Effect of postnatal photoperiod on DNA methylation dynamics in the mouse brain

Journal Pre-proofs Research report Effect of postnatal photoperiod on DNA methylation dynamics in the mouse brain Nozomu Takaki, Tatsuhiro Uchiwa, Mitsuhiro Furuse, Shinobu Yasuo PII: DOI: Reference:

S0006-8993(20)30081-0 https://doi.org/10.1016/j.brainres.2020.146725 BRES 146725

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Brain Research

Received Date: Revised Date: Accepted Date:

20 January 2019 26 October 2019 10 February 2020

Please cite this article as: N. Takaki, T. Uchiwa, M. Furuse, S. Yasuo, Effect of postnatal photoperiod on DNA methylation dynamics in the mouse brain, Brain Research (2020), doi: https://doi.org/10.1016/j.brainres. 2020.146725

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1

Effect of postnatal photoperiod on DNA methylation dynamics in the mouse brain

2 3

Nozomu Takaki, Tatsuhiro Uchiwa, Mitsuhiro Furuse, Shinobu Yasuo

4 5

Laboratory of Regulation in Metabolism and Behavior, Faculty of Agriculture, Kyushu

6

University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan

7 8

Corresponding author: Shinobu Yasuo, Ph.D.

9

Laboratory of Regulation in Metabolism and Behavior, Faculty of Agriculture, Kyushu-

10

University

11

744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan

12

Phone/Fax: +81-92-802-4597

13

E-mail: [email protected]

14 15

Abbreviations: 5-hmC, 5-hydroxymethylcytosine; 5-mC, 5-methylcytosine; DG, dentate

16

gyrus; DNMT, DNA methyltransferase; HC, hippocampus; LD, long-day; OB, olfactory

17

bulb; P, postnatal days; SD, short-day; SSC, saline sodium citrate; TET, ten-eleven

18

translocation; ZT, Zeitgeber time.

1

19

Abstract

20

Season of birth influences the onset of psychiatric diseases in mammals. Recent studies

21

using rodent models have revealed that photoperiod during early life stages has a strong

22

impact on affective and cognitive behaviors, neuronal activity, and hippocampal

23

neurogenesis/astrogenesis in later life. The present study examined the effect of postnatal

24

photoperiod on global DNA methylation and hydroxymethylation dynamics in the mouse

25

brain. Male mice born under short-day (SD) conditions were divided into SD and long-

26

day (LD) groups on the day of birth. Temporal expression of DNA methyltransferases

27

(DNMT1/3a) with 5-methylcytosine (5-mC) levels, as well as protein levels of ten-eleven

28

translocation (TET) 2 with 5-hydroxymethylcytosine (5-hmC) levels, were analyzed from

29

postnatal day 4 (P4) to P21. Levels of 5-hmC in all hippocampal areas were higher in the

30

LD group than in the SD group at P21, with a positive correlation between 5-hmC levels

31

and TET2 levels throughout the experimental period. Inconsistent results were observed

32

between DNMT1/3a mRNA levels and 5-mC levels. On the other hand, in the OB, mRNA

33

levels of DNMT1 and DNMT3a were slightly lower in the LD group similar to 5-mC

34

levels, but TET2 and 5-hmC levels were not influenced by the photoperiod. In conclusion,

35

postnatal exposure of mice to LD conditions induces an increase in TET2-dependent

36

DNA hydroxymethylation in the hippocampus, which might be involved in the long-term

37

effects of postnatal photoperiod on neurogenesis and affective/cognitive behaviors.

