Circadian clock regulator Bmal1 gates axon regeneration via Tet3 epigenetics in mouse sensory neurons

Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs. However, the underlying mechanisms remain unclear. Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion. By applying conditional deletion of Bmal1 in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications. Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis. Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-of-day effect on axon growth potential. Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration.

Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs.However, the underlying mechanisms remain unclear.Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion.By applying conditional deletion of Bmal1 in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications.Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis.Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-ofday effect on axon growth potential.Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration.
Unlike injury in the central nervous system (CNS), peripheral nerve injury triggers axon regeneration, but the repair process is often slow and incomplete.A crucial step for successful nerve repair is to accelerate axon regeneration 1 , but the gene regulatory mechanisms that enable injured neurons to rapidly switch into a growth state remain poorly defined 2 .Emerging evidence also implicates neuroimmune interactions in augmenting axon regeneration; [3][4][5][6] how neuronal intrinsic and extrinsic injury and stress responses are orchestrated is unclear.Such knowledge may help advance new strategies to enhance nerve repair, with translational relevance for CNS injury.
Dorsal root ganglia (DRG) sensory neurons represent a preferred system to study axon regeneration, as their central and peripheral axon branches respond differently to axotomy.While central lesion (CL) after spinal cord injury leaves DRG neurons in a state that is refractory to axon regeneration, peripheral lesion (PL) triggers a regenerative response that enables regeneration of not only peripheral but also central branches 7,8 .This so-called "conditioning lesion" effect is transcription dependent 9 , thus presenting a starting point to investigate regulatory mechanisms of regeneration-associated genes (RAGs) 2,10,11 .
Works from our laboratory and others have demonstrated that conditioning lesion initiates a genome-wide reconfiguration of histone modifications and DNA methylation, thus adding a layer of epigenetic control for robust and fine-tuned gene regulation 2,10,[12][13][14][15] .Tet methylcytosine dioxygenase 3 (Tet3) has emerged as a critical regulator of axon regeneration in conditioned DRG neurons.Tet3 catalyzes the iterative oxidation of methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), formylcytosine (5fC), and carboxylcytosine (5caC), which is then converted back to 5C by base excision repair (BER) 16 .We previously showed that PL induces an upregulation of Tet3, but not Tet1 or Tet2, in axotomized DRG neurons along with global 5hmC gains 17 .DNA demethylation is required for the conditioning effect, as Tet3 silencing reduces global 5hmC levels and diminishes axon regrowth capacity 18 .Aside from being an intermediate of the DNA demethylation cascade, 5hmC is also a stable DNA base with unique gene regulatory functions in its own right.Notably, 5hmC is highly abundant in the CNS, accounting for ~40% of modified cytosines, mostly in gene body regions [19][20][21][22] , and is pivotal for neuronal differentiation 22,23 .While these earlier studies showed the necessity of Tet3 and DNA demethylation for axon regeneration, the regulators of Tet3 induction and how Tet3 selects the target sites for DNA demethylation remain unresolved.It is also unclear if Tet3 augmentation and the resultant 5hmC gain can promote regenerative capacity.
Here, in search for transcription factors (TFs) that may affect Tet3-5hmC responsiveness to axonal injury, we performed an unbiased screen for over-represented TF binding motifs within the differentially hydroxymethylated regions (DhMRs) of conditioned DRG.Intriguingly, we identified an enrichment of the binding motif for Bmal1, a central regulator of the circadian clock 24 .By applying conditional gene deletion in conjunction with in vitro and in vivo models of axon regrowth, we demonstrated that neuronal knockout of Bmal1 enhanced axon regeneration partly through a Tet3-dependent mechanism.Transcriptomics profiling revealed that the Bmal1 regulon after peripheral axotomy not only concerned axon regrowth, but also stress responses, energy homeostasis, and neuroinflammation.Interestingly, Tet3 and 5hmC displayed diurnal oscillations in DRG neurons, corresponding to a time-of-day effect on axon growth potential.Our study thus raises awareness of the time-of-day effect on neural regenerative responses, with ramifications for experimental designs and translational strategies for neural repair.

