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Sleep Architecture in Mice Is Shaped by the Transcription Factor AP-2Β

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Sleep Architecture in Mice Is Shaped by the Transcription Factor AP-2Β Genetics: Early Online, published on September 2, 2020 as 10.1534/genetics.120.303435 Title: Sleep architecture in mice is shaped by the transcription factor AP-2β Authors: Ayaka Nakai1,2, Tomoyuki Fujiyama1, Nanae Nagata1, Mitsuaki Kashiwagi1, Aya Ikkyu1, Marina Takagi1, Chika Tatsuzawa1, Kaeko Tanaka1, Miyo Kakizaki1, Mika Kanuka1, Taizo Kawano1, Seiya Mizuno3, Fumihiro Sugiyama3, Satoru Takahashi3, Hiromasa Funato1,4, Takeshi Sakurai1,5,6, Masashi Yanagisawa1,6,7,8, Yu Hayashi1,9＊ Affiliations: 1) International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, 305-8575, Japan 2) PhD Program in Neuroscience, Comprehensive Human Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, 305-8575, Japan 3) Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, 305-8575, Japan 4) Department of Anatomy, Faculty of Medicine, Toho University, Tokyo, 143-8540, Japan 5) Faculty of Medicine, University of Tsukuba, Tsukuba, 305-8575, Japan 6) Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, 305-8575, Japan 7) Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 8) R&D Center for Frontiers of MIRAI in Policy and Technology, University of Tsukuba, Tsukuba, 305-8575, Japan 9) Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507, Japan *Correspondence to: [email protected] 1 Copyright 2020. Abstract The molecular mechanism regulating sleep largely remains to be elucidated. In humans, families that carry mutations in TFAP2B, which encodes the transcription factor AP-2β, self-reported sleep abnormalities such as short-sleep and parasomnia. Notably, AP- 2 transcription factors play essential roles in sleep regulation in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Thus, AP-2 transcription factors might have a conserved role in sleep regulation across the animal phyla. However, direct evidence supporting the involvement of TFAP2B in mammalian sleep was lacking. In this study, by using the CRISPR/Cas9 technology, we generated two Tfap2b mutant mouse strains, Tfap2bK144 and Tfap2bK145, each harboring a single-nucleotide mutation within the introns of Tfap2b mimicking the mutations in two human kindreds that self-reported sleep abnormalities. The effects of these mutations were compared with those of a Tfap2b knockout allele (Tfap2b-). The protein expression level of TFAP2B in the embryonic brain was reduced to about half in Tfap2b+/- mice and was further reduced in Tfap2b-/- mice. By contrast, the protein expression level was normal in Tfap2bK145/+ mice but was reduced in Tfap2bK145/K145 mice to a similar extent as Tfap2b-/- mice. Tfap2bK144/+ and Tfap2bK144/K144 showed normal protein expression levels. Tfap2b+/- female mice showed increased wakefulness time and decreased non-rapid eye movement sleep (NREMS) time. By contrast, Tfap2bK145/+ female mice showed an apparently normal amount of sleep but instead exhibited fragmented NREMS, whereas Tfap2bK144/+ male mice showed reduced NREMS time specifically in the dark phase. Finally, in the adult brain, Tfap2b-LacZ expression was detected in the superior colliculus, locus coeruleus, cerebellum, and the nucleus of solitary tract. These findings provide direct evidence that TFAP2B influences NREMS amounts in mice and also show that different mutations in Tfap2b can lead to diverse effects on the sleep architecture. Introduction Sleep is a physiological state widely observed in animals. However, the molecular pathways involved in regulating sleep and to what the extent they are conserved among vertebrates and invertebrates remain largely unknown. In humans, natural short sleepers exhibit a reduced amount of time in sleep without reporting daytime fatigue (reviewed in (Shi et al. 2017)). In cases where the natural short sleep trait is hereditary, it is expected that the identification of the causal gene might lead to the understanding of the molecules that control sleepiness. In addition, recent studies using invertebrate animal models suggest that sleep-regulatory mechanisms are at least partly conserved at the molecular 2 level between vertebrates and invertebrates (reviewed in (Miyazaki et al. 2017)). For example, a gain-of-function mutation in the salt-inducible kinase 3 leads to a drastic increase in the amount of non-rapid eye movement sleep (NREMS) in mice, whereas its orthologue in the nematode Caenorhabditis elegans promotes developmentally-timed sleep and the orthologue in the fruit fly Drosophila melanogaster promotes total sleep (Funato et al. 2016). Thus, studies using invertebrate animal models are also expected to lead to the