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Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs. ; Novo Nordisk Foundation Center for Protein Research is supported fi nancially by the Novo Nordisk Foundation (Grant NNF14CC0001) and the Danish Cancer Foundation for a grant to G.M. T.H.S. is supported by the Ministerio de Economía y Competitividad (MINECO) (BFU2015-68354, Ayudas para incentivar la incorporación estable de doc- tores (IED) 2015 and institutional funding through the Centres of Excellence Severo Ochoa award and from the CERCA Programme of the Catalan Government), S.S.B. by a predoctoral fellowship from Fundacio La Caixa and M.V.P. by a Severo Ochoa FPI predoctoral fellowship (MINECO). P.R. is supported by the Marie Sk ł odowska-Curie European Training Network (ETN) " TEMPERA. We would like to thank the Protein Production Facility Platform at CPR for the excellent technical assistance, M. Orozco at IRB Barcelona for helping with the initial modelling analysis and also thank the PRO-MS Danish National Mass Spectrometry Platform for Functional Proteomics and the CPR Mass Spectrometry Platform for instrument support and assistance.

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies. ; A.D. and E.G. were Marie-Curie Early Stage Researchers supported by the European Union's Horizon 2020 research and innovation program (675585 Marie-Curie ITN "SymBioSys") to J.S.R. A.D. was funded by German Federal Ministry of Education and Research (Bundesministerium fur Bildung und € Forschung BMBF) MSCoreSys research initiative research core SMART-CARE (031L0212A). This work was further supported by the JRC for Computational Biomedicine which was partially funded by Bayer AG, and the Medical Research Council (MC_UU_12022/6 to C.F. and M.S.). The Novo Nordisk Foundation Center for Protein Research is supported by Novo Nordisk Foundation grant number NNF14CC0001. J.V.O. was funded by a grant from Danish Council for Independent Research (8020-00100B) to partly support K.B.E. who was also supported in part by the Lundbeck Foundation (R193-2015-243). R.K. ...