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Quantitative genetic-interaction mapping in mammalian cells

Abstract

Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed 11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map. Comparison of the GI and protein-protein interaction (PPI) data revealed that pairs of genes exhibiting positive GIs and/or similar genetic profiles were predictive of the corresponding proteins being physically associated. The mammalian GI map identified pathways and complexes but also resolved functionally distinct submodules within larger protein complexes. By integrating GI and PPI data, we created a functional map of chromatin complexes in mouse fibroblasts, revealing that the PAF complex is a central player in the mammalian chromatin landscape.

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Figure 1: An overview of the mammalian E-MAP pipeline.
Figure 2: Characteristics and quality control of GI data set.
Figure 3: Comparison of GI and PPI data sets.
Figure 4: A module map of chromatin-related genes.
Figure 5: Validation of observed GIs with an orthogonal phenotypic readout.

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Acknowledgements

We thank members of the Bandyopadhyay, Panning and Krogan laboratories for constructive discussions, J. Forrest (TTP Labtech) for providing the Acumen eX3 cytometer, J. DeRisi for allowing us access to the uPrint 3D printer, G. Tzertzinis (NEB) for providing high-concentration ShortCut RNase III, and T. Fazzio for technical advice. This work has been supported by US National Institutes of Health (GM082250, GM081879, AI090935, AI091575 to N.J.K., P50CA58207 to S.B. and GM085186 to B.P.); Defense Advanced Research Projects Agency (HR0011-11-C-0094 to N.J.K.) and University of California San Francisco Program for Breakthrough Biomedical Research to N.J.K. and B.P.

Author information

Authors and Affiliations

Authors

Contributions

A.R., D.T., G.C., S.B., B.P. and N.J.K. designed the project; A.R. and D.T. performed the experiments; A.R., D.T., M.S., G.L.N. and S.B. analyzed the data; and A.R., D.T., M.S., G.C., S.B., B.P. and N.J.K. wrote the paper.

Corresponding authors

Correspondence to Sourav Bandyopadhyay, Barbara Panning or Nevan J Krogan.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Tables 1,4,5 (PDF 13178 kb)

Supplementary Table 2

Significant GIs observed (−2 ≤ S ≥ 2). (XLSX 34 kb)

Supplementary Table 3

Oligonucleotides used to generate esiRNAs. (XLSX 69 kb)

Supplementary Dataset 1

Raw data before scoring GIs. (ZIP 278 kb)

Supplementary Dataset 2

Scored and clustered GI data in Treeview format. (ZIP 212 kb)

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Roguev, A., Talbot, D., Negri, G. et al. Quantitative genetic-interaction mapping in mammalian cells. Nat Methods 10, 432–437 (2013). https://doi.org/10.1038/nmeth.2398

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