Environmental circadian disruption re-programs liver circadian gene expression

Circadian gene expression is fundamental to the establishment and functions of the circadian clock, a cell-autonomous and evolutionary-conserved timing system. Yet, how it is affected by environmental-circadian disruption (ECD) such as shiftwork and jetlag, which impact millions of people worldwide, are ill-defined. Here, we provided the first comprehensive description of liver circadian gene expression under normal and after ECD conditions. We found that post-transcription and post-translation processes are dominant contributors to whole-cell or nuclear circadian proteome, respectively. Furthermore, rhythmicity of 64% transcriptome, 98% whole-cell proteome and 95% nuclear proteome is re-written by ECD. The re-writing, which is associated with changes of circadian cis-regulatory elements, RNA-processing and protein trafficking, diminishes circadian regulation of fat and carbohydrate metabolism and persists after one week of ECD-recovery.


Main Text:
The circadian clock, a cell-autonomous timing system, is driven by an evolutionary conserved transcription-translation feedback loop. It has evolved as a mechanism for organisms to synchronize daily cycles of internal biological rhythms with external environmental conditions by modulating the expression of thousands of clock-controlled genes (1)(2)(3)(4). Under environmental circadian disruption (ECD) conditions such as jetlag, social jetlag and shiftwork, the natural harmonic alignment is its molecular underpinnings and functional implications, we systematically interrogated changes in the circadian gene expression processencompassing transcription, translation and post-translationat theomics level in mouse livers after one-week ECD recovery (13) in comparison to standard circadian (STD) conditions ( fig. S1).
Integrated circadian gene expression. The circadian gene expression process had been intensively 15 investigated, resulting in a thorough understanding of circadian transcription regulation from chromatin opening, cis-trans regulator interaction to transcript abundance (14)(15)(16). However, at the post-transcriptional and post-translational levels our understanding of the process is sparse (17)(18)(19)(20). To fill this gap as well as inquire the state of gene expression under the STD control condition, we performed RNA-seq of wholecell mRNAs and mass spectrometry analysis of whole-cell and nuclear proteins in the same set of mouse 20 livers, which were entrained under STD and collected under circadian conditions ( fig. S1).

Topography of circadian gene expression:
Among 21061 quantified transcript time series, we found 5502 (26%) exhibit a rhythmic pattern of abundance with the peaks spreading throughout the cycle. Transcripts of all core clock genes including Clock, Arntl, Per1/2/3, Cry1/2, Csnk1d/e and Nr1d1/2 were quantified in the RNA-seq. Their patterns of abundance were validated by RT-qPCR (Figs 1A-B, S2A) and 25 are consistent with previous studies (15,21). At the protein level, we quantified 7,314 circadian time series across whole-cell and nuclear compartments. Processing and results of mass-spec were validated in terms of enrichment of known mouse liver nuclear proteins, Pearson correlation between compartments, consistence of patterns of circadian protein abundance between mass-spec and published studies (22,23) and enrichment of known circadian associated functions (figs. S2B-E). In the whole-cell circadian 30 proteome, 285 (3.9%) were rhythmic. These proteins exhibit a bimodal distribution of peak abundance concentrating around CT7 and CT17. In the nuclear compartment, 678 (9.3%) were rhythmic with a distinct phase distribution from that of the whole-cell proteome, assuming a unimodal pattern centering around CT20 (Fig. 1B). These proteomes are enriched with cellular and molecular processes such as endosome transport, autophagy and response to hypoxia ( fig. S2E). This is the first comprehensive and coherent 35 description of daily abundance of whole-cell transcripts, whole-cell proteins and nuclear proteins in the same set of tissues under circadian condition.
Differential rhythmicity of transcripts and proteins: The rhythmicity of a transcript abundance has routinely been used to infer circadian biological function, but it is protein that mostly performs the gene function. We thus asked how faithfully transcript rhythmicity is recapitulated in protein rhythmicity. To 40 minimize the effect of difference in detection levels between RNA-seq and Mass Spectrometry on the analysis, we firstly selected for genes of which transcript(s), whole-cell protein(s) and nuclear protein(s) were all quantified in the circadian transcriptome and circadian proteomes, and then examined their relationship in time and space. We found 6937 of such genes, constituting 33% all quantified transcripts and 95% all quantified proteins (Figs. 1C-D). Within this sub-population, less than 5% rhythmic transcripts transcription (Arntl, Pgm2l1, Gtf2h2, Txndc9, Polr1d, Polr2c) or metabolism (Gc, Cyp4a14, Gpat4, Acot12) exhibit differential circadian rhythmicity at transcript and protein levels ( Fig 1G). These results indicate a profound dissociation in circadian rhythmicity of transcripts and 5 proteins and thus warrant extra caution in inferring a circadian function from rhythmic transcripts at individual or -omics levels. sub-cellular localizations, which are active and tightly regulated processes (26), affect circadian protein rhythmicity, 25 thereby circadian outputs, we examined the overlap between whole-cell and nuclear circadian proteomes. 94% nuclear rhythmic 30 proteins are not rhythmic at the whole cell level (Fig. 1H). The rhythmicity of these proteins must be acquired post-translationally as they 35 are not rhythmic at the wholecell level, making contribution from transcriptional, posttranscriptional and 40 translational controls unlikely. Additionally, only 14% of whole-cell rhythmic proteins might be allocated to the rhythmicity in nuclear 45 compartment (Fig. 1I). The remainders, 86%, have to be attributed to rhythmicity in other compartments, reconstituting their rhythmicity 50 at the whole-cell level. These results indicate the circadian    (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 29, 2023. ; https://doi.org/10.1101/2023.08.28.555175 doi: bioRxiv preprint nuclear proteome mostly arises at the post-translational level and suggest the existence of compartmental specific circadian proteomes, a less studied area in the field (27), not only in the nucleus but also in other organelles.
