Review

Development of the Circadian Core

Machinery in Mammals

Yasuhiro Umemura and Kazuhiro Yagita

Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto, 602-
8566, Japan

Correspondence to Kazuhiro Yagita: Fax: þ81 75-241-1499 kyagita@koto.kpu-m.ac.jp

https://doi.org/10.1016/j.jmb.2019.11.026
Edited by Achim Kramer

Abstract
The emergence of circadian molecular oscillation is observed as a gradual process during the development in
mammals. Pluripotent stem cell differentiation cultures recapitulate this process, whereas reprogramming into
an undifferentiated state reverses it. These findings indicate that the circadian clock is tightly coupled to the
state of cellular differentiation. The state of the circadian core machinery in nonrhythmic cells may be different
from that in rhythmic cells. In this review, we describe the circadian rhythm development during ontogeny in
mammals and focus on the molecular mechanisms that suppress circadian molecular oscillations during early
development and in pluripotent stem cells. We also discuss the biological implications of repressing cellular
circadian oscillation in nonrhythmic cells.
© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://crea
tivecommons.org/licenses/by-nc-nd/4.0/).

Introduction CRY in the nucleus and cytoplasm [5]. Once the

The mammalian circadian clock regulates diverse
physiological functions in the whole body, such as
the sleepewake cycle, hormone release, body
temperature, and metabolism. The central pace-
maker of the circadian clock is the suprachiasmatic
nucleus (SCN) in the hypothalamus. The SCN tunes
the peripheral tissue clocks in anticipation of the
cyclic environmental changes of the rotating earth.
Each organ and also each cell, in addition to cultured
cell lines, are endowed with an intrinsic cellular
circadian clock [1e4]. The molecular principle of the
cellular circadian clock is driven by transcriptional/
translational feedback loops (TTFLs) composed of a
subset of circadian clock genes. At the core of the
TTFLs are two transcriptional factors, BMAL1 (also
called ARNTL) and CLOCK. BMAL1 and CLOCK
form a heterodimer that actively works in the
induction of gene transcription by binding to E-box
enhancer elements. Their induced gene expression
includes their negative regulators Period (Per1, 2, 3)
and Cryptochrome (Cry1, 2). The core circadian
clock proteins assemble into a larger than 1-MDa
macromolecular complex, which include PER and
BMAL1eCLOCK heterodimer forms a complex with
PER and CRY proteins, it loses its transactivation
activity. PERs and CRYs are reported to inhibit the
inducible gene expression by promoting the dis-
sociation of the BMAL1eCLOCK heterodimers from
DNA [6]. In addition, BMAL1eCLOCK heterodimers
also induce the expression of Rev-erba (Nr1d1) and
Rev-erbb (Nr1d2), whose proteins repress Bmal1
expression by binding to the RORE promoter
element [7,8]. This process accrues the delay in
gene expression of the clock components, and this
delay in the TTFLs leads to circadian rhythmicity.
Other proteins are involved in the regulation of this
process and have been featured in this special issue
and other reviews [9,10].
Circadian Development During
Ontogenesis
Although almost all somatic cells are equipped
with an intrinsic circadian clock, it has been reported
that some cell types, including zygotes, the early
embryo, and germline cells, do not have a robust

