The CoREST complex regulates multiple histone modifications temporal-specifically in clock neurons

Epigenetic regulation is important for circadian rhythm. In previous studies, multiple histone modifications were found at the Period (Per) locus. However, most of these studies were not conducted in clock neurons. In our screen, we found that a CoREST mutation resulted in defects in circadian rhythm by affecting Per transcription. Based on previous studies, we hypothesized that CoREST regulates circadian rhythm by regulating multiple histone modifiers at the Per locus. Genetic and physical interaction experiments supported these regulatory relationships. Moreover, through tissue-specific chromatin immunoprecipitation assays in clock neurons, we found that the CoREST mutation led to time-dependent changes in corresponding histone modifications at the Per locus. Finally, we proposed a model indicating the role of the CoREST complex in the regulation of circadian rhythm. This study revealed the dynamic changes of histone modifications at the Per locus specifically in clock neurons. Importantly, it provides insights into the role of epigenetic factors in the regulation of dynamic gene expression changes in circadian rhythm.


Introduction
Studies in recent decades have revealed the core molecular mechanism that controls biological rhythms.The first molecular clock gene, Period (Per), was identified through a genetic screen of mutants generated in Drosophila melanogaster [1].In Drosophila clock neurons, the protein complexes composed of CLOCK/CYCLE (CLK)/(CYC) and PERIOD/TIMELESS (PER)/(TIM) form a negative feedback loop [2].Studying the regulatory mechanisms of the core clock genes within this regulatory loop is of great significance for improving our understanding of the molecular mechanisms of the biological clock.
Histone modification plays an important role in the regulation of circadian rhythm.H3K9me3 and H3K27me3 have been found to modify mammalian clock genes and their downstream genes [3][4][5].Moreover, histone acetylation and ubiquitination are also important in circadian rhythm regulation [6][7][8][9][10].However, most studies investigating the interplay between histone methylation/acetylation and clock regulation have been conducted in liver tissue or mammalian cell lines.The roles of histone methylation/acetylation in the core clock regulatory circuit of clock neurons are still unclear.
The analysis of the molecular features of clock neurons shows that they possess a unique pool of expressed genes.In a previous study, clock neuronspecific expressed genes were analysed using microarray technology [11].However, the epigenetic characteristics of clock neurons remain unknown.Recently, the development of the mini-INTACT (isolation of nuclei tagged in specific cell type) method has provided a rapid way to isolate neurons for

Drosophila activity monitor-based method for circadian rhythm measurement
For all activity measurements, flies were kept in a 12 h light/12 h dark (12:12 LD) cycle at 25°C.Flies (3−5 days old) were individually loaded into detection tubes (length, 65 mm; inner diameter, 5 mm) containing standard cornmeal fly food at one end and a cotton stopper placed at the other end.The circadian rhythm was measured using the DAM (Drosophila activity monitor) System (Trikinetics, MA), which counted the infrared beam crossings of individual flies in each tube every 1 min.All circadian rhythm tests were carried out on male flies unless otherwise specified.Flies were entrained in the detection tube at 25°C for 72−96 h in a 12 h light/12 h dark cycle.Subsequently, data were collected in dark conditions for at least 5 days using the DAM System.The analyses of circadian rhythm were carried out using faasX software (obtained from the website https://trikinetics.com) and MATLAB (MathWorks, Natick, MA).

Circadian behaviour assessment of temperature-mediated phase shift
The temperature cycle (TC) experiment was conducted following the method described by Gentile et al. [23].Initially, flies were synchronized to three light/dark (LD) cycles at 25°C.Subsequently, the temperature was reduced to 16°C for 6 h, followed by 12 h at 25°C and 12 h at 16°C TC for 6 days.The initial TC was then modified by delaying the temperature rise by 6 h, and flies were tested to resynchronize with the shifted TC for another 6 days.The entrainment index (EI) was calculated as the ratio of total activities occurring during a 6 h window to the activities occurring during the entire warm phase [23].A value close to 1 indicates that most of the activity occurred within the specified window, indicating entrainment.Based on the inspection of the average activity profiles of the control flies, a 6 h window for the main temperature-synchronized activity was defined as ZT15-ZT20.5 (displayed as a red dotted line in figure 1a,b; ZT, zeitgeber time) [23].