38 39

Keywords: DNA methyltransferase; Ten-eleven translocation; Hippocampus; 5-

40

Methylcytosine; 5-Hydroxymethylcytosine

2

41

1. Introduction

42

Seasonal changes in photoperiod influence various physiological functions and

43

behaviors in mammals, including reproduction, energy homeostasis, neuroplasticity, and

44

affective/cognitive behaviors (Dardente et al., 2019; Prendergast and Nelson, 2005;

45

Walton et al., 2011). In addition, gestational and/or postnatal photoperiod has enduring

46

effects on physiological and behavioral phenotypes; pre- and/or postnatal exposure to

47

short-day (SD) condition increases anxiety- and/or depression-like behaviors in Siberian

48

hamsters (Pyter and Nelson, 2006), rats (Toki et al., 2007), and C3H mice (Green et al.,

49

2015). Our previous studies demonstrated that C57BL/6J mice are a useful strain for the

50

study of photoperiodic regulation in affective behaviors, evidenced by the photoperiodic

51

changes in depression-like behaviors and related functions (Otsuka et al., 2012; Otsuka

52

et al., 2014). We have further clarified that photoperiod during early life stages altered

53

prepulse inhibition and depression-like behavior in adult C57BL/6J mice (Takai et al.,

54

2018). The critical window in rodents appears to be the early postnatal period (Takai et

55

al., 2018; Uchiwa et al., 2016), which is equivalent to the third trimester of pregnancy in

56

humans in terms of brain development (Clancy et al., 2007). The hippocampus represents

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one of the photoperiod-programmable regions, given that pre- and postnatal photoperiod

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alter the proliferation of neural stem cells and astrogenesis in the dentate gyrus (DG), as

59

well as expression of glucocorticoid receptors in later life (Takai et al., 2018; Toki et al.,

60

2007). These reports suggest that epigenetic mechanisms are involved in the long-term

61

effect of photoperiod over life stages, which may be related to the association between

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birth season and the onset of psychiatric diseases, including schizophrenia, bipolar

63

disorder, and seasonal affective disorder in humans (Foster and Roenneberg, 2008).

64

DNA methylation is one of the major epigenetic modification systems and has

3

65

important roles in neurodevelopmental or psychiatric diseases. For example, inhibition of

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DNA methylation in the hippocampus demonstrated anti-depressant-like effects (Sales et

67

al., 2011), and DNA methylation on peripheral blood DNA was associated with traits of

68

autism (Wong et al., 2014), and schizophrenia (Dempster et al., 2011). DNA methylation

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occurs by the addition of a methyl group to C5 position of cytosine to form 5-

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methylcytosine (5-mC), catalyzed by a family of DNA methyltransferases (DNMTs). De

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novo methylation is catalyzed by DNMT3a and DNMT3b, while DNMT1 maintains the

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pattern of methylation when DNA duplicates (Johnson et al., 2012). Alternately, ten-

73

eleven translocation (TET) proteins (TET1, TET2, and TET3) are involved in the DNA

74

demethylation

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hydroxymethylcytosine (5-hmC), 5-formylcytosine and 5-carboxylcytosine (Wu and

76

Zhang, 2017). Approximately 60 - 80% of CpG sites in the mammalian genome are

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modified to 5-mC, and 5-hmC accounting for roughly 40% of the modified cytosine in

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the brain (Kriaucionis and Heintz, 2009; Smith and Meissner, 2013). Methylation of CpG

79

islands plays a role in silencing of transcription and heterochromatin formation (Smith

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and Meissner, 2013). Recent studies further clarified that 5-hmC is not only an

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intermediate of demethylation process, but also has specific functions, such as promotion

82

of transcription and chromatin accessibility via a 5-hmC-binding protein (Mellén et al.,

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2012). Epigenetic modification by 5-hmC is associated with neurodevelopment and

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diseases; 5-hmC levels in the hippocampus are decreased in patients with Alzheimer's

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disease (Chouliaras et al., 2013), and 5-hmC levels in the cerebellum are inversely

86

correlated with the levels of methyl-CpG-binding protein 2, a protein responsible for Rett

87

syndrome (Szulwach et al., 2011).

