Research Articles: Behavioral/Cognitive Neuronal Myocyte-specific enhancer factor 2D (MEF2D) is required for normal circadian and sleep behavior in mice https://doi.org/10.1523/JNEUROSCI.0411-19.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.0411-19.2019 Received: 20 February 2019 Revised: 8 July 2019 Accepted: 10 August 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 the authors 1 Title: Neuronal Myocyte-specific enhancer factor 2D (MEF2D) is required for 2 normal circadian and sleep behavior in mice 3 4 Abbreviated Title: Neuronal MEF2D and sleep behavior in mice 5 6 Jennifer A. Mohawk1,2, Kimberly H. Cox1, Makito Sato2,3, Seung-Hee Yoo1, Masashi 7 Yanagisawa2,3, Eric N. Olson4, Joseph S. Takahashi1,2* 8 9 Affiliations: 10 1 Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, 11 TX 75390-9111, USA 12 13 2 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, 14 TX 75390-9111, USA 15 16 3 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, 17 TX 75390-9111, USA 18 19 4 Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, 20 University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 21 22 23 * To whom correspondence should be addressed: [email protected] 24 25 Number of pages: 25 26 Number of figures: 6 27 Number of tables: 1 28 Number of extended data figures: 0 29 Number of words for abstract: 186 30 for introduction: 616 31 for discussion: 1141 32 33 Conflict of Interest Statement: The authors declare no competing financial interests. 34 35 Acknowledgements: This work was supported by the FIRST program from JSPS and 36 the WPI program from Japan's MEXT (M.Y.); NIH grants AR-067294 and HL-130253 37 and the Robert A. Welch Foundation grant 1-0025 (E.N.O.); and 38 NIH grants MH078024 and AG045795 (J.S.T.). M.Y. was, and J.S.T. is an Investigator 39 in the Howard Hughes Medical Institute. The authors would like to thank Dr. Rhonda 40 Bassel-Duby for contributing the Mef2d knockout and floxed mice, and Dr. Robert 41 Greene for suggesting the analysis in Figure 6g. 42 43 J.A. Mohawk’s current address: 2951 Gentle Creek Trail, Prosper, TX 75078. M. 44 Sato’s and M. Yanagisawa’s current address: International Institute for Integrative 45 Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba 305-8575, Japan. S-H. 46 Yoo’s current address: Department of Biochemistry and Molecular Biology, The 47 University of Texas Health Science Center at Houston, 6431 Fannin St., Houston, TX 48 77030, USA. 49 ABSTRACT 50 51 The transcription factor, myocyte enhancer factor-2 (MEF2), is required for 52 normal circadian behavior in Drosophila; however, its role in the mammalian circadian 53 system has not been established. Of the four mammalian Mef2 genes, Mef2d is highly 54 expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus, a region critical 55 for coordinating peripheral circadian clocks. Utilizing both conventional and brain- 56 specific Mef2d knockout (Mef2d-/-) mouse lines, we demonstrate that MEF2D is 57 essential for maintaining the length of the circadian free-running period of locomotor 58 activity and normal sleep patterns in male mice. Crossing Mef2d-/- with Per2::luc 59 reporter mice, we show that these behavioral changes are achieved without altering the 60 endogenous period of the master circadian oscillator in the SCN. Together, our data 61 suggest that alterations in behavior in Mef2d-/- mice may be the result of an effect on 62 SCN output, rather than an effect on timekeeping within the SCN itself. These findings 63 add to the growing body of evidence that MEF2 proteins play important roles in the 64 brain. 65 66 67 SIGNIFICANCE STATEMENT 68 69 These studies are the first to show a role for MEF2 proteins in the brain outside 70 of the hippocampus, and our findings suggest that these proteins may play diverse roles 71 in the central nervous system. It is important to continue to build on our understanding 72 of the roles of proteins acting in the SCN, because SCN dysfunction underlies jet lag in 73 humans and influences the response to shift work schedules, which are now known as 1 74 risk factors for the development of cancer. Our work on MEF2D could be the basis for 75 opening new lines of research in the development and regulation of circadian rhythms. 