Quantitative real-time PCR
Total RNA was extracted from 30 heads using TRIzol Reagent (TIANGEN, no.DP4−02).Reverse transcription and real-time PCR (RT-PCR) were performed using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time; TakaRa, no.RR047A) and SuperReal PreMix Plus (SYBR Green; TIANGEN, no.FP205−02) following the manufacturer's instructions.All experiments were performed using the StepOne Real-Time PCR system (Applied Biosystem, Foster, CA).Quantification was 2 royalsocietypublishing.org/journal/rsob Open Biol.14: 230355 performed using the ΔΔCT method.Unpaired two-tailed Student's t-test (Prism GraphPad) was used to compare the differences between genotypes.All primers used are listed in electronic supplementary material, table S1.All quantitative RT-PCR analyses were performed with three biological replicates.

Cell culture and transient transfection
S2 cells were cultured in serum-free insect cell culture medium (HyClone, no.SH30278.02) at 25°C.Transient transfection was performed using X-tremeGENE HP DNA Transfection Reagent (Roche, no.06366236001), following the manufacturer's protocol.

Plasmid constructions 2.7.1. Cloning of pAC-CN fusion plasmids
The N-terminal part of mCerulean (CN) was amplified via PCR from the pGWAAV-CMV-PSD95-mCerulean using the forward primer EcoRI-CN F and reverse primer NotI-CN R [24].Plasmid pAC-mGFP was digested with EcoRI-NotI, and the CN fragment was inserted, resulting in pAC-CN.The CoREST-RF (CoREST long form) was amplified via PCR from the cDNA of fly heads using the forward primer NotI-CN-CoREST-RF F and the reverse primer HindIII-CN-CoREST-RF R. Plasmid pAC-CN was digested with NotI-HindIII, and the CoREST-RF fragment was inserted, resulting in pAC-CN-CoREST-RF.LSD1, HDAC1 and KDM4A were also cloned into the pAC-CN vector using the same procedure, respectively.All primers used are listed in electronic supplementary material, table S1.

Cloning of pAC-CC fusion plasmids
The C-terminal portion of mCerulean (CC) was amplified via PCR from the pGWAAV-CMV-PSD95-mCerulean using the forward primer NotI-CC F and reverse primer HindIII-CC R [24].Plasmid pAC-mGFP was digested with NotI-HindIII, and the CC fragment was inserted, resulting in pAC-CC.The CoREST-RF was amplified via PCR from the cDNA of fly heads using the forward primer EcoRI-CoREST-RF-CC F and reverse primer NotI-CoREST-RF-CC R. Plasmid pAC-CC was digested with EcoRI-NotI, and the CoREST-RF fragment was inserted, resulting in pAC-CoREST-RF-CC.E(Z), HDAC1 and KDM4A were also cloned into the pAC-CC vector using the same procedure, respectively.All primers used are listed in electronic supplementary material, table S1.

Bimolecular fluorescence complementation
The bimolecular fluorescence complementation (BiFC) analysis was performed following the method described by Bischof et al. [24].Briefly, the mCerulean partial sequences encoding amino acid residues 1−173 (CN) or amino acid residues 173−238 (CC) were used to construct plasmids containing fusion genes.Fusion gene plasmids were transfected into S2 cells.After 48 h of transfection, cells were washed three times with phosphate-buffered saline (PBS).The samples were analysed using confocal microscopy (Leica SP8).

