Genome-Scale CRISPR Screens Identify Novel Genes That Regulate Erythropoietin Production and Secretion

Abstract

Erythropoietin (EPO) is a plasma glycoprotein that binds to bone marrow erythroid progenitors, stimulating their proliferation and differentiation. In adult humans, EPO is secreted into the circulation by hepatocytes and specialized kidney peritubular fibroblasts, contributing approximately 20% and 80% to the total plasma EPO levels, respectively. EPO production is induced by hypoxia due to increased stability of the transcription factor hypoxia-inducible factor (HIF). However, HIF leads to increased expression of genes that promote angiogenesis and oncogenesis, which raises concerns about clinical strategies that promote EPO production via increased HIF levels. Therefore, we seek to identify novel HIF-independent regulators of EPO production as well as regulators of EPO secretion. To identify novel regulators of EPO secretion, we generated a reporter HEK293T cell line stably expressing EPO fused to GFP, and as an internal control, alpha-1-antitrypsin (A1AT) fused to mCherry. We demonstrated that both EPO and A1AT are efficiently secreted from the reporter cell line, and that treatment with Brefeldin A (which disrupts ER-to-Golgi transport) results in the intracellular accumulation of both proteins, verifying the integrity of the secretion machinery in these cells. The reporter cell line was mutagenized using a CRISPR/Cas9 knockout library, which delivers SpCas9, a puromycin resistance cassette, and a pooled collection of 123,411 single guide RNAs targeting almost every coding gene in the human genome. We isolated cells with normal mCherry but increased or decreased GFP fluorescence. This strategy enabled the identification of genes that specifically affect EPO but not A1AT levels, thereby ruling out genes that affect global secretion. We identified and validated SURF4 as an ER cargo receptor that is required for the efficient secretion of EPO. Deletion of SURF4 leads to EPO accumulation within the ER, while over-expression of SURF4 results in enhanced efficiency of EPO secretion. To identify novel regulators of EPO production, we generated a reporter HEP3B cell line with homozygous insertion of coding sequences for p2A and eGFP at the endogenous EPO locus. This HEP3B EPO-p2A-eGFP cell line expresses equivalent levels of EPO and eGFP proteins, both translated from the same mRNA molecule. Using a similar strategy as above, we performed a genome-scale CRISPR knock-out screen with and without HIF activation to identify genes that regulate EPO production independent of HIF. This screen was followed by a secondary validation screen targeting the top 1,255 candidate regulators of EPO production nominated by the genome-scale screen. These studies identified ZNF574 as a novel HIF-independent regulator for EPO production. In normoxia, deletion of ZNF574 resulted in increased EPO mRNA and protein levels. Under hypoxic conditions, deletion of ZNF574 led to increased EPO production, more so than in wildtype cells exposed to hypoxia. RNA sequencing analysis showed that ZNF574 deletion did not significantly impact the expression of most HIF-regulated genes. These findings suggest that ZNF574 regulates EPO production without activating the HIF pathway. In summary, we have identified and validated novel regulators of EPO production and secretion. These findings have important implications for several anemia disorders, particularly for anemia of chronic kidney disease, which results from impaired EPO production.