76 77 INTRODUCTION 78 79 The circadian clock is essential to the proper timing and regulation of behavior 80 and physiology. In mammals, a master pacemaker in the suprachiasmatic nucleus 81 (SCN) of the ventral hypothalamus coordinates circadian clocks throughout the 82 organism (Welsh et al., 2010; Albrecht, 2012; Herzog et al., 2017). The circadian time- 83 keeping mechanism is composed of a transcriptional autoregulatory feedback loop. At 84 the core of this loop, CLOCK and BMAL1 drive transcription of Period (Per1, Per2) and 85 Cryptochrome (Cry1, Cry2), whose protein products, in turn, dimerize, translocate back 86 into the nucleus, and repress the activity of CLOCK and BMAL1, thereby inhibiting their 87 own expression. In addition, accessory loops interact with the core clock loop, forming a 88 web of modulators contributing to the refinement of the oscillatory network and, 89 ultimately, circadian biological outputs (Shearman et al., 2000; Ueda et al., 2005; 90 Takahashi, 2017). 91 The transcription factor myocyte enhancer factor-2 (MEF2) has been linked to 92 the maintenance of normal circadian behavior in Drosophila (Blanchard et al., 2010; 93 Sivachenko et al., 2013). Overexpression of Mef2 in clock neurons leads to lengthening 94 of the free-running period of locomotor activity and weak behavioral rhythms in flies. 95 Repressing Mef2 activity has a similar effect, with flies displaying weakly rhythmic or 96 arrhythmic patterns of locomotor activity (Blanchard et al., 2010). However, the role of 97 MEF2 in the mammalian circadian system has not been established. 2 98 In vertebrates, there are four Mef2 genes: Mef2a, Mef2b, Mef2c, and Mef2d. 99 These genes display both differential and overlapping expression patterns throughout 100 the body, but are most abundantly expressed in muscle (both skeletal and cardiac) 101 (Black and Olson, 1998) and brain (Leifer et al., 1993; Ikeshima et al., 1995; Lyons et 102 al., 1995). Their protein products play vital roles in muscle development and stress- and 103 activity-dependent signaling (Shalizi and Bonni, 2005; Potthoff and Olson, 2007; 104 Dietrich, 2013). In neurons, MEF2 targets a diverse set of genes involved in synapse 105 development and remodeling (Flavell et al., 2006; Flavell et al., 2008), while loss of 106 MEF2 has been associated with neuronal apoptosis (Mao et al., 1999; Li et al., 2001; 107 Liu et al., 2003; Akhtar et al., 2012). Both in vitro and in vivo studies have shown that 108 MEF2 activation or overexpression can lead to the reduction of excitatory synapses in 109 hippocampal neurons (Barbosa et al., 2008; Pfeiffer et al., 2010). Interestingly, while 110 prenatal loss of Mef2c in the brain has been shown to impair hippocampal-dependent 111 learning in mice (Barbosa et al., 2008; Akhtar et al., 2012; Rashid et al., 2014), 112 postnatal loss of Mef2c had no such behavioral effect (Adachi et al., 2016), and 113 overexpression of MEF2 in discrete regions within the hippocampus disrupts memory 114 formation (Cole et al., 2012; Rashid et al., 2014). These findings suggest a complex role 115 for MEF2 proteins in the development and function of the hippocampus, but the impact 116 of loss of MEF2 on non-hippocampal dependent behaviors has not been thoroughly 117 investigated. 118 Global deletion of Mef2a or Mef2c is lethal in mice, while Mef2d null mice are 119 viable (Arnold et al., 2007; Kim et al., 2008; Omori et al., 2015) and Mef2d is highly 120 expressed in the SCN(Lein et al., 2007). Therefore, utilizing both a conventional and 3 121 brain-specific Mef2d KO, we began our investigation of the role of MEF2 proteins in the 122 regulation of mammalian circadian behavior. We demonstrate that Mef2D is essential 123 for maintaining the length of the circadian free-running period of locomotor activity. Loss 124 of Mef2D leads to a lengthening of the behavioral period and to disruption and 125 fragmentation of sleep patterns without altering the endogenous period of the master 126 circadian oscillator. Importantly, these effects are specific to Mef2D, as loss of either 127 Mef2A or Mef2C in the brain had no effect on locomotor free-running period. 128 129 MATERIALS AND METHODS 130 131 132 Animals. 133 Mef2d-/- mice (Arnold et al., 2007; Kim et al., 2008) and wild-type littermates on a 134 (129SvEv/C57BL/6J) mixed genetic background were derived from heterozygote 135 crosses. Brain-specific Mef2a, Mef2c, and Mef2d heterozygote and knockout mice (and 136 floxed, cre-negative controls) were derived from crossing CamKIIa-iCre mice (Casanova 137 et al., 2001) with Mef2a/c/dfx/fx triple-floxed mice (Barbosa et al., 2008; Liu et al., 2014; 138 Adachi et al., 2016) and intercrossing to obtain the desired genotypes. For 139 bioluminescence recordings to assess circadian rhythms in the SCN and peripheral 140 tissues, Mef2d-/- mice were crossed with mice carrying the Per2::luc reporter (Yoo et al., 141 2004).