Chromatin immunoprecipitation to detect clock binding
Chromatin immunoprecipitation (ChIP) of adult heads was performed as previously described [25], with minor modifications.Twenty-five heads were collected in 450 µl PBS on ice.For cross-linking, 6.02 µl 37% formaldehyde was added, followed by incubation at room temperature for 10 min.Chromatin was sonicated for 2.5 min on ice (settings were 10 s on, 30 s off, high power).The sheared chromatin had an average length of 0.1-0.5 kb.Rabbit anti-GFP (Invitrogen, no.G10362) was used for immunoprecipitation.Fold enrichment was calculated by the ΔΔCT method.All ChIP analyses were repeated three times as independent biological replicates (refer to electronic supplementary material, table S1 for primer sequences).

CoREST long isoform was required for the regulation of circadian rhythm
In a candidate screen aimed at identifying epigenetic regulators of circadian rhythm, we discovered that mutations of CoREST resulted in circadian rhythm defects.The two insertional mutations of CoREST, CoREST MI08173 and CoREST EY14216 , resulted in a decrease in rhythmicity percentages to 62.8% and 60.6% respectively, compared with w 1118 control (figure 2a,d).Moreover, both mutants exhibited significantly reduced power values (figure 2e).Female flies carrying CoREST MI08173 and CoREST EY14216 mutations also exhibited identical circadian rhythm defects as observed in male flies (figure 2f,h,j).Furthermore, the weak enhancement of circadian phenotypes in the CoREST MI08173 /CoREST EY14216 double mutant (female; both genes are on the X chromosome) (figure 2i,k) suggested that these two alleles likely share similar mechanisms in causing circadian phenotypes.Hence, these results support the role of CoREST as a regulator of circadian rhythm.
A previous study has shown that CoREST has two major splicing forms with different functions, which are represented by RC and RF (figure 2l) [20].the long form of CoREST was referred to as CoREST-RF.While, the N-terminus truncated isoform was CoREST-RC, which contains an extra sequence at the 5′UTR (figure 2l).CoREST EY14216 and CoREST MI08173 alleles are insertional mutations of CoREST, inserted in the 5′ and 3′ of the CoREST coding region, respectively (figure 2l).As a result, CoREST EY14216 only affects RF instead of RC (figure 2m).On the other hand, CoREST MI08173 affects both the RF and the RC isoforms (figure 2m).
To determine which splicing form was crucial in the circadian phenotypes caused by CoREST MI08173 and CoREST EY14216 alleles, we conducted genetic interaction experiments.Our findings revealed that the expression of RF driven by tim-Gal4 was able to rescue the circadian phenotypes caused by both alleles (figure 2n,o).In contrast, the expression of RC driven by tim-Gal4 failed to rescue the phenotypes caused by CoREST MI08173 and CoREST EY14216 (figure 2n,o).Consistently, specific RNAi knockdown of RC did not result in noticeable circadian phenotypes (figure 2p,q).The knockdown of CoREST RF in clock neurons using tim-Gal4 resulted in a decrease in the percentage of rhythmicity to 77.1% (figure 2p).Moreover, the power value was also significantly reduced in tim-Gal4/UAS-CoREST RF RNAi (figure 2q).These results indicated that the long-form RF was the major isoform responsible for the circadian phenotypes.
In conclusion, CoREST long isoform was essential for regulating the circadian rhythm.In our subsequent experiments, we primarily used the CoREST MI08173 allele as it exhibits stronger effects compared to the other allele.

