Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

Abstract

Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map such essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-of-function and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor surface levels. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~400 SLC genes, for TNFR1-mediated and FAS-mediated but not TRAIL-R1-mediated responses. Mechanistically, we demonstrate that loss of SLC39A7 resulted in augmented ER stress and impaired receptor trafficking, thereby globally affecting downstream signaling. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9-based synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1, which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-of-function and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting further investigation of their individual contribution and potential role in pathological conditions.

Epistasis-driven identification of SLC25A51 as a regulator of human mitochondrial NAD import

“…Despite the high degree of functional redundancy displayed by transporters 46 , we show here that informative patterns of genetic interactions can be systematically derived for this family, allowing the generation of testable hypotheses for the de-orphanization of poorly characterized SLCs. Of all approaches that we have used in the past, including proteomics 47 , 48 , drug resistance 16 , 49 , and cell biological readouts 50 , 51 , genetic interactions among SLC has the potential to be one of the most powerful, as data points contribute to construct the evidence. As in a puzzle with a limited set of pieces, if all components are evaluated, individual genes may become implicated by exclusion principles.…”

Zinc transporters and their functional integration in mammalian cells

“…Mice lacking ZIP7 in the intestinal epithelium triggered ER stress in proliferative progenitor cells with a significant increase of apoptotic cell death ( 196 ). In human KBM7 myeloid leukemia cells, loss of ZIP7 resulted in augmented ER stress and impaired tumor necrosis factor receptor trafficking to the surface membrane ( 197 ). Emerging data suggests a general protective role of ZIP7 against ER stress.…”

Location, location, location: A compartmentalized view of TNF-induced necroptotic signaling

“…Much attention has been put toward expanding our understanding beyond the core actions of RIPK1, RIPK3, and MLKL. For example, many screens have been performed to identify additional mediators of necroptosis (27,28,31,(42)(43)(44)(45)(46)(47). Although these efforts have uncovered myriad interactors, posttranslational modifications, and epigenetic regulators that control necroptotic signaling, little overlap exists between the regulators identified in genetic screens (Fig.…”