88

process,

catalyzing

the

conversion

from

5-mC

into

5-

Developmental changes in the expression of DNMTs in the brain have been reported

4

89

in rats. The expression levels of DNMTs were highest during the first week of life and

90

decreased gradually until 3 weeks postnatally, although global DNA methylation levels

91

did not follow this expression pattern (Simmons et al., 2013). It was also reported that

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neuronal cells in mice acquire 5-hmC from postnatal neurodevelopment through

93

adulthood (Szulwach et al., 2011). Environmental factors such as caregiver maltreatment

94

and dietary conditions alter 5-mC and 5-hmC levels in the hippocampus, amygdala, or

95

thalamus (Doherty et al., 2016; Weng et al., 2014) suggesting that DNA methylation

96

dynamics may be additionally modified by early postnatal photoperiod. Therefore, in this

97

study we analyzed the effect of early postnatal photoperiod on the mRNA or protein levels

98

of DNMTs and TETs, as well as global 5-mC and 5-hmC levels in the C57BL/6J mouse

99

brain. For the analysis of TETs, we focused on TET2 due to the strong impact of TET2

100

in neurogenesis and memory (Gontier et al., 2018).

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2. Results

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2.1.

The mRNA expression of DNMT1 and DNMT3a

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Expressions of both DNMT1 and DNMT3a were most prominent in the hippocampus

104

and olfactory bulb (OB) as observed by in situ hybridization (Fig. 1A). Therefore, we

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measured their mRNA levels in these particular regions. Overall, the mRNA levels of

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DNMT1 in the hippocampus and OB were lower in the long-day (LD) group compared

107

with its levels in the SD group (Fig. 1B). A statistically significant difference was detected

108

in the OB (F(1,17)=13.73, p < 0.01), and a trend was observed in the DG (F(1,17)=4.42,

109

p = 0.0506). Moreover, the effect of age was significant in all regions (DG: F(2,17)=6.42,

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CA1: F(2,16)=10.83, OB: F(2,17)=8.83, p < 0.01, CA3: F(2,16)=5.62, p < 0.05); the

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mRNA levels of DNMT1 decreased with increasing age (Fig. 1B). No significant

112

interaction between photoperiod and age was detected in any of the regions examined.

113

The mRNA levels of DNMT3a in the hippocampus and OB were also lower in LD group

114

compared with its levels in the SD group (Fig. 1B). The main effect of photoperiod was

115

detected in the CA3 (F(1,17)=11.00, p < 0.01) and OB (F(1,17)=6.74, p < 0.05). However,

116

neither the effect of age nor the interaction between photoperiod and age were significant

117

in any of the regions analyzed. The mRNA levels of neither DNMT1 nor DNMT3a in the

118

hippocampus and OB in the 21-day-old (P21) mice were affected by the photoperiod (data

119

not shown).

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2.2.

Global levels of 5-mC and 5-hmC

122

5-mC was clearly detected by immunostaining in the DG and OB of P4 and P10 mice.

123

However, in CA1 and CA3 at the same ages, 5-mC levels were too weak to analyze (Fig.

124

2A). Moreover, weak 5-mC signals were observed in the brains of P21 mice (data not

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125

shown). Therefore, we quantified 5-mC signals only in the DG and OB of P4 and P10

126

mice. Our results demonstrated that there was no significant difference in 5-mC levels in

127

the DG between mice of the LD and SD group (Fig. 2B). However, 5-mC levels in the

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OB were significantly lower in the mice under LD condition compared with those under

129

SD (F(1,19)=4.92, p < 0.05, Fig. 2B). Additionally, the effect of age on 5-mC levels was

130

significant in the DG (F(1,19)=6.24, p < 0.05), but not in the OB. No significant

131

interaction was detected between photoperiod and age.