CoREST regulates circadian rhythm by modulating the expression of Per
Previous studies have reported that CoREST mediates the binding of epigenetic factors on chromatin [16,17,26].To investigate the mechanism of CoREST function in circadian rhythm, we examined the binding profile of epigenetic factors on core clock regulators, Per, Clk, Tim and Cyc.In modENCODE database (http://www.modencode.org/),we identified significant binding of KDM4A, LSD1 and HDAC1 on the Per locus (electronic supplementary material, figure S1A) [27].Previous data have shown that LSD1 and HDAC1 are binding proteins of CoREST [16,17,26].It has also been observed that LSD1 interacts with E(Z) [28,29].Moreover, previous studies have shown that mutation and overexpression of Per result in strong and weak rhythmic phenotypes, respectively [1,30].Therefore, we hypothesized that CoREST regulates circadian rhythm by controlling the activity of these factors at the Per locus.
To test our hypothesis, we examined the effects of CoREST mutation on the expression of Per.First, we detected the relative expression level of Per in CoREST MI08173 .We found that under constant darkness (CT) conditions, the peak of Per expression in w 1118 was located at CT12, while this peak was shifted to CT16 in CoREST MI08173 (figure 3a).Compared with the w 1118 , the overall oscillation pattern of Per expression in CoREST MI08173 was significantly enlarged (figure 3a; w 1118 JTK_amplitude = 0.449, p < 0.0001; CoREST MI08173 JTK_amplitude = 1.500, p = 0.0008) [31].Under the LD condition, the peaks of Per expression of w 1118 and CoREST MI08173 were at ZT16 (figure 3b).Compared with the w 1118 , the overall oscillation pattern of Per expression in CoREST MI08173  was also significantly enlarged (figure 3b; w 1118 JTK_amplitude = 4.631, p < 0.0001; CoREST MI08173 JTK_amplitude = 5.460, p = 0.0001).Consistently, the variation of Per expression level from ZT8 to ZT16 was steeper compared with the w 1118 (figure 3b).The protein level of Per was also influenced by CoREST.The detection of PER protein at various time points demonstrated alterations in its protein level (figure 3c,d).These results imply that CoREST influences the expression pattern of Per.In addition, we also constructed CoREST MI08173 /Per 01 double mutant (female) to reduce the amplitude of Per and performed behavioural assays.The results showed that the CoREST MI08173 /Per 01 double mutant could partially rescue the circadian phenotype caused by CoREST MI08173 (figure 3e,f).Based on these results, we concluded that CoREST regulates circadian rhythm by modulating Per expression.

Identification of genetic and physical interactions between CoREST and histone modification factors KDM4A, LSD1, E(Z) and HDAC1
To further validate the hypothesis that CoREST regulates the circadian rhythm by recruiting epigenetic factors (including KDM4A, LSD1, E(Z) and HDAC1) at the Per locus, we conducted genetic interactions tests between CoREST and these histone modification factors.We examined the circadian rhythm phenotypes of various mutants and double mutants.The results showed that the mutations in KDM4A and HDAC1 resulted in mild rhythmic defects, respectively (with the percentage of rhythmicity at 88.5% in KDM4 KO /+ and 96.6% in HDAC1 303 /+) while, the mutation in E(Z) did not result in any rhythm defects (percentage rhythmicity in E(Z) 63 /+ was 100%) (electronic supplementary material, figure S1b, f).We deduced that the weaker rhythm phenotype occurred because none of these mutants could be homozygous, and only a subset of them could fulfil the organism's required function.Consistently, the knockdown of LSD1, KDM4A and HDAC1 in clock neurons resulted in mild defects in the percentage of rhythmicity (electronic supplementary material, figure S1e).In contrast, the power value significantly decreased, thereby confirming our conjecture (electronic supplementary material, figure S1f).These results suggest that HDAC1, KDM4A and LSD1 are all involved in the regulation of circadian rhythm.
CoREST MI08173 ; KDM4A KO /+double mutant (percentage of rhythmicity was 75.9% in the double mutant compared to 62.8% in CoREST MI08173 ) did not significantly rescue or enhance the percentage of rhythmicity of CoREST MI08173 (figure 4a,e).However, CoREST MI08173 ; E(Z) 63 /+double mutant (percentage of rhythmicity was 85.3% in the double mutant compared to 62.8% in CoREST MI08173 ) showed the rescued phenotypes of CoREST MI08173 in the percentage of rhythmicity (figure 4b,e).There was also a clear enhancement in the power value (figure 4f).
CoREST MI08173 ; HDAC1 303 double mutant (percentage of rhythmicity was 47.8% in the double mutant compared to 62.8% in CoREST MI08173 ) showed an enhanced phenotype of CoREST MI08173 in the percentage of rhythmicity (figure 4c,e).CoREST MI08173 ; tim-Gal4/+; UAS-LSD1 RNAi/+ (percentage rhythmicity of 48.4% compared with 54.8% in CoREST MI08173 ; tim-Gal4/+) showed a trend towards an enhanced phenotype of CoREST MI08173 in the percentage of rhythmicity and power value (figure 4d,f).These results indicate that these factors are potential circadian regulators that mediate the function of CoREST in clock neurons.