0022-2836/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creati
vecommons.org/licenses/by-nc-nd/4.0/). Journal of Molecular Biology (2020) 432, 3611e3617
3612
circadian clock [11e13]. In recent decades, a
gradual circadian development during ontogeny
has been reported [14e17]. In the mouse, embryonic
tissues, such as the heart and liver, show circadian
rhythms at around E13e18 [18e21]. Although the
mouse SCN at E13.5 displays no circadian oscilla-
tions, the circadian rhythm of the SCN develops at
E14.5 [22]. Human circadian rhythms also emerge
gradually. The circadian rhythm of body temperature
emerges immediately after birth in full-term infants
[23], followed by the hormonal circadian rhythms
between 3 and 6 months of age [23], with the
sleepewake cycle becoming more stable after 6
weeks of life [24]. Interestingly, in zebrafish, the
circadian molecular oscillation is developed within
one day of fertilization [25]. The different circadian
clock formation periods may be attributed to species-
specific developmental processes.
Circadian Clock Development Tightly
Coupled with Cellular Differentiation
State
Recently, it has been reported that, despite mouse
pluripotent stem cells such as embryonic stem (ES)
cells having no discernible circadian molecular
oscillations of TTFLs, in vitro differentiation of ES
cells presents a circadian oscillation of clock gene
expression cell-autonomously [26,27]. Reprogram-
ming of somatic cells using Yamanaka factors (Oct3/
4, Klf4, Sox2, and c-Myc) [28] to produce induced
pluripotent stem (iPS) cells results in the disappear-
ance of the established circadian rhythm, suggesting
that the circadian clock development is tightly
coupled with the cellular differentiation state
(Fig. 1) [26].
During cell differentiation, genomewide epigenetic
modifications occur, and lineage-specific gene reg-
ulatory networks are established. Each undifferen-
tiated cell is considered to follow a lineage-specific
pathway to differentiate adequately as described in
Waddington's epigenetic landscape [29]. Almost all
differentiated cells ultimately establish the TTFLs of
Fig. 1. Circadian clock development is tightly coupled to cellular differentiation. The core TTFLs of the circadian
molecular oscillation in ES cells are not detectable, but the in vitro differentiation of ES cells induces a cell-autonomous
robust circadian oscillation. The circadian oscillation disappears after reprogramming to iPS cells.
Development of Circadian Machinery
the core circadian clock. The core circadian clock
gene network regulates the diverse lineage- and
tissue-specific gene expression as a circadian out-
put. Consequently, higher-order physiological func-
tions are acquired in the whole body. For example,
the circadian clock in the mouse embryonic heart
gradually develops from E10 after organogenesis,
and a circadian molecular oscillation is observed
from E17 [30]. In young adults, clock-controlled
heart-specific genes are expressed [31]. It is also
considered that other tissues acquire a core circa-
dian molecular oscillation during ontogeny, at which
point clock-controlled tissue-specific output genes
are expressed. It is still unknown how tissue-specific
gene expression becomes under the control of the
core circadian clock after cell differentiation. The
regulation may be related to the several circadian
regulation of mRNA transcripts. Circadian regula-
tions reside not only in the process of transcription
but also mRNA translation and modification such as
ploy-A modification, RNA methylation, and RNA-
editing [32e34]. Recently, it was reported that most
mRNAs of mouse forebrain are oscillating exclu-
sively in synapse likely by mRNA transport from
cytosol to synapse, and the mRNA localization and
the local translation are related to sleep/wake cycle
[35,36].
Molecular Mechanisms of Circadian
Clock Development During Ontogenesis
In vitro differentiated mouse ES and iPS cells
require an approximately 14-day culture time for
circadian clock development, and the discernible
and robust circadian oscillation does not emerge
immediately after the loss of pluripotency
[26,30,37,38]. It is suggested that additional
mechanisms are necessary for the development of
the circadian clock after cell differentiation. Our
previous work [20,26,30,37e39] has proposed a
two-step mechanism as a working hypothesis for
circadian clock development during ontogeny [30].
The first step is the cell-lineage determination
Development of Circadian Machinery 3613

process from an undifferentiated to a differentiated This stepwise differentiation process may be

state. The second step is a CLOCK protein rate-
limiting TTFL network maturation process.
CLOCK protein expression is suppressed posttran-
scriptionally in the first step, and this posttranscrip-
tional suppression gradually subsides during the
second step [30]. The gradual upregulation in the
expression of CLOCK proteins is concomitant with
the increase in the amplitude of circadian molecular
oscillation. NPAS2, a paralog of CLOCK that can
compensate for CLOCK function [40,41], is hardly
expressed in undifferentiated mouse cells [30];
therefore, posttranscriptional suppression of
CLOCK proteins is one of the key mechanisms of
the development of the circadian clock. We have
previously reported that Dicer/Dgcr8-dependent
miRNAs-mediated repression and Clock mRNA
export retardation are the main mechanisms of
CLOCK posttranscriptional suppression. These sup-
pressive mechanisms may be gradually canceled
during the second step of the circadian clock
development [30].
required for the development of circadian molecular
oscillation [30,37]. Perturbations of the differentiation
process by genetic modifications, such as DNA
methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b)
knockouts, c-Myc overexpression, or Kpna2 over-
expression, prevent the development of the circa-
dian molecular oscillation. The perturbation of the
normal differentiation process in the first step may
inhibit cells from starting the developmental upregu-
lation of CLOCK protein expression in the second
step [30,37].
Interestingly, mouse multipotent germline stem
(mGS) cells [42], a type of pluripotent stem cells, also
present posttranscriptional suppression of CLOCK
protein expression and circadian molecular oscilla-
tion development similarly to ES cells and iPS cells
during in vitro differentiation (Fig. 2) [30]. Further-
more, we recently suggested that posttranscriptional
suppression of CLOCK proteins also occurs in
human early-stage development by analysis using
human iPS cells [39]. Human iPS cells require longer