132

Further, 5-hmC was clearly detected by immunostaining in the hippocampus and OB

133

of P4, P10, and P21 mice (Fig. 3A). 5-hmC levels in the DG and CA3 were not

134

significantly different between LD and SD groups, while those in CA1 were significantly

135

higher in the LD group compared with those in the SD group (F(1,28) =6.28, p < 0.05,

136

Fig. 3B). Post-hoc analysis clarified that 5-hmC levels in the LD group were significantly

137

higher compared with those in the SD group in all hippocampal regions of P21 mice (p <

138

0.01; Fig. 3B). Moreover, the effect of age on 5-hmC levels was significant in all

139

hippocampal regions (DG: F(2,29)=11.32, CA1: F(2,28)=40.67, CA3: F(2,29)=60.49, p

140

< 0.001); 5-hmC levels decreased with increasing age in the hippocampus, whereas they

141

increased with increasing age in the OB (Fig. 3B). Furthermore, significant interactions

142

between photoperiod and age were detected in all hippocampal regions (DG:

143

F(2,29)=6.31, CA3: F(2,29)=8.43, p < 0.01; CA1: F(2,28)=4.63, p < 0.05). The levels of

144

5-hmC in the OB were not significantly different between LD and SD groups at any age

145

(Fig. 3B).

146 147 148

2.3.

TET2 protein levels

TET2 immunosignals were clearly detected in the hippocampus and OB (Fig. 3A).

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Signals in all regions were not significantly different between LD and SD groups (Fig.

150

3B). The main effect of age on TET2 levels was significant in all brain regions examined

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(DG: F(2,28)=17.59, CA1: F(2,27)=46.51, CA3: F(2,29)=62.71, p < 0.001; OB:

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F(2,30)=3.60, p < 0.05). Similar to 5-hmC levels, TET2 levels decreased or increased

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with increasing age in the hippocampus and OB, respectively (Fig. 3B). A statistically

154

significant interaction between photoperiod and age was detected in all hippocampal

155

regions (DG: F(2,28)=4.22, CA1: F(2,27)=4.97, p < 0.05; CA3: F(2,29)=7.62, p < 0.01).

156

Post-hoc analysis demonstrated that TET2 levels were significantly lower in the LD group

157

compared with those in the SD group in the DG and CA3 of P4 mice (DG: p < 0.05, CA3:

158

p < 0.01).

159

The photoperiod- and age-dependent trends were similar for 5-hmC and TET2 levels;

160

therefore, we sought to analyze the correlation between them. Indeed, we found strong

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correlations between TET2 and 5-hmC levels in the DG (p < 0.0001, r = 0.709, Fig. 3B),

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CA1 (p < 0.0001, r = 0.892, Fig. 3B), and CA3 (p < 0.0001, r = 0.938, Fig. 3B). On the

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other hand, the correlation between TET2 levels and 5-hmC levels in the OB was rather

164

weak (p < 0.05, r = 0.403, Fig. 3B).

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3. Discussion

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The present study demonstrated, for the first time, that early postnatal photoperiod

167

alters DNA methylation and hydroxymethylation dynamics in the hippocampus and OB

168

in a region-specific manner. Furthermore, exposure of mice to LD conditions induces an

169

increase in the DNA hydroxymethylation marker (5-hmC) in the hippocampus and a

170

decrease in the DNA methylation marker (5-mC) in the OB. Importantly, these changes

171

reflect the changes in the mRNA expression or protein levels of regulatory enzymes.

172

Hippocampal TET2 and 5-hmC levels showed a positive correlation, as observed in the

173

comparison between young and aged mice (Gontier et al., 2018) and the promotor region

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of miR-137 between sedentary and exercise groups in aged mice (Jessop and Toledo-

175

Rodriguez, 2018). In the OB, the mRNA levels of DNMT1 and DNMT3a were lower in

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the LD group, similar to that of 5-mC levels, although the physiological consequences

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remain unclear. DNA methylation status in the OB is linked to odor location memories

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in injured mice (Tajerian et al., 2019), suggesting a modulatory role of postnatal

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photoperiod in sensory systems.