In clock neurons, multiple histone modifications at the Per locus were affected by CoREST
We deduce that if CoREST recruits these epigenetic factors at Per locus to regulate the circadian rhythm, the relevant histone modifications should rely on CoREST.Therefore, we conducted ChIP experiments in the context of clock neurons to investigate whether the histone modifications at the Per locus were altered owing to CoREST mutation.We analysed H3K27me3, regulated by E(Z) [32,33], H3K4me2 regulated by LSD1 [34], H3K27ac regulated by HDAC1 [35], H3K9me3 regulated by KDM4A and LSD1 [34,36] and H3K36me3 regulated by KDM4A [36,37] in the clock neurons of w 1118 and CoREST MI08173 using CNS-ChIP (see §2) (figure 5a).
We found that at the Per locus, the negative control IgG showed no change.In contrast, H3K27me3 significantly increased at ZT16 compared with ZT8 in the control group (electronic supplementary material, figure S3b, d).After the CoREST mutation, there was a significant increase in the level of H3K27me3 (figure 5b,c), indicating that CoREST negatively regulates this modification.E(Z) is a positive regulator of H3K27me3 at both ZT8 and ZT16 (electronic supplementary material, figure S3c ,  d).These results suggest that the CoREST negatively regulates E(Z) in clock neurons, which is consistent with the results of the genetic interaction between CoREST and E(Z) mutants (figure 4b,e, f).
H3K4me2 levels were found to be higher at ZT16 than at ZT8 in the Per locus in the control group (figure 5d,e).The level of H3K4me2 was significantly higher in the CoREST MI08173 mutant (figure 5d,e), indicating a negative regulation by CoREST.The core of the CoREST complex contains LSD1, a histone modification enzyme that demethylates histone H3K4-me2 and -me1 residues [14][15][16][17].Therefore, these results suggest that the CoREST positively regulates LSD1 in clock neurons.
The levels of H3K27ac showed little change at the Per locus between ZT16 and ZT8 in control (electronic supplementary material, figure S3E and F).After CoREST mutation, the level of H3K27ac significantly increased (figure 5f,g), indicating a (q) Summary of the interactions between the proteins that make up the CoREST complex.'+' indicates physical interaction between two proteins.'−' indicates that there is no physical interaction between two proteins.
negative regulation by CoREST.HDAC1 was the negative regulator of H3K27ac at both ZT8 and ZT16 (electronic supplementary material, figure S3e , f).Consequently, CoREST positively regulates HDAC1, which is consistent with the results of genetic interaction between CoREST and HDAC1 mutants (figure 4c,e,f).
At the Per locus, H3K9me3 levels were significantly higher at ZT16 compared with ZT8 in the control group (electronic supplementary material, figure S3G and H).After the CoREST mutation, H3K9me3 levels decreased significantly at ZT8, indicating that CoREST positively regulates H3K9me3 (figure 5h).However, there was no effect on H3K9me3 levels at ZT16 when the CoREST function was lost (figure 5i), indicating that the CoREST regulation of H3K9me3 was time dependent.To further explore whether the temporal specificity was owing to the time-dependent function of KDM4A, we examined H3K9me3 in KDM4A mutants.The results showed that KDM4A primarily acts as a negative regulator of H3K9me3 at ZT8, while it had no effect on H3K9me3 at ZT16 (electronic supplementary material, figure S3G and H).In addition, it has been reported that LSD1 facilitates H3K9me3 modification [34].These results suggest that CoREST negatively regulates KDM4A, but positively regulates LSD1 in clock neurons, which is consistent with the results of genetic interaction analysis between CoREST, KDM4A and LSD1 mutants (figure 4e,f).
H3K36me3 levels are higher at ZT16 than at ZT8 in the Per promoter in the control group (electronic supplementary material, figure S3I and J).After the CoREST mutation, there was a significant increase in H3K36me3 levels at ZT8 and a significant decrease at ZT16 (figure 5j,k).For H3K36me3, KDM4A acted as a positive regulator at ZT8 but a negative regulator at ZT16 (electronic supplementary material, figure S3i, j).Consequently, at both ZT8 and ZT16, CoREST negatively regulates KDM4A, which is consistent with the situation observed with H3K9me3.Similar to H3K9me3, the temporal-specific function of CoREST may be attributable to the time-dependent role of KDM4A.This conjecture is supported by the rescue of the CoREST MI08173 power value by the CoREST MI08173 ; KDM4A KO /+double mutant (figure 4a,e,f).
To investigate whether CoREST regulates the cycling of histone modifications at the period locus in the clock neurons, we examined the H3K27me3 levels in clock neurons of both CoREST mutants and the controls.Consistent with the mRNA levels, we observed oscillations with a much higher amplitude in the mutants, reaching a peak at ZT16 (electronic supplementary material, figure S3k).
In conclusion, these results demonstrate that CoREST regulates circadian rhythm.This regulation was to some extent dependent on histone-modifying factors, such as KDM4A, LSD1, E(Z) and HDAC1, along with their corresponding histone modifications.Moreover, the temporal-specific regulation of H3K9me3 and H3K36me3 by CoREST can be attributed to the temporal-specific function of KDM4A.