Fig. 2. CLOCK protein expression is upregulated and PER proteins translocate from the cytoplasm to the nucleus
during in vitro differentiation, correlating with the emergence of the “24-h” oscillation. (A) Morphology of ES cells,
mGS cells, and iPS cells. Scale bars ¼ 100 mm. (B) The circadian oscillations are emerged gradually in the pluripotent stem
cells during in vitro differentiation [30]. Representative bioluminescence traces in undifferentiated pluripotent stem cells
carrying Bmal1-luc reporters (top panels) and averaged bioluminescence traces in vitro differentiated pluripotent stem cells
at day 7, 14, and 28. Data are shown with s.e.m. (n ¼ 4e6). (C) The suppression of CLOCK protein expression and the
cytoplasmic localization of PER are observed in the undifferentiated pluripotent stem cells [30,32]. Immunofluorescence
study of endogenous CLOCK and PER1 proteins in ES cells, mGS cells, iPS cells, and 28-day differentiated ES cells.
Arrowheads indicate feeder cells. Scale bars ¼ 25 mm. Part of data were adapted and modified from Umemura et al. [30].
3614
differentiation times in culture for the development of
the circadian molecular oscillations [39]. This may be
related to the difference in the gestation periods
between mouse and human.
The circadian oscillation suppression in the first
step cannot be rescued by CLOCK overexpression
[30], suggesting that other mechanisms are
involved. Although further elucidation is needed to
understand the molecular mechanisms, the core
clock proteins and genes may be in different states
between normal rhythmic cells with circadian clock
and nonrhythmic cells. The state of most core clock
molecules in nonrhythmic cells seems to have
inhibitory effects on the cellular circadian oscillation.
Although Per2 gene expression is relatively low in
mouse ES cells, most core clock genes are
transcribed at similar expression levels in the
rhythmic differentiated cells. As reported previously,
the knock-down of Clock and Npas2 expression
induced that E-box-driven gene expressions such as
Nr1d1 and Dbp were inhibited in the somatic cells
[43] , as seen in Bmal1 knockout mice [44]. However,
these clock genes are expressed at almost similar
levels between ES cells and the rhythmic differen-
tiated cells. This discrepancy may arise from the
different epigenetic state of overall genome between
ES cells and the differentiated cells or E-box-binding
transcriptional factors such as MYC, which is highly
expressed in ES cells. On the other hand, it was
reported about the effect of dominant-negative
BMAL1 (BMAL1-DN) overexpression in ES cells
[26]. BMAL1-DN has the lack of the C-terminus and
strongly suppresses the BMAL1/CLOCK-mediated
transcription [45]. Although in NIH3T3 cells with the
circadian molecular oscillation, the BMAL1-DN over-
expression indicated the suppression of the clock
gene sets including Dbp, the BMAL1-DN suppres-
sive effect in ES cells is not observed in the clock
gene set. This result supports the CLOCK posttran-
scriptional suppression in ES cells.
The other components of clock also seem to be
regulated for the suppression of clock molecular
oscillation not at the level of transcription but at the
level of translation and/or the protein modification.
PER1 proteins in mouse pluripotent stem cells such
as ES cells, iPS cells, and mGS cells, are exclusively
localized to the cytoplasm (Fig. 2) [37]. In normal
rhythmic differentiated cells, PERs and CRYs can
shuttle dynamically between the nucleus and cyto-
plasm independently, but they are mainly localized
to the nucleus. Some modifications, such as
phosphorylation and ubiquitination, of these nega-
tive regulators modulate the subcellular dynamics
and protein stability [46e52]. PER2 is phosphory-
lated by CK2 and GSK3beta, and these modifica-
tions change its subcellular localization [50,53]. In
addition, PER-CRY binding induces the accumula-
Development of Circadian Machinery
tion of the complex in the nucleus and stabilizes PER
proteins [54e56]. In future, it will be necessary to
elucidate whether these molecular mechanisms that
occur in rhythmic cells also function in nonrhythmic
cells, such as ES cells.
CLOCK Suppression in Human
Epigenetic Cancers
Interestingly, we found that posttranscriptional
suppression of CLOCK proteins is also observed in