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A previous study reported that exposure of adult animals to different photoperiods

181

alters DNMTs expression in Siberian hamsters, in which DNMT3a expression in the

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hypothalamus was higher in hamsters under LD condition than in those under SD

183

(Stevenson, 2017). Another study using adult male and female Siberian hamsters

184

demonstrated that DNMT3a expression in the testis and uterus was higher in animals

185

exposed to SD than in those exposed to LD, with higher global 5-mC levels in the testis

186

(Lynch et al., 2016). The present study is the first to demonstrate the effect of early

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postnatal photoperiod on DNA methylation/hydroxymethylation associated measures,

188

including DNMTs expression. Furthermore, the results observed in the hippocampus and

9

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OB were different from those of other tissues in animals exposed to photoperiods during

190

adulthood. Taken together, the effect of photoperiod on the expression of DNMTs and 5-

191

mC levels appears to be brain-region specific and, on the other hand, to depend on the

192

organ, age, and sex examined in each study.

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The physiological impact of the early postnatal photoperiod-dependent modulation of

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DNA methylation/hydroxymethylation is not clear in the present study. One possible

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explanation is the influence on hippocampal neurogenesis and related behaviors in later

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life, which have been shown to be modulated by early postnatal photoperiod in mice, rats,

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and hamsters (Green et al., 2015; Pyter and Nelson, 2006; Takai et al., 2018; Toki et al.,

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2007). Deficiency of dietary methyl-donors in adolescence resulted in impaired learning

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and memory, and decreased expression of glutamate receptors with specific CpG

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hypermethylation (Tomizawa et al., 2015). In adult rats bred for low response to novelty,

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increased dietary methyl-donor content ameliorated anxiety-like behaviors and decreased

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immobility in the forced swim test (McCoy et al., 2017). In addition, 5-hmC abundance

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increased with neural stem cell differentiation, accompanied by increased TET2 protein

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levels (Li et al., 2017). Thus, age-dependent changes in TET2 and 5-hmC levels in the

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hippocampus are positively associated with neurogenesis and cognitive functions

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(Gontier et al., 2018; Jessop and Toledo-Rodriguez, 2018). Notably, the present study

207

clarified higher levels of 5-hmC and TET2 in the hippocampus of the LD group,

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compared with those in the SD group, which is in agreement with the higher levels of

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neurogenesis in animals bred under LD (Takai et al., 2018). Mechanisms underlying

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postnatal photoperiod-induced changes in 5-hmC/TET2 levels remain unclear. TET2

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proteins can be regulated by calpains, a family of calcium-dependent proteases (Wang

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and Zhang, 278). Calpain activity in hippocampal cells is modulated by glucocorticoid

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and retinoic acid signaling (Roumes et al., 2016), both which are under the control of

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photoperiod (Otsuka et al., 2012; Dardente et al., 2019), suggesting a possible role in the

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regulation of TET2 protein levels.

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One of the cellular functions of DNA methylation/hydroxymethylation system is

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transcriptional regulation. At the molecular level, our previous study showed that early

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postnatal photoperiod alters the expression of a glucocorticoid receptor (Nr3c1) in the

219

hippocampus (Takai et al., 2018). The present study revealed that postnatal photoperiod

220

induced changes in DNA hydroxymethylation in the hippocampus. These data suggest

221

that

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methylation/hydroxymethylation dynamics as a consequence of photoperiodic conditions,

223

given that DNA methylation of Nr3c1 is highly sensitive to stress and glucocorticoid, a

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photoperiod-controlled hormone (Otsuka et al., 2012). For example, repetitive restrain

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stress on rats decreased global methylation levels in the hippocampus with decreased

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expression of Nr3c1 (Makhathini et al., 2017), and acute stress increased 5-hmC levels

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of Nr3c1 promoter in the mouse hippocampus (Li et al., 2015). Embryonic exposure to

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high dose of corticosterone increased DNA methylation of the hypothalamic Nr3c1 gene

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in the chicken (Ahmed et al., 2014). However, we have no data about the effect of early

230

postnatal photoperiod on methylation/hypermethylation levels in specific genes, nor

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genomic distribution of their markers, which needs to be studied in future.

the

altered

expression

of

Nr3c1

may

be

caused

by

DNA

232

In the present study, expression of DNMT1 and DNMT3a and 5-mC levels in both the

233

hippocampus and OB decreased with age. These observations are in congruence with the