identification of key molecules that contribute to sleep regulation across the animal phyla. In this study, we focused on the transcription factor AP-2β (TFAP2B). TFAP2B is a member of the AP-2 transcription factor family, which is involved in diverse processes including cell proliferation, differentiation, and apoptosis at both developmental and mature stages (reviewed in (Eckert et al. 2005)). In mice, Tfap2b knockout pups die shortly after birth due to renal failure(Moser et al. 1997, 2003). Tfap2b is also crucial for limb formation and differentiation of several neuronal cells (Seok et al. 2008; Jin et al. 2015; Seki et al. 2015). In humans, mutations in TFAP2B lead to Char syndrome (MIM 169100) (Satoda et al. 2000). Char syndrome is a congenital disease characterized by patent ductus arteriosus, facial dysmorphism, and hand anomalies (Davidson 1993). Notably, two human kindreds with Char syndrome, kindred 144 and 145, self-reported abnormalities in sleep, namely parasomnia (sleep-walking) and short-sleep, respectively (Mani et al. 2005). The two kindreds harbor a mutation in the introns of TFAP2B, thus implying the involvement of TFAP2B in sleep regulation. Moreover, in the nematode Caenorhabditis elegans, the orthologue APTF-1 is crucial for specification of the sleep-promoting neuron RIS, and aptf-1 mutants exhibit severely reduced quiescence during both developmentally-timed sleep and stress-induced sleep (Turek et al. 2013, 2016; Konietzka et al. 2020). In addition, in the fruit fly Drosophila melanogaster, knockdown of neuronal TFAP-2 results in reduced night time sleep (Kucherenko et al. 2016). These findings, in combination with recent studies supporting that the molecular mechanisms of sleep are conserved between mammals and invertebrates, suggest that mammalian TFAP2B is involved in sleep regulation. However, in case of mammals, conclusive evidence was lacking because the direct effect of mutating this gene was unknown. In this study, we tested whether sleep is affected in mice deficient for Tfap2b. Furthermore, considering that the human kindreds 144 and 145 self-reported totally different sleep abnormalities, we generated two mutant mouse strains, each carrying a mutation in Tfap2b that mimicked the mutation in the human kindreds 144 and 145 by utilizing CRISPR/Cas9 genome editing, and examined their effects on the sleep architecture. 3 Methods Animals. All animal experiments were approved by the institutional animal care and use committee of the University of Tsukuba. All animals were maintained according to the institutional guidelines of the animal facilities of the Laboratory of Animal Resource Center, University of Tsukuba. Mice were maintained and bred in International Institute for Integrative Sleep Medicine under 12-hour light/dark cycle and free access to food and water. Male and female mice of 10-13 weeks of age were used for sleep analyses. To obtain Tfap2b+/- mice, Tfap2btm1a(EUCOMM)Wtsi/+ mice from KOMP (Knockout Mouse Project) (Skarnes et al. 2011) were crossed with mice carrying the Ayu1-cre allele (Niwa et al. 1993) to remove a genomic DNA fragment containing Tfap2b exon3 and a neomycin resistant cassette flanked by loxP. The Tfap2btm1a(EUCOMM)Wtsi allele contains an IRES:lacZ trapping cassette with Engrailed (En2) splice acceptor sequences immediately after Tfap2b exon2 and is followed by a poly-A transcription termination signals. F1 offspring were then maintained by crossing with C57BL/6J mice. Generation of Tfap2bK144 and Tfap2bK145 mice by CRISPR/Cas9 technology The Tfap2bK144 point mutation (c.601+5G>A) and the Tfap2bK145 point mutation (c.822-1G>C) were introduced to the mouse genome by CRISPR/Cas9 technology as previously reported with some modifications (Sato et al. 2018). The guide RNA (gRNA) was synthesized and purified using the GeneArt Precision gRNA Synthesis Kit (ThermoFisher). The sequences of the gRNA were 5‘-GTCAGTCATTAAAAAAGGTA-3’ for Tfap2bK144 and 5‘-TTTTCGATTTGGCTCTGCAG-3’ for Tfap2bK145. The donor oligonucleotides for Tfap2bK144 and Tfap2bK145 were both 101 bases (Integrated DNA Technologies), in which the desired point mutations were introduced. Pregnant mare serum gonadotropin and human chorionic gonadotropin were intraperitoneally injected into female C57BL/6J mice (Charles River Laboratories), and unfertilized oocytes were collected from their oviducts. We then performed in vitro fertilization with these oocytes and sperm from male C57BL/6J mice. The gRNA, donor oligonucleotide, and GeneArt Platinum Cas9 Nuclease (ThermoFisher) were electroporated into zygotes using a NEPA 21 electroporator (NEPAGNENE). After electroporation, embryos developed to the two-cell stage in KSOM- AA medium. The next day, the electroporated
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