ECD re-writes the circadian transcriptome. After ECD we found 3445 (16%) transcripts exhibiting a circadian rhythmic pattern of abundance among 21061 quantified circadian time series. In response to ECD, 5 3118 STD rhythmic transcripts lose their rhythmicity (LORR) while 1061 non-rhythmic transcripts acquired rhythmicity (GORR), constituting 56.7% and 30.8% of all rhythmic transcripts under the same condition, respectively. There are 2384 STD rhythmic transcripts that retain rhythmicity (RORR), regardless of whether there is a change in phase or amplitude of the oscillation. Each of these rhythmic populations displays a distributed pattern of peak abundance throughout the cycle (Fig 2A-C). Collectively, there are 10 6,563 rhythmic transcripts, of which 64% switched rhythmicity in response to ECD (Fig. 2D). The circadian transcriptome is thus re-written by ECD.
Among core clock transcripts, the rhythmicity of Arntl, Per1/2, Cry1 and Nr1d1 showed a perturbation in phase or amplitude, but none exhibits a marked loss or gain of rhythmicity (Fig. 2E, fig. S3), suggesting potential contribution of factor(s) other than the core clock machinery to the re-writing. To seek clues for 15 such regulator(s), we compared enrichment of cis regulatory elements within 20Kb upstream of transcription start sites in STD-rhythmic and ECD-rhythmic populations using iRegulon (28). At the top of    . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 29, 2023. ; https://doi.org/10.1101/2023.08.28.555175 doi: bioRxiv preprint the list for the STD-rhythmic population (5502 transcripts) is "ARNTL-DBD", a motif for DNA binding domain of ARNTL(or BMAL1) which is a key transcription activator of the circadian feedback loop (29). Such enrichment is therefore expected and a validation of the analysis. Among the top 5 STD enriched elements, 4 harbor the E-box (CACGTG), a known dominant circadian cis-element (30). In ECD-rhythmic population, the highest enriched element is not a known circadian cis-element. E-box comes at 3 rd to 5 th 5 positions. Thus, re-writing of circadian rhythmic transcripts associates with a reduction of BMAL1 binding concurrence with an increase in binding of a tobe-identified transcription factor(s) (Fig. 2F).
10 Circadian whole-cell and nuclear proteomes are re-written by ECD. After ECD, we quantified 5317 protein time series 15 across both whole-cell and nuclear compartment.  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 29, 2023. ; https://doi.org/10.1101/2023.08.28.555175 doi: bioRxiv preprint distributed throughout a large part of the cycle. 633 of 678 STD rhythmic proteins (93.4%) lost their rhythmicity (LORNE) while 314 proteins acquired rhythmicity (GORNE) in response to ECD. There are 992 proteins, in total, that exhibit a rhythmic pattern of abundance in the nuclear compartment, of which 95% switched their rhythmicity after ECD. (Figs. 3B, D, E). Collectively, 12.7%, 23.8% and 33.5% proteins are rhythmic under STD, ECD or both conditions, respectively (Fig. 3F). 5 Such high magnitudes of change in rhythmicity at the transcript and protein levels were unexpected. To see if loosening the stringency for rhythmic calling would have a significant impact on the magnitudes, we re-performed the analysis using one of the highest recall (RAIN) (31) or most popular (JTK-Cycle)(32) algorithm. Both showed insignificant and mild changes in the magnitude of re-writing of the proteomes or transcriptome, respectively (fig. S4). These observations suggested that ECD re-writes the entire circadian 10 gene expression system from transcription to posttranslation.
The increase in protein rhythmicity at the whole- 15 cell level, both quantity (from 3.9% to 17%) and synchrony (dispersed to discrete bimodal phase distribution), after ECD 20 compared to STD is intriguing (Fig. 1B- G   3  6  9  12  15  18  21  24  3  6  9  12  15  18  21  24  3  6  9  12  15  18  CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 29, 2023. ; https://doi.org/10.1101/2023.08.28.555175 doi: bioRxiv preprint observed. In agreement with this interpretation, resilience of processes such as inflammation, glomerular and tubular injuries is compromised after ECD (6,33,34). We thus propose that the gain of rhythmicity reflects not a strengthening of the system but a losing of resilience to perturbations.