CoREST complex regulates CLK binding at the Per locus
A previous study has shown that CLK is the main transcriptional factor of Per [38].Epigenetic modifications often regulate gene expression through transcription factors.Therefore, we investigated whether CoREST regulates the binding of CLK to the Per gene locus.The expression level of Per at CT16 or ZT16 was significantly higher than that at CT4 or ZT8 (figure 3a,b).Consistent with this, the CLK binding at the Per locus of CT16 or ZT16 was more abundant compared to that at CT4 or ZT8 (figure 6a,b).In CoREST mutants, compared to the control group, the CLK binding at the Per locus of CT16 or ZT16 increased, while it decreased at CT4 or ZT8 (figure 6a,b), indicating that the presence of the CoREST complex inhibited CLK binding at CT16 and ZT16 while enhancing CLK binding at CT4 and ZT8.Therefore, the overall effect of CoREST complex regulation on the Per locus is to maintain the variation of CLK binding and the oscillation of Per expression within a narrow range.
In conclusion, we have a model illustrating the regulation of the CoREST complex on histone modification at the Per locus (figure 6c).Through direct interactions between CoREST-RF and HDAC1, CoREST-RF and LSD1, LSD1 and E(Z), HDAC1 and KDM4A, CoREST positively regulates LSD1 and HDAC1, while negatively regulates E(Z) and KDMA.Under the influence of these histone-modifying factors, CoREST interactively regulates multiple histone modifications in a time-dependent manner.At ZT8, the absence of CoREST led to a substantial elevation in H3K4me2 and H3K27ac, a significant decrease in H3K9me3, and an increase in H3K27me3 and H3K36me3, ultimately resulting in a decrease in Per expression (figures 3b and 6c).While, at ZT16, the loss of CoREST resulted in a significant elevation in H3K27me3, a mild decrease in H3K36me3, a significant increase in H3K4me2 and H3K27ac, and no significant change in the repressive mark H3K9me3.These combined effects contributed to an increase in Per expression (figures 3b and 6c).Notably, at ZT16, both the gene body and promoter of Per exhibited a substantial repressive modification, H3K9me3, which likely accounted for the subsequent trough in Per expression (figures 3b,5i and 6c).

