former studies to systematically probe the genetic regulators of ACE2 surface abundance in HuH7 cells.…”

“…Multiple CRISPR screens have implicated cholesterol regulation as an important mediator of host cell interactions with SARS-CoV-2 (Daniloski et al, 2021;Hoffmann et al, 2021;Schneider et al, 2021;Wang et al, 2021). In addition to canonical SREBP regulators, we also noted the identification of several genes in these studies that we had recently identified in a screen for regulators of low-density lipoprotein (LDL) uptake (Emmer et al, 2021). These included RAB10 and multiple components of the exocyst complex (Hoffmann et al, 2021;Schneider et al, 2021;Wang et al, 2021).…”

“…Investigations of ACE2 protein expression have been relatively limited and confounded by uncertain specificity of different commercial ACE2 antibodies. Recently, we engineered ACE2-overexpressing and ACE2-deleted cell lines and performed systematic testing of a panel of commercial antibodies by flow cytometry, finding only 2 of 13 to exhibit specificity and sensitivity for ACE2 surface protein (Sherman and Emmer, 2021). Unexpectedly, we found that multiple isogenic cell lines demonstrated heterogeneity of endogenous ACE2 expression, suggesting that they may serve as a simplified model to dissect the molecular pathways that govern ACE2 expression.…”

“…To identify functional regulators of ACE2 surface abundance, we first defined a list of candidate genes from several different sources (Figure 1A, Supplemental Table 1). These included modifiers of SARS-CoV-2 cytopathic effect in recently reported CRISPR screens (Daniloski et al, 2021;Gordon et al, 2020;Heaton et al, 2020;Hoffmann et al, 2021;Wei et al, 2021), genes in proximity to human GWAS loci associated with COVID-19 susceptibility (Ganna, 2021) (Supplemental Table 2), genes we previously identified by RNA-seq whose correlation was associated with ACE2 expression in sorted HuH7 cells (Sherman and Emmer, 2021), candidates identified in our own pilot genome-wide CRISPR screens for ACE2 abundance (Figure S1, Supplemental Tables 3-4), and a set of hypothesis-driven manually selected genes. In total, we targeted 833 genes with 15 gRNA per gene.…”