Post-transcription and post-translation are dominant contributors to circadian proteomes under ECD. Given that post-transcription and post-translation are the major contributors to protein rhythmicity 5 under STD, we asked how ECD would affect those contributions. We examined overlaps between the circadian transcriptome, circadian whole-cell proteome and circadian nuclear proteome after ECD as performed for STD. After ECD, we found 5028 transcripts of which whole-cell protein or nuclear protein were also quantified. Within the 5028 sub-population, 73% rhythmic whole-cell proteins don't have rhythmic transcripts (Figs. 4 A-E). This percentage is substantially higher than the 52% under STD. In the 10 nuclear protein rhythmic sub-population, 86% are not rhythmic at the whole-cell protein level (Figs. 4F-G). Thus, under ECD post-transcriptional and post-translational processes are dominant contributors to protein rhythmicity at both whole-cell and nuclear compartment levels.
To seek further evidence for contribution of post-transcription to circadian whole-cell proteomes, we examined two properties of rhythmicity, amplitude and phase, in populations of genes in which both 15 transcripts and whole-cell proteins are rhythmic under STD or ECD. In gene expression process, transcripts, which have an average half-life of 4.8 min (35), are produced before proteins. Therefore, a rhythmic gene population with more contribution from transcription would have higher ratio of genes with the phase of transcript leading the phase of protein to genes with the phase of transcript lagging the phase of protein.
We found the ratio to be ~4:1 under STD but ~ 1:1 after ECD (Fig. 4H). Another prediction is that a 20 rhythmic gene population with higher contribution from protein rhythmicity would have higher ratio of protein's amplitude to transcript's amplitude. The amplitude ratio is ~1:4 under STD and increases to 9:1 after ECD (Fig. 4I). These results indicated that transcription contributes more to protein rhythmicity under STD than ECD while post-transcription contributes more to protein rhythmicity after ECD than STD. These are additional pieces of evidence for a higher contribution of post-transcription to protein rhythmicity after 25 ECD.
ECD reprograms circadian molecular functions. To assess the impact of ECD on circadian functions, we performed gene ontology enrichment analysis of rhythmic whole-cell proteins after ECD alone or in comparison with STD. Beside of known ECD-associated pathways such as C-type lectin receptor, Toll-like receptor or GnRH (36)(37)(38), the ECD rhythmic population is also enriched with post-transcriptional and post- 30 translational processes such as mRNA processing, translation initiation, intracellular protein transport and MAPK/Ras signaling. Interestingly, there is an apparent day-night functional partitioning. Posttranslational processes such as vesicular transport and protein localization peak during the nighttime while post-transcriptional and translational initiation processes peak during the daytime (Fig. 5A). In response to ECD, the enrichment of several STD-enriched processes is diminished such as acyl-CoA and di/tri- 35 carboxylic acid metabolic processes while several others became highly enriched including posttranscription processing and intra-cellular protein transport. Among proteins undergoing significant change in rhythmicity are regulators of these processes, some of which were implicated in circadian dys/functions, such as PRMT5 (39) Given the central role of liver in metabolism, we took a closer look at the effect of ECD on metabolic functions. Analyzing the enrichment and network interaction of metabolic terms, we found that fatty acid and carboxylic acid metabolic processes lost most of their enrichment while amino acid metabolic processes, which were also enriched under STD, significantly increase in enrichment (Figs. 5D-E). These 45 results suggest there is a change in prioritizing of circadian regulation from carbohydrate and fat to protein metabolism in response to ECD. The lost in timing of fat and carbohydrate metabolism might underlie, at least in part, ECD-associated metabolic disorders.
Interestingly, a previous study showed that mice, after being subjected to the same ECD and recovery paradigm, exhibited normal body temperature and running-wheel behavior under diurnal (LD) 50 condition (34). Together with this observation, our results suggested organisms who experienced ECD might exhibit normal daily behavioral rhythms but not fully recover at the molecular level, at least after a . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 29, 2023. ; https://doi.org/10.1101/2023.08.28.555175 doi: bioRxiv preprint short period of recovery time. The discordance in recovery at molecular and behavioral levels might indicate a diversion or a masking of behaviors by light. We favor the later scenario as these behaviorally "normal"    . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 29, 2023. ; https://doi.org/10.1101/2023.08.28.555175 doi: bioRxiv preprint mice are very susceptible to physiological challenges (33,34).
In summary, our study showed that environmental circadian disrupted re-programs the circadian gene expression system and molecular circadian functions, which are not recovered, at least at the molecular level, after one week of recovery. Additionally, it also provided the first comprehensive documentation of changes in the circadian gene expression process in response to environmental circadian disruption, laying doi:10.1126/science.1226339.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 29, 2023. ; https://doi.org/10.1101/2023.08.28.555175 doi: bioRxiv preprint