The ability of Drosophila to be entrained by environmental changes is limited by CoREST mutation
To further investigate the function of CoREST-dependent epigenetic regulatory machinery in Drosophila, we examined the adaptation of the circadian rhythm of the CoREST mutant and the control group to changing conditions.Specifically, we tested the temperature entrainment condition at 16°C for 12 h followed by 12 h at 25°C TCs.The results revealed a significant reduction in the entrainment index for the CoREST mutations (figure 1a,c), indicating a decreased ability to adapt to environmental changes.It has been previously reported that synchronization to low TCs in DD requires Per in ventral lateral neurons [23].The altered expression pattern of Per caused by CoREST mutations may explain the inability of CoRESTMI 08173 to adapt to environmental changes.In conclusion, these data collectively indicate that CoREST mutation limits the ability of Drosophila to be entrained by temperature.

Discussion
In this study, we investigated the mechanism of CoREST regulation in circadian rhythm.By studying the regulation of the CoREST complex on circadian rhythm, we revealed that CoREST regulates HDAC1, LSD1, E(Z) and KDM4A and their corresponding histone modifications at the Per locus specifically in clock neurons.Interestingly, we found dynamic changes in histone modifications at different time points of the Per locus, suggesting that histone modification plays an important role in regulating gene expression oscillation.More importantly, we found differential effects of the same factor on the Per locus at different time points, such as the effect of KDM4A on H3K9me3 and H3K36me3 at ZT8 and ZT16.Understanding the mechanism behind this time dependence poses an intriguing problem.
The regulatory relationships identified in this study are consistent with or provide an explanation for phenotypes previously reported in the literature.The discovery of CoREST's function on E(Z) explains the previous report that CoREST negatively regulates H3K27me3 in Drosophila follicle cells [19].The discovery of CoREST's role on LSD1 and E(Z) also explains its involvement in regulating the H3K4me2, H3K9me3 and H3K27me3, as reported in a previous study [18].Previous reports have indicated that H3K36me3 levels increase alongside transcription to inhibit repeated transcription [39].Specifically, H3K36me3 on the gene body correlates with transcriptional activity.Conversely, the presence of H3K36me3, along with H3K9me3, at the regulatory region maintains a repressive state of gene expression [40].Our study demonstrates a positive correlation between H3K36me3 on the gene body of the Per locus and transcriptional levels, while a negative correlation is observed between H3K36me3 at the regulatory region and transcriptional levels (figure 5j,k).All of these pieces of evidence support the effectiveness of the techniques employed in this study.
Histone modifications, as a feedback regulation mechanism, maintain the dynamic expression of Per in a relatively narrow range.Transcription is mainly driven by CLK.This study shows that the level of inhibitory histone modification is closely related to the Per transcriptional level in w 1118 flies.There is a time delay between the mRNA level and the levels of epigenetic modifications.This phenomenon is similar to what we observe for the mRNA level and protein level of clock genes.At ZT8, when the expression level is relatively low, the inhibitory histone modifications are also low.This low level of inhibitory histone modifications at the Per locus facilitates the subsequent enhancement of the transcriptional level.On the contrary, at ZT16, when the expression level is relatively high, the inhibitory histone modifications are high.This high level of inhibitory histone modifications at the Per locus dampens its subsequent transcription.The function of CoREST complex is to maintain histone modifications of Per locus.Our data indicate that except for H3K9me3 at ZT16, the other four modifications can be altered by CoREST mutation.Other mechanisms are involved in maintaining H3K9me3 at ZT16.The collective effects of multiple histone modifications at the Per locus can influence the recruitment of CLK and transcription.The increase in activating histone modifications promote CLK binding and transcription, and vice versa (figure 6c).The question then remains: how is the initial state established with both a relatively high level of H3K9me3 and a relatively high CLK binding and transcription level?Feedback mechanisms are probably involved in this process.
One interesting discovery in this study is the temporal-specific role of CoREST in regulating H3K9me3 and H3K36me3.Further evidence from our study demonstrates that this is attributed to the temporal-dependent function of KDM4A.Regarding H3K9me3, the lack of impact from KDM4A or CoREST loss at ZT16 may be owing to high levels of H3K9me3 at this stage or the involvement of other mechanisms maintaining its level.Additionally, other similar mechanisms have been observed to maintain H3K9me3 levels in mammals [3].As for H3K36me3, the contrary effects at ZT8 and ZT16 resulting from KDM4A or CoREST loss could be a consequence of different histone modification states, diverse expression patterns of interacting factors, or varying responses from redundant homologues during these time points.Investigating the underlying mechanism behind this phenomenon would be of great interest, although we are currently lacking the necessary reagents to conduct further experiments.This study serves as yet another example highlighting the involvement of the CoREST complex in timely dynamic transcriptional regulation.A previous report by Y.H.'s lab demonstrated the role of CoREST in activity-dependent transcription regulation in memory [20].
The tests of physical interactions among the factors mentioned in this study have limitations.The lack of an in vivo context means that the results shown here only provide evidence of the possibility of interaction, rather than the actual states of the protein complexes.As previously mentioned, the CoREST complex may exhibit dynamic behaviour at different time points in terms of temporal-specific regulation.The model in figure 6c may only capture a limited state of the CoREST complex.The inclusion of certain components, particularly E(Z) or KDM4A, could be subject to dynamic regulation by other factors.The genetic relationship discovered in this study is limited in this regard.It would be intriguing to further investigate the components and functions of the CoREST complex at various time points in clock neurons.