Bmal1 binding motif is enriched in DhMRs of conditioned DRG
To explore the functional relevance of DNA hydroxymethylation in axon regeneration, we previously performed genome-wide mapping of 5hmC modifications in axotomized DRG, which identified 1,036 DhMRs specific for peripheral lesion (PL, regenerative state) as compared to dorsal column lesion (DCL, non-regenerative state) or no injury 17 (Fig. 1a).Among the PL-DhMRs (each ~200 bp), 561 (54%) displayed 5hmC gain and 475 (46%) loss, both predominantly localized in gene body and intergenic regions rather than promoters and CpG islands (Fig. 1b), suggesting a complex role in gene regulation.
We next analyzed the PL-DhMRs with 5hmC gain or loss separately, which identified shared (e.g., ARNT, NPAS1) but also distinct enriched bHLH-PAS binding motifs (Fig. S1b).Notably, many of these bHLH-PAS TFs belonged to the RAGs induced by the conditioning lesion, implicating a role in axon regeneration (Fig. S1c).Indeed, activation of HIF1α (a bHLH-PAS TF) through intermittent hypoxia promotes axon regrowth of DRG neurons 29 .

Conditioning lesion enhances chromatin accessibility at DhMRs
Since DNA methylation alters chromatin landscape 30 , we next examined a recent ATAC-seq dataset on DRG before and after PL 14 .This revealed that PL led to increased chromatin accessibility at DhMRs (more prominent for the ones with 5hmC gain) and transcription start site (TSS) of the associated genes (Fig. S1d).Our previous work has shown that genes harboring PL-DhMRs are enriched for pathways linked to axon regeneration (e.g., CREB and Neurotrophin) 17 , here we analyzed the genes displaying 5hmC gain or loss separately, which showed that the genes in the 5hmC gain category (n = 988) concerned epithelial cell proliferation and differentiation, organ regeneration, and lymphocyte co-stimulation, while the genes in the 5hmC loss category (n = 834) mainly concerned metabolism (e.g., response to thyroid hormone, amino acid biosynthetic, glucose homeostasis) and cytoskeletal reorganization (Fig. S1d; Supplementary Data 2).In this context, it is noteworthy that as an intermediate of the DNA demethylation cascade, 5hmC gain or loss does not strictly predict transcriptional gain or loss 17 .
Tet3 has two isoforms due to alternative transcriptional start sites [31][32][33] .We thus examined their relative abundance in DRG, which showed that the short form (Tet3-S), which lacks the CXXC DNAbinding domain, is the predominant form (Fig. 1e).Hence, Tet3 recruitment to target genomic loci for DNA hydroxymethylation upon peripheral axotomy might involve cooperation with TFs.
We next focused on Bmal1, an essential and the only nonredundant core regulator of the mammalian circadian clock; 34 its function in axon regeneration is unknown.We first examined the PL-DhMRs containing Bmal1 binding motifs (n = 341) and the associated genes (n = 616, identified by GREAT 35 ) (Fig. 1f, g, Supplementary Data 3).Pathway enrichment analysis showed that these genes featured well-known pro-regenerative pathways, e.g., IGF-1, BMP, NGF, Axon guidance, and PTEN 36,37 (Fig. 1g).Bmal1 is known as a pioneer-like TF (i.e., modulating chromatin structure to recruit other TFs) 38,39 , we thus re-examined the DRG ATAC-seq dataset, which indeed revealed that PL resulted in increased chromatin accessibility at these DhMRs and the TSS of the associated genes (Fig. 1h), consistent with epigenetic changes in regenerating DRG (Fig. 1f).
We wondered whether there might be a potential protein-protein interaction between Tet3 and Bmal1.Co-immunoprecipitation (co-IP) with a specific antibody against Tet3 [40][41][42] demonstrated pull down of Tet3 along with Bmal1 in Neuro-2a cells and in DRG lysates (Fig. S2a, b).Unlike in Neuro-2a lysate with a predominant band of Tet3 at ~185 kDa detected in Western blotting (WB) (Fig. S2c), in whole DRG lysates WB showed a dominant band of Tet3 at ~80 kDa, while Tet3 pull down resulted in a faint band at ~185 kDa and multiple bands at lower molecular weights (Fig. S2b), which may reflect proteolytic processing of Tet3 43 .Attempts to reverse co-IP with a specific antibody against C-terminal region of Bmal1 failed to pull down either endogenous or overexpressed Tet3 in Neuro-2a cells (Fig. S2c), possibly related to antigen masking from Tet3 binding to the C-terminal domain of Bmal1 which contains a conserved transactivation domain involved in binding to transcriptional regulators 44,45 .