234

report using rats, in which expression of DNMTs and 5-mC levels gradually decreased

235

from birth to P21 in the hippocampus (Simmons et al., 2013). The present study also

236

showed an age-dependent decrease in 5-hmC and TET2 protein levels in the hippocampus,

11

237

in line with findings of a previous study, showing that 5-hmC and TET2 levels decreased

238

with age, at least until the age of 18 months, and affected neurogenesis and memory in

239

mice (Gontier et al., 2018). On the other hand, we uncovered unexpected results in the

240

OB, in which 5-hmC and TET2 levels increased with age. Brain area-specific patterns of

241

age-dependent 5-hmC and TET2 levels may be related with different timing of postnatal

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synaptogenesis and neurogenesis between the hippocampus and the OB; synaptogenesis

243

and neurogenesis in the DG is most active between P7 and P30 (O'Kusky et al., 2000;

244

Steward and Falk, 1986), while the OB grows steadily in size from birth to maturity, until

245

the age of 12 weeks (Pomeroy et al., 1990).

246

In conclusion, here we demonstrated that the photoperiod during early postnatal life

247

differentially alters DNA methylation and hydroxymethylation systems in the mouse

248

hippocampus and OB. In the hippocampus, the photoperiod possibly alters global 5-hmC

249

levels by modulating TET2 protein levels. In the OB, global 5-mC levels were regulated

250

by the photoperiod and similar changes were observed in the expression of DNMTs.

251

These changes may have long-term effects on neurogenesis and affective/cognitive

252

behaviors.

253 254

Conflict of interest

255

The authors declare that they have no conflict of interest.

256 257

Acknowledgement

258

This study was supported by Grants-in-Aid for Challenging Exploratory Research

259

(No.17K19914) to S.Y. from the Japanese Society for the Promotion of Science.

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4. Experimental procedure

261

4.1.

Animals

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The study was conducted according to the Guidelines for Animal Experiments by the

263

Faculty of Agriculture in Kyushu University, following the law No. 105 and Notification

264

No. 6. All animal experiments reported here were conducted in accordance with the as

265

well as All experiments were approved by Animal Care and Use Committee of Kyushu

266

University (A28-069-0). Mice were maintained in a light-tight box placed in a room at

267

the temperature of 25 ± 1°C. Water and standard diet for laboratory rodents (MF, Oriental

268

Yeast, Tokyo, Japan) were available ad libitum and were replenished at least once a week.

269

Male and female 6-week-old C57BL/6J mice obtained from Japan SLC, Inc. were

270

separately housed in groups of 3 or 4 animals under SD (6 h light - 18 h dark, 6L18D) for

271

more than 3 weeks. Thereafter they were mated under SD and the offsprings with their

272

mothers and littermates were randomly separated into 2 groups on the day of birth: SD

273

and LD (18 h light - 6 h dark, 18L6D) groups. Both groups included offsprings from 2 -

274

4 litters in each age. This study used the SD condition, not 12L12D, for acclimation and

275

control for the following reasons: 1) the shifts from 12L12D to SD and from 12L12D to

276

LD are opposite to each other with regard to the photoperiodic response and have been

277

suggested to involve distinct mechanisms; thus, it would be difficult to compare the

278

outcomes (Yasuo et al., 2010); 2) 12L12D was interpreted as a stimulatory LD condition

279

in F344 rats (Tavolaro et al., 2015) and, thus, the effect of LD would not be precisely

280

evaluated. Therefore, we used SD as the control condition and focused on the stimulatory

281

effect of LD.

