Dynamic Circadian Protein–Protein Interaction Networks Predict Temporal Organization of Cellular Functions

et al. (2013) Dynamic Circadian Protein-Protein Interaction Networks Predict Temporal Organization of Cellular Functions. PLoS Genet 9(3): e1003398. doi:10.1371/journal.pgen.1003398 Dynamic Circadian Protein-Protein Interaction Networks Predict Temporal Organization of Cellular Functions Thomas Wallach 0 Katja Schellenberg 0 Bert Maier 0 Ravi Kiran Reddy Kalathur 0 Pablo Porras 0 Erich E. Wanker 0 Matthias E. Futschik 0 Achim Kramer 0 John B. Hogenesch, University of Pennsylvania, United States of America 0 1 Laboratory of Chronobiology, Charite -Universita tsmedizin , Berlin, Germany, 2 Sysbiolab , University of Algarve , Faro, Portugal, 3 Max Delbr u ck Center for Molecular Medicine, Berlin , Germany Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (,24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner. - Funding: This study was supported by grants of the BMBF (NGFN1/2, GO-Bio), EU (EuroSpin and SynSys), as well as the Helmholtz Association (MSBN, HelMA) to EEW; the Portuguese Fundacao para a Cie ncia e a Tecnologia to MEF and RKRK (IBB/CBME, LA; SFRH/BPD/70718/2010); and the Deutsche Forschungsgemeinschaft (SFB740, SFB618) to AK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Circadian clocks are endogenous oscillators conserved in nearly all living organisms that drive ,24 hour cycles in physiology and behavior. In mammals, the circadian oscillator is composed of interconnected transcriptional translational negative and positive feedback-loops which generate circadian rhythms at the molecular level. Within this gene-regulatory network, a precise timing of gene expression, proteinprotein interactions (PPIs) as well as posttranscriptional and posttranslational modifications is essential for sustaining circadian rhythms with normal dynamics [13]. The interaction between the transcription factors CLOCK and BMAL1, which has been discovered in a yeast-two-hybrid (Y2H) screen [4], is crucial for the activation of the Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2) genes. PER and CRY proteins form large complexes that inhibit their own transcription by binding directly to the CLOCK/BMAL1 complex during the late night [5]. Circadian rhythms in gene expression are pervasive 210% of the transcriptome in a given tissue is under circadian control [6,7]. Consequently, also a large fraction of the proteome is thought to be regulated in a time-of-day dependent manner, although systems-wide studies of circadian protein abundance rhythms are still rare (however, see [8]). Cellular functions are increasingly recognized to be regulated by protein complexes or modules [9], thus PPIs and their timing are predicted to be crucial. In most cases, in which PPIs exert a regulatory function, such interactions are transient and occur only under specific conditions, e.g. as a response to a signal, after binding of a co-factor or when the expression of one or both partners is induced in response to a changing cellular condition. Circadian clock regulation of cellular functions via PPIs can be accomplished by restricting important interactions to specific times of the day. In the circadian oscillator, many of the known PPIs also happen predominantly at specific times of the day, e.g. PER/CRY complexes bind to CLOCK/ BMAL1 in the late night to inhibit transactivation [5]. Here, the temporal binding profile correlates with the abundance profiles of PER and CRY proteins. While these examples demonstrate the fundamental importance of precisely timed PPIs for the circadian clockwork, we are still far from a comprehensive view of the PPI network among circadian oscillator proteins and their dynamics. Furthermore, the extent of a regulation of circadian output processes via time-of-day dependent PPIs is largely unknown. To elucidate unknown regulatory mechanisms within the circadian clockwork we have systematically mapped PPIs among 46 circadian components using high-throughput Y2H interaction experiments. We have identified 109 so far uncharacterized interactions and have successfully validated a sub-fraction via coimmunoprecipitation experiments in human cells. Among the novel PPIs we have identified modulators of CLOCK/BMLA1 function indicating a role for protein phosphatase 1 (PPP1) in the dynamic regulation of BMAL1 abundance. Furthermore, to Circadian clocks are endogenous oscillators that drive daily rhythms in physiology, metabolism, and behavior. In mammals, circadian rhythms are generated within nearly every cell; and, although dysfunction of circadian clocks is associated with various diseases (including diabetes and cancer), the molecular mechanisms linking the clock machinery with output pathways are little understood. Since essentially all biological processes depend on proteinprotein interactions, we investigated here on a systems-wide level how time-of-day-specific proteinprotein interactions contribute to the temporal organization of cellular physiology. We constructed a circadian interactome using experimentally generated proteinprotein interaction data and made this network dynamic by the incorporation of time-of-day-dependent expression data. Interestingly, systematic genetic network perturbation (RNAi and overexpression) suggests a crucial role for circadian components involved in dynamic interactions. Systems analysis of a global network revealed that interacting proteins are in the liver significantly more expressed at similar daytimes likely to restrict regulatory interactions to specific circadian phases within cells. Overall, circadian proteinprotein interactions are predicted to dynamically connect important cellular processes (signal transduction, cell cycle, etc.) usingvery often protein modules with components co-expressed (...truncated)