Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity

Masao Doi,Kenji Sakimura,Yukari Takahashi,Hiroshi Kiyonari,Yoshiaki Yamaguchi,Iori Murai,Jean-Michel Fustin,Choogon Lee,Hitoshi Okamura,Hiroyuki Shimatani,Kazuhiro Yagita,Hida Hayashi,Manabu Abe,Yuta Atobe,Nobuya Koike

doi:10.1038/s41467-019-10532-2

Abstract

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E′-box cis-element in the promoter region of the core clock gene Per2. The Per2 E′-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E′-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E′-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.

Highlights

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive
We used the piggyBac (PB) transposase tool for genome engineering because it allows for seamless removal of the PBflanked marker cassette (PB-Neo) from the host genome after the mutation is introduced (Fig. 1a)
Conventional methods that rely on the Cre/loxP or Flp/FRT system leave behind a single loxP- or FRT-derived ectopic sequence after marker excision

Summary

Introduction

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. The rhythm generating mechanism of the circadian clock involves clock genes, which regulate their own transcription in a negative transcription-translation feedback loop[4,5,6] In this canonical feedback model, non-coding cis-elements are fundamental to both initiating and closing the loop[7,8,9]. The Per[2] E′-box sequence (5′–CACGTT–3′) located near the putative transcription initiation site[17] (–20 to –15) has been demonstrated to be the principal circadian cis-element that is sufficient to induce oscillating levels of reporter transcription via the mouse Per[2] minimal promoter[17,18] The importance of this particular cis-element is further implied by the high degree of DNA sequence conservation in its flanking region between humans and mice[17] and by extremely enriched clock protein binding to this element[19]. Available genome-wide ChIPseq data highlight the predominant peak of clock protein binding activity around this E′-box (see Supplementary Fig. 1)

Methods

Results

Conclusion