Conditioning lesion downregulates Bmal1 in DRG neurons
To investigate the relevance of Bmal1 in axon regeneration, we first examined how axonal injury might affect the expression of interconnected clock genes of the translational/transcriptional feedback loops that control Bmal1 expression and transcriptional activity (Fig. 2a).To this end, we performed RNA-sequencing (RNA-seq) to profile differentially expressed genes (DEGs) in DRG in response to PL (Supplementary Data 4).We found that Bmal1 (a.k.a.Arntl) was significantly downregulated at 1 day post-injury (1 dpi) relative to no injury (Fig. 2b), in agreement with a published DRG RNA-seq dataset 14 .The result was corroborated by qRT-PCR analysis (Fig. 2c).Surveying a recent single cell RNA-seq dataset 46 revealed that 8 out of the 9 subclusters of DRG neurons downregulated Bmal1 at 24 h post-PL (Fig. S3a).
Bmal1 (the β subunit) heterodimerizes with Clock (the α subunit) to form a transcriptional complex that binds to Enhancer (E)-box motifs to regulate a large repertoire of clock-controlled genes (CCGs) as well as its own feedback regulators (Fig. 2a).These include CRY and PER, both negative regulators of Bmal1 transcriptional activity 47 , as well as REV-ERBα/β, two orphan nuclear receptors constituting a second feedback loop, exerting negative effect on Bmal1 transcription 48 .We found that Per1 and Cry2, but not Cry1, were upregulated after PL (Fig. 2b, c).Time course analysis showed that Bmal1 expression was downregulated at 12 h post-injury, but returned  to baseline level by 36 h, while Per1 was upregulated at both time points (Fig. 2d).
We next conducted immunofluorescence (IF) staining, which confirmed diminished Bmal1 nuclear immunosignals in conditioned DRG neurons at 1 dpi as compared to no injury (Fig. 2e).Of note, WB did not reveal a significant reduction of Bmal1 protein levels in DRG tissue lysates at 12 or 24 h post-injury (Fig. S3b), possibly due to a high abundance of glial cells (neurons representing only 12.5% of total cells in DRG 6 ).IF for Tet3 and 5hmC showed that both were increased in axotomized DRG neurons at 1 dpi (Fig. S3c-e), in agreement with previous works from the Ming laboratory and our own 17,18 .Concordantly, Tet3 mRNA levels were higher after PL, and scRNA-seq confirmed Tet3 induction in all DRG neuronal subtypes (Fig. S3a, c).Hence, conditioning lesion of DRG affects the expression of molecular oscillators, with Bmal1 activity transiently suppressed as evidenced by its reduced nuclear levels and induction of Per1 and Cry2, both negative regulators of Bmal1 transcriptional activity 47 .

Bmal1 inhibition enhances neurite outgrowth
To understand the functional significance of Bmal1 downregulation by the conditioning lesion, we first tested the effect of silencing Bmal1 in Neuro-2a cells (Fig. S4a), which led to enhanced neurite outgrowth (Fig. 3a), whereas forced over-expression of Bmal1 reduced neurite elongation but not neurite initiation, evidenced by comparable proportions of axon-bearing cells (Figs. 3b, S4b).Likewise, in induced neurons derived from human embryonic stem (hES) cells wherein the clock was operational 49 , pharmacological stabilization of PER and CRY (both negative regulators of Bmal1) 50,51 also enhanced neurite outgrowth (Figs.3c, S4c, d).These culture results support an inhibitory role of Bmal1 for axon growth.
To study the function of Bmal1 in vivo, we generated Bmal1 fl/fl Thy1-CreER T2 mice for neuron-specific, tamoxifen-inducible conditional knockout (cKO) of Bmal1.This strategy circumvents developmental phenotypes of Bmal1 deletion with the Nestin-Cre line, which results in neurodegeneration 52 .As the Thy1-CRE ERT2 /EYFP (SLICK-H) transgenic line drives pan-neuronal expression of both CreER T2 recombinase and EYFP 53,54 , we first confirmed uniform expression of EYFP in DRG neurons with fluorescence imaging of DRG tissue sections and primary DRG neuron cultures (Fig. S5a, b).
Next, we conducted neurite outgrowth assays with primary DRG neurons, which gauge axon regrowth potential, as the dissociation process results in axotomy (Fig. 3d).Aligned with the above results, DRG neurons from Bmal1 cKO mice extended longer neurites than controls at 22 h in vitro (mean 311 ± 10 vs. 260 ± 7 µm) (Fig. 3e).Similar results were also obtained with primary DRG neurons from Bmal1 Nes-Cre mice (Fig. S6a).The proportion of neurite-bearing neurons was not different between genotypes, signifying comparable axon initiation capacity (Fig. S6b).