282

For in situ hybridization, the offsprings at P4, P7, P10 and P21 (n = 3 - 4, including

283

offsprings from more than 2 litters for each age) were euthanized by isoflurane gas and

13

284

decapitated at Zeitgeber time (ZT; ZT0 corresponds to light onset) 2. The brains were

285

collected and frozen with dry ice and stored in -80 °C until analysis. Because it was

286

difficult to quickly remove the skull of P4, P7, and P10 mice, their brains were frozen

287

with the skull. For immunohistochemistry, the offsprings at P4, P10 and P21 (n = 5 - 6,

288

including offsprings from more than 2 litters for each age) were euthanized by isoflurane

289

gas and decapitated at ZT2, and the brains were incubated with 4% paraformaldehyde for

290

12 h and cryoprotected with 20% sucrose at 4 °C. The brains were embedded in OCT

291

compound and frozen quickly. For both analyses, only male offsprings were used because

292

it is reported that the effect of early postnatal environment on DNA methylation in the

293

hippocampus is different between male and female rats (Doherty et al., 2016), and in the

294

previous studies on the effect of postnatal photoperiod male animals were often used

295

(Takai et al., 2018; Toki et al., 2007; Uchiwa et al., 2016). Therefore, the results of the

296

present and previous studies can be compared appropriately.

297 298

4.2.

In situ hybridization

299

Sagittal brain sections (18 μm-thick) were prepared using a cryostat (Leica, Wetzler,

300

Germany). Antisense and sense oligonucleotide probes for each gene (antisense: DNMT1:

301

5′-GGCCCACAGCGTTTCCGAGATGCCTGCTTGGTGGAATCCTTCCGA-3′;

302

DNMT3a:

303

GCCTCCAATGAAGAGTGGGTGCTCCAGGGTGACATTGAGGCTCCC-3′)

304

labeled with [33P]dATP (PerkinElmer, MA, US) by using terminal deoxynucleotidyl

305

transferase (Invitrogen, CA, US) at 37 °C for 2 h. The sections were fixed using 4%

306

paraformaldehyde in phosphate buffered saline for 10 min, and hybridization was carried

307

out overnight at 42 °C with 750,000 cpm of 33P-labeled probe for each slide. Two high-

5′-

14

were

308

stringency post-hybridization washes were performed at 55 °C. The sections were air-

309

dried and exposed to an X-ray film (Kodak, Tokyo, Japan) for 2 weeks with 14C-labeled

310

standards (American Radiolabeled Chemicals, MO, US). Sense probes detected no

311

signals for either DNMT1 or DNMT3a. We also performed the analysis for DNMT3b and

312

TET2 expression, but signals were too weak to analyze (data not shown).

313

Relative optical densities of signals were measured using a computed image analysis

314

system (ImageJ, National Institutes of Health, http://imagej.nih.gov/ij) and were

315

converted into the relative radioactivity units (Bq/mg) by using the 14C-labeled standards.

316

Two sections including the hippocampus and OB were selected from each animal for each

317

gene, and signals in the DG, CA1, CA3, and OB were analyzed. In each section,

318

background values obtained from adjacent brain areas that did not exhibit a hybridization

319

signal were subtracted. Signals from two sections were averaged in each animal.

320 321

4.3.

Immunohistochemistry

322

Sagittal cryostat sections (20 μm-thick) including the hippocampus and OB were

323

prepared. For immunostaining of 5-mC and 5-hmC, the sections were dried and soaked

324

sequentially in 1.5% hydrogen peroxide in methanol for 10 min, 50% formamide/2 ×

325

saline sodium citrate (SSC) at 65 °C for 30 min, 2 × SSC for 5 min, 1 N hydrochloric acid

326

at 37 °C for 30 min, and 0.1 M boric acid (pH 8.5) for 10 min. The sections were then

327

incubated with 10% normal goat serum for 30 min. Thereafter, the sections were

328

incubated overnight with rabbit monoclonal anti-5-mC antibody (1:800, Cell Signaling

329

Technology, Danvers, MA) or rabbit polyclonal anti-5-hmC antibody (Active Motif,

330

Carlsbad, CA) diluted in buffer containing bovine serum albumin (1%). Subsequently,

331

the sections were incubated with biotinylated goat anti-rabbit IgG (1:100) antibody and

15

332

ExtrAvidin-Peroxidase (1:100) for 1 h each. Immunosignals were visualized using

333

diaminobenzidine. For immunostaining of TET2, we used rabbit polyclonal anti-TET2

334

(1:500, Abcam, Cambridge, UK) antibody as a primary antibody, and the immunostaining

335

was performed as described above, except for eliminating the formamide, SSC, HCl, and

336

boric acid steps.