Figure 1 .
Figure 1.CoREST is required to synchronize with low temperature cycles in DD. (a,b) Actograms.Flies were initially synchronized to 3 LD cycles at 25°C, followed by two at 16°C and 25°C temperature cycles (TCs) in DD, each TC was delayed by 6 h compared to the previous regime, and subsequently released to DD at 25°C.Cyan and pink areas indicate 16 and 25°C, respectively.n = 16.Red dots indicate activity peaks.The red rectangle frames a 6 h window for the main temperature-synchronized activity.(c) Plotting of the EI.Data information: Statistical differences were measured using unpaired two-tailed Student's t-test.

Figure 5 .
Figure 5. CoREST regulating histone modification on the Per genomic locus in clock neurons.(a) Primers used in (b)-(k).(b,c) ChIP experiments showing the relative enrichment (% Input) of H3K27me3 on the Per gene locus in CoREST MI08173 and w 1118 .(b) H3K27me3 was weakly upregulated in CoREST MI08173 at ZT8. (c) H3K27me3 was strongly upregulated in the CoREST MI08173 mutant at ZT16. (d,e) ChIP experiments showing the relative enrichment (% Input) of H3K4me2 on Per gene locus in CoREST MI08173 and w 1118 .(d) H3K4me2 was strongly upregulated in CoREST MI08173 at ZT8. (e) H3K4me2 was also strongly upregulated in the CoREST MI08173 mutant at ZT16. (f,g) ChIP experiments showing the relative enrichment (% Input) of H3K27ac on Per gene locus in CoREST MI08173 and w 1118 .(f) H3K27ac was upregulated in the CoREST MI08173 mutant at ZT8. (g) H3K27ac was also upregulated in the CoREST MI08173 mutant at ZT16. (h,i) ChIP experiments showing the relative enrichment (% Input) of H3K9me3 on the Per gene locus in CoREST MI08173 and w 1118 .(h) H3K9me3 was downregulated in the CoREST MI08173 mutant at ZT8. (i) H3K9me3 was invariant in the CoREST MI08173 mutant at ZT16. (j,k) ChIP experiments showing the relative enrichment (% Input) of H3K36me3 on the Per gene locus in CoREST mutant CoREST MI08173 and w 1118 control.(j) H3K36me3 was upregulated in Per promoter but downregulated in Per gene body in the CoREST MI08173 mutant at ZT8. (k) H3K36me3 was weakly downregulated in the CoREST MI08173 mutant at ZT16.Data information: Statistical differences were measured using unpaired two-tailed Student's t-test.The significance levels were represented as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.If the p-value was greater than 0.05, it was not displayed in the figures.