The neurite length of Bmal1-deficient DRG neurons did not reach that of conditioned DRG neurons with prior PL (Fig. 3e).This indicated that the conditioning effect also operates through Bmal1-independent mechanisms.However, Bmal1 deletion could further augment the conditioning effect, evidenced by even longer axons of conditioned Bmal1 cKO DRG neurons than conditioned controls at 10 h post-seeding (mean 329 ± 144 vs. 278 ± 121 µm) (Fig. 3f).On the other hand, Bmal1 over-expression in primary DRG neurons did not cause significant reduction of neurite outgrowth, unlike in Neuro-2a cells (Fig. S6c, and see Fig. 3b), suggesting that the conditioning effect can bypass or overcome the Bmal1 block.The growth promoting effects of Bmal1 deletion were also confirmed in adult DRG explant cultures, which allow analysis after a longer growth period of 4 days after seeding (Fig. 3g).
We next compared in vivo axon regeneration in a sciatic nerve crush injury model (Fig. 4a).IF staining for SCG10, a well-established axon regeneration marker that specifically labels regenerating but not uninjured sensory axons 14,18,29,58 , showed that Bmal1 cKO mice displayed accelerated axonal regrowth at 1 dpi and 3 dpi, with ~50% longer SCG10 + axons than in control (Bmal1 fl/fl or Bmal1 fl/+ ) that received identical tamoxifen injection (Fig. 4b-e).Regeneration index calculation confirmed the enhanced axon regenerative phenotype of Bmal1 cKO animals (Fig. 4c, e).These results were corroborated in Bmal1 Nes-Cre cKO mice (Fig. S6d, e).We also examined re-innervation of the epidermal layer of the glabrous skin of the foot pad after sciatic nerve injury (Fig. 4f).By 21 dpi, whole mount IF for the neuronal marker PGP9.5 revealed that cutaneous reinnervation area reached over twice as large in Bmal1 cKO mice as in controls (Fig. 4f, g).This also held true for regenerated axon branches labeled by neurofilament-H (NF-H) + (Fig. 4h), signifying a higher abundance of maturing axons in the reinnervated epidermis 59 .Together, these results supported an inhibitory role of Bmal1 in axon regeneration and that the conditioning lesion operates partly by suppressing the Bmal1 blockade (Fig. 4i).

Bmal1 ablation augments Tet3 and 5hmC induction in conditioned DRG neurons
To pinpoint the mechanisms by which Bmal1 restricts axon regeneration, we tested the hypothesis that Bmal1 gates 5hmC responsiveness to axonal injury.We first examined uninjured DRG neurons and observed higher baseline levels of Tet3 and 5hmC in Bmal1-deficient DRG neurons relative to controls (Fig. S7a, b).Upon PL, their upregulation appeared more pronounced in Bmal1 cKO DRG neurons than controls at 3 dpi (Fig. 5a, b), a finding also observed in primary DRG neurons (axotomized during dissociation) (Fig. S7c).The phenotypes were sustained at 21 dpi after PL, signifying long-lasting epigenetic changes with Bmal1 deletion (Fig. S7d, e).
Given the global 5hmC elevation in Bmal1-deficient DRG neurons, we initially anticipated induction of a wide range of RAGs, but surveying RAGs in uninjured DRGs from Bmal1 cKO vs. control mice revealed no significant upregulation of well-known RAGs such as Atf3 (a marker of conditioning lesion 60 ), neurotrophins (Bdnf, Ngf, Ntf3), Gal, or Gap43 (Fig. 5c).We thus posited that Bmal1 deletion and the concomitant Tet3-5hmC epigenetic changes may prime DRGs for regenerative responses to axonal injury.Indeed, in axotomized DRGs at 1 day after PL, we detected increased transcription of Bdnf, Gal, and Npy in Bmal1 cKO DRGs relative to controls (Fig. 5c), while IF staining verified higher protein level of NPY in mutant DRG neurons at 3 dpi (Fig. 5d).In line with the IF results shown in Fig. 5a, Tet3 transcription was higher in Bmal1 cKO DRG (Fig. 5c).Of note, not all RAGs were affected, e.g., GAP43 or ATF3 immunosignals appeared comparable between genotypes at baseline or 3 dpi (Fig. S8a-c).