some types of human epigenetic cancers, such as
Wilms tumor and malignant rhabdoid tumor [57].
This suggests not only the disruption of the circadian
clock in cancer but also that epigenetic cancers
might undergo deviation from the normal cellular
differentiation process. Conversely, it could reflect
the pathophysiological significance of CLOCK pro-
tein suppression. Although the relationships
between cancer and circadian clock disruption
have been reported in several studies [58e67], the
viewpoint of cellular differentiation coupled to the
circadian clock may be useful to further investigate in
these settings.
Carrier Proteins of the Circadian Clock
Proteins
It is important to regulate the subcellular localiza-
tion of circadian clock molecules in the 24-h
oscillation period. Many types of carrier proteins
have been identified as regulators of the subcellular
localization of clock proteins. These carrier proteins
recognize nuclear localization signals in the clock
proteins and transport them from the cytoplasm to
the nucleus. KPNB1, also known as importin subunit
beta-1, is associated with KPNAs (importin subunit
alpha) and translocates the carrier substrates from
the cytoplasm to the nucleus. KPNB1 interacts
mainly with PERs and mediates PER and CRY
nuclear transport [68]. Other carrier proteins,
TNPO1, facilitate the nuclear localization of PER1,
but not PER2 [69]. More recently, it was reported that
noncoding RNAs are also related to the localization
of clock proteins [70].
One member of the Kpna family, Kpna2, has
been identified as an importin alpha subunit, and
its expression is abundant in undifferentiated cells,
such as ES cells. KPNA2 are reported to regulate
the differentiation state of cells by promoting
OCT3/4 nuclear entry and preventing OCT6
nuclear entry, leading to cellular differentiation
[71,72]. Recently, we found that Kpna2 over-
expression during in vitro differentiation of ES
Development of Circadian Machinery
cells has inhibitory effects on the development of
the circadian clock and the nuclear localization of
PERs. However, the binding interaction between
KPNA2 and PERs is considered to be very weak.
Therefore, KPNA2 overexpression during in vitro
differentiation of ES cells may inhibit the process of
cellular differentiation and indirectly affect the
abnormal localization of PER proteins to the
cytoplasm [37].
Biological Significance of Arresting the
TTFLs
Clock genes and proteins in nonrhythmic cells
may be regulated for arresting TTFLs, suggesting
that circadian oscillation is disadvantageous in the
early developmental stages. Although the biologi-
cal significance has not yet been elucidated in
mammals, in the fruit fly, Drosophila melanogaster,
Clock (Clk) overexpression induces developmental
lethality [73], and Clk posttranscriptional regulation
is essential for adequate development [74]. This
suggests that regulation of CLOCK expression is
essential not only for the circadian clock emer-
gence but also for the developmental process as a
whole.
Interestingly, in mammals, circadian clock arrest
may be not only due to defective TTFLs by
different states of circadian clock molecules.
Although it is believed that the maternal circadian
rhythm entrains the fetus [15,17,18,75], in our
study, gene expression was not rhythmic in the
mouse heart at E10e12, suggesting that maternal
circadian clock entrainment in the embryo is
prevented by unknown mechanisms in early
development [30]. This suggests the presence of
multilayered mechanisms that suppress both the
TTFLs of the circadian clock and the maternal
clock-entrained gene cycles during early develop-
ment and might highlight further the biological
significance of the suppression of the circadian
clock in early developmental stages.
Conflict of interest statement
This work was supported in part by grants-in-aid
for scientific research from the Japan Society for the
Promotion of Science to Y.U. (17KT0129) and K.Y.
(18H02600 and 19K22516).
Received 15 October 2019;
Received in revised form 26 November 2019;
Accepted 26 November 2019
Available online 10 January 2020
3615
Keywords:
cellular differentiation;
posttranscriptional regulation;
CLOCK;
circadian rhythm;
cellular circadian clock
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