337

For the quantification of 5-mC and 5-hmC signals, optical densities of each signal were

338

analyzed using ImageJ. Two sections from each animal were selected for hippocampus

339

and OB and the optical densities in the DG, CA1, CA3, and OB were analyzed. In each

340

section, background values obtained from adjacent brain areas were measured and

341

subtracted from the values of target areas. The signal levels measured in two sections

342

were averaged for each animal, and expressed as the percentage of the maximum value

343

of all animals.

344 345

4.4.

Statistics

346

Data were analyzed by two-way ANOVA designating photoperiod and age as the two

347

factors. When significant interaction between photoperiod and age was detected, post hoc

348

analysis was performed using Bonferroni’s multiple comparison test. Correlation analysis

349

between TET2 and 5-hmC levels was performed using Pearson’s correlation coefficient

350

with Fisher’s z transformation. Values were considered significantly different at p < 0.05.

351

For the in situ hybridization data, P4, P7, and P10 mice were analyzed separately from

352

P21 mice, because their sampling methods were different (with or without skull). t-Test

353

was used to analyze the data in the P21 mice. All analyses were performed using

354

Graphpad Prism version 7.02 for Windows (GraphPad Software Inc., San Diego, CA).

16

355

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Figure legends

531

Figure 1

532

Effect of postnatal photoperiod on the expression of DNA methyltransferases (DNMTs)

533

in the mouse hippocampus (HC) and olfactory bulb (OB). (A) Representative

534

autoradiographs of in situ hybridizations for DNMT1 and DNMT3a in postnatal day 4

535

(P4), P7, and P10 mice. (B) Temporal expressions of DNMT1 and DNMT3a mRNAs in

536

the hippocampal dentate gyrus (DG), CA1, CA3, and in the OB of mice postnatally

537

exposed to short-day (SD) and long-day (LD) conditions. Means ± standard error of mean

538

(n = 3 - 4). *p < 0.05, **p < 0.01, two-way ANOVA. Scale bars: 1 mm.

539 540

Figure 2

541

Effect of postnatal photoperiod on 5-methylcytosine (5-mC) levels in the mouse

542

hippocampal dentate gyrus (DG) and olfactory bulb (OB) at postnatal day 4 (P4) and P10.

543

(A) Representative images of immunostainings against 5-mC. (B) Temporal changes in

544

5-mC levels in the DG and OB in mice postnatally exposed to short-day (SD) and long-

545

day (LD) conditions. Means ± standard error of mean (n = 5 - 6). *p < 0.05, two-way

546

ANOVA. Scale bars: 50 μm.

547 548

Figure 3

549

Effect of postnatal photoperiod on 5-hydroxymethylcytosine (5-hmC) and protein levels

550

of ten-eleven translocation (TET) 2 in the hippocampus (HC) and olfactory bulb (OB) of

551

postnatal day 4 (P4), P10, and P21 mice. (A) Representative images of immunostainings

552

against 5-hmC and TET2. (B) Temporal changes in 5-hmC levels and TET2 protein levels

553

in the hippocampal dentate gyrus (DG), CA1, and CA3 and in the OB in mice postnatally

25

554

exposed to short-day (SD) and long-day (LD) conditions. The relationship between 5-

555

hmC and TET2 levels is also illustrated. Means ± standard error of mean (n = 5 - 6). *p

556

< 0.05, **p < 0.01, two-way ANOVA. #p < 0.05, ##p < 0.01, Bonferroni’s multiple

557

comparison test. Scale bars: 100 μm.

558



Postnatal photoperiod altered DNA methylation system in mouse brain.

559



Hippocampus and olfactory bulb showed different dynamics of related enzyme levels.

560



These changes may relate to long-term effects on neurogenesis and behaviors.

561

26

562

27

563

28

564

29