To assess if Tet3 is required for the axon promoting effect of Bmal1 deletion, we first took advantage of C35, a newly characterized cell-permeable inhibitor of Tet catalytic activity 61 .Exposure to C35 reduced 5hmC levels in DRG neurons (Fig. S8d) and attenuated axon elongation phenotype of Bmal1 cKO neurons in a dose-dependent manner (Fig. S8e, f).We next tested Tet3 knockdown by siRNA (Fig. S8g), which similarly reduced 5hmC levels and the growth advantage of Bmal1 cKO DRG neurons (Figs.5e, f, S8h).Hence, the promoting effect of Bmal1 deletion requires Tet3.
Since Bmal1 ablation can increase reactive oxygen species (ROS) 52 , which may restrict Fe 2+ availability required for Tet3 catalytic activity [62][63][64] , we tested the effect of N-acetylcysteine amide (NACA), a ROS inhibitor with free radical scavenging property 65 .We found that while NACA treatment led to a modest increase of 5hmC in control DRG neurons, the effect was more pronounced in Bmal1 cKO neurons (Fig. 5g).This suggested that even though Tet3 was upregulated in Bmal1 cKO DRG neurons, there appears to be a built-in safeguard mechanism via ROS-mediated inhibition of Tet3 catalytic activity.Neurite outgrowth assay showed that NACA treatment further augmented the pro-growth phenotype of Bmal1 cKO (Fig. 5h).In aggregate, Bmal1 acts as an inhibitor of axon regeneration by limiting Tet3-5hmC gains; Bmal1 ablation mimics the conditioning lesion in lifting this epigenetic block partly in a Tet3-dependent manner, and the promoting effect can be potentiated by anti-ROS scavenger to unleash Tet3 catalytic activity (Fig. 5i).
The upstream regulators of the Tet3-5hmC regulon as predicted by IPA with positive activation z-scores included CREB1, NGF, as well as RBPJ and THRB (the two TFs with binding motifs enriched in PL-DhMRs, see Fig. 1c), while those with negative activation z-scores included TCF4 (Wnt pathway), PPARGC1A (an integrator of energy metabolism and circadian clock 66 ), and NR3C1 (a novel circadian regulator 67 ) (Fig. 6d).For validation, we examined the expression of βcatenin, a transcription factor in the Wnt pathway and TCF4 interacting protein 68 .We found that at 3 dpi, while the overall expression levels appeared comparable, the membrane localization of β-catenin appeared higher in Bmal1 cKO DRG neurons than in controls, signifying inactivation of Wnt pathway, in line with bioinformatic results (Fig. 6e) and reported inhibitory role of Wnt in axon regeneration 69 .IF also verified that at 3 dpi, Bmal1 cKO DRG neurons contained higher nuclear levels of pCREB S133 , a converging effector downstream of cAMP, MAPK, PI3K/ATK, and mTOR signaling (Fig. 6f).
We next analyzed PL-DhMRs with or without Bmal1 binding motif separately (Figs.S9a, S10a; Supplementary Data 6-8).For both categories, close to half displayed 5hmC gain, 40% 5hmC loss, and 10% both.The two groups of PL-DhMRs both showed increased chromatin accessibility after PL, as did the associated genes near TSS (see Fig. 1h and Fig. S10b).Of the predicted Tet3-5hmC regulon containing Bmal1 binding motif (n = 129 RAGs, possibly direct targets of Bmal1) or not (n = 223 RAGs, likely indirect targets of Bmal1 via Tet3), both shared enriched pathways for axon growth, circadian rhythm, and DNA methylation/transcriptional repression 69 (Figs.S9b, c, S10c, d), further supporting a link of circadian clock and genome-wide reconfiguration of DNA methylome after axonal injury.Top enriched pathways for those containing Bmal1 binding motif included HIF1α, mTOR, and autophagy (Fig. S9c).We further conducted IPA graphical summary analyses, which applies machine learning to prioritize and connect biological pathways and infer novel relationships 70 .This highlighted that the Tet3-5hmC regulon with Bmal1 binding motif featured cytoskeletal organization, formation of cellular protrusion, KLF6 (a known axon regeneration promoting TF [71][72][73][74] ), while those without Bmal1 binding motif featured CREB1, IL1B, and immune response of macrophages (Figs.S9d, S10e).Of note, Bmal1 itself was part of the downregulated Tet3-5hmC regulon with Bmal1 binding motif, while Tet3 was part the regulon without Bmal1 binding motif, suggesting selfregulatory loops in response to PL (Supplementary Data 6 and 7).

