Loss of function genetic screens reveal MTGR1 as an intracellular repressor of β1 integrin-dependent neurite outgrowth
Introduction
Despite significant advances in our understanding of the molecules that contribute to axonal growth, progress in overcoming the failure of central nervous system axons to regenerate after injury has been disappointing. Among the many factors that contribute to the poor regeneration of injured CNS axons are: reduced intrinsic growth capacity of the injured neurons, inability to overcome inhibitory molecules in the region of the injury, and failure to respond to growth promoting molecules in the region of the injury. As axonal growth is not hindered during development, it is reasonable to hypothesize that these properties are present in the developing neuron, but are lost or suppressed in the adult (Spencer and Filbin, 2004, He and Koprivica, 2004, Neumann et al., 2002).
Importantly, manipulations of the molecular machinery of the damaged neuron in the adult can enhance growth, indicating that these properties can be reinstated (Neumann and Woolf, 1999, Neumann et al., 2002, Filbin, 2003). Indeed, in recent years several small molecules and proteins that either promote or inhibit the growth of neurites and axons have been identified. Among these are guidance and signaling molecules (e.g. cAMP), secreted growth promoting and inhibitory molecules, including neurotrophins, netrins, slits, ephrins, semaphorins and myelin-associated proteins (Tessier-Lavigne and Goodman, 2000, Tessier-Lavigne and Goodman, 1996, Schnorrer and Dickson, 2004, Nakamura et al., 1998, Arevalo and Chao, 2005, Neumann and Woolf, 1999, Neumann et al., 2002).
With a view to providing a more extensive catalogue of the molecular contribution to various complex processes, attention has turned to genetic screens (Nijman et al., 2005, Paddison et al., 2004). For example, several comprehensive screening methods to study regulators of neuronal function have been described in drosophila, C. elegans and zebrafish (Hivert et al., 2002, Hua et al., 2005, Runko and Kaprielian, 2004, Shao et al., 2005). Here we describe the results of two functional genetic screens for repressors of neurite outgrowth. We generated and screened a genome-wide retroviral GFP-genetic suppressor element (GSE) library and a large-scale lentiviral siRNA library targeting 8500 genes. The screen integrated a highly sensitive and comprehensive functional and array-based analysis. Most of the targets that we identified fell into five major categories: receptors, proteoglycans, kinases adaptor proteins and transcription factors. In this paper we report on the contribution of MTGR1 (myeloid translocation gene-related protein-1) and GFI1 (growth factor independence-1), both of which belong to the same ETO/MTG8 (myeloid translocation gene on 8q22) protein complex (Hock and Orkin, 2006, McGhee et al., 2003, Amann et al., 2005). This complex is a transcriptional repressor that regulates proliferation and differentiation of hematopoietic and stem cells. We demonstrated that the MTGR1 and GFI1 negatively regulate β1-integrin-dependent elongation of neurites in human SH-SY5Y neuroblastoma cells.
Section snippets
Cell culture
SH-SY5Y cells were obtained from the ATCC and maintained in DMEM/F12 media (50:50; Gibco) supplemented with 1% non-essential amino acids (Gibco), 15% heat-inactivated fetal calf serum, FCS (HyClone), 100 μg/mL penicillin and 100 μg/mL streptomycin at 37 °C, 5% CO2. To induce differentiation, cells were plated on laminin-coated plates (Becton Dickinson Labware) and treated with 0–50 μM trans-retinoic acid (RA) (Sigma) for 36–72 h. Phoenix cells were obtained from the ATCC with permission of G. Nolan
Results
Fig. 1C illustrates the strategy that we followed in the first screen. We generated a library from SH-SY5Y cells, which is a sympathetic nervous system-derived, clonal human neuroblastoma cell line. We chose SH-SY5Y cells for several reasons. SH-SY5Y cells exhibit a distinct neuronal phenotype when grown on laminin and treated with neurotrophic factors or retinoic acid (Pahlman et al., 1981, Kaplan et al., 1993, Jalava et al., 1992, Jalava et al., 1993, Jamsa et al., 2004). Differentiated
Discussion
In this paper we describe a powerful, high throughput genetic strategy that uses a functional genetic screen of two large-scale libraries in human neuronal cells. The success of this screening approach is illustrated by our discovery that the MTG8(ETO)/MTGR1 protein complex is a significant contributor to the molecular mechanisms that underlie and regulate β1 integrin-dependent neurite elongation/outgrowth in SH-SY5Y cells. These novel primary and secondary screens establish a powerful system
Acknowledgments
We thank Alex Chenchik and Michail Makhanov (System Biosciences) for help with the lentiviral siRNA libraries, Valerie Vincent for help with the Cellomics analysis and Ariadne Genomics for help with bioinformatics. This work was supported by NIH grants NS14627 and 48499 and an Opportunity Award from the Sandler Program in Basic Sciences at UCSF.
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