Neuronal Bmal1 ablation affects immune response in axotomized DRG
Immune pathways enrichment in the predicted Tet3-5hmC regulon in conditioning lesion implies potential non-autonomous effects of neuroepigenetics.We thus first surveyed the expression of immuneassociated genes in uninjured DRGs.Consistent with only a priming effect of Bmal1 deletion, qRT-PCR showed no significant change of Ccl2, Il1b, Fcgr3a (encoding CD16), Mrc1 (CD206), Mki67 (proliferation marker) or Fn1 (matrix protein fibronectin) transcription in uninjured DRGs between genotypes (Fig. S11a).One exception was Il6, which was upregulated in mutant DRG.Since IL6 is a target of REV-ERB repressive activity 75 , its upregulation aligned with the observed downregulation of REV-ERBs in Bmal1-deficient DRGs (see Fig. S5d).By contrast, conditioned Bmal1 cKO DRGs at 1 dpi showed higher expression of Csf1 (colony stimulating factor 1, a potent regulator of macrophage activation), but lower expression of Il1b, while metabolism related gene Ppargc1a was slightly downregulated (Fig. 6g).
We observed higher levels of IBA1 (a marker upregulated in activated macrophages) and CD206 (a marker of pro-repair/anti-inflammatory macrophages) in conditioned but not uninjured Bmal1 cKO DRGs than control counterparts, but phagocytosis marker CD68 showed no overt changes between genotypes (Figs.6h, i, S11a-c).Taken together, Bmal1 cKO appeared to affect neuroimmune interactions after axotomy.

Bmal1 regulon concerns xenobiotic, lipid, and energy metabolism
To gain a more comprehensive view of Bmal1-regulated genes during axon regeneration, we performed additional RNA-seq on semi-purified primary DRG neurons (axotomized during dissociation) at 28 h after seeding.Principal component analysis (PCA) showed a clear segregation of three samples from Bmal1 cKO vs. control cohorts (both received tamoxifen) (Fig. 7a).We confirmed a significant downregulation of Bmal1 in mutant samples (Fig. S12a).Hierarchical clustering revealed more upregulated than downregulated DEGs in Bmal1 cKO vs. control (Figs.7b, S12b), in line with global Tet3-5hmC elevation and thereby transcriptional activation by Bmal1 ablation.The Bmal1 regulon in regenerating DRG neurons (n = 625 DEGs, cutoff log 2 FC of 0.25, P < 0.01, Supplementary Data 9) included cytoplasmic proteins, membrane proteins, secreted factors, and nuclear factors (Fig. 7c), and they concerned not only axon growth (axon guidance, growth factor signaling), but also stress responses (e.g., Xenobiotic detoxification, Oxidative stress response, DNA damage repair, Autophagy, Unfolded protein response), lipid metabolism, and notably immune signaling (Fig. 7d).
To identify Bmal1 regulon potentially regulated via Tet3-5hmC epigenetics, we intersected the DEGs with PL-DhMR associated genes, yielding 85 overlapping genes (Fig. 7e; Supplementary Data 10).IPA of these genes showed positive activation z-scores for the canonical pathways of Phagosome formation, G-protein coupled receptor signaling, CREB signaling in neurons, HIF1α, and ID1 signaling, while Wnt/ β-catenin showed a negative activation z-score (Fig. 7e, f).About 42% of them (36 genes) harbored Bmal1 binding motif (Supplementary Data 11), and they concerned axon growth (BDNF, Myc, RAC/PAK/p38), vascularization (HIF1α, VEGF, Angiopoietin receptor Tie2), as well as energy homeostasis and metabolism (Ghrelin regulation of energy homeostasis, Polyamine metabolism, Methionine De Novo and Salvage pathway, Vitamin D Receptor pathway) (Fig. 7g).Altogether, through Tet3-5hmC dependent and -independent mechanisms, Bmal1 regulates a large repertoire of genes related to axon guidance and growth, but also pathways not well studied in the context of conditioning lesion.
Resonating with a repressive role of Bmal1 on Tet3 expression, we observed diurnal changes of Tet3 and 5hmC in DRG neurons, higher at ZT12-15 than ZT0-3 (Fig. 8b-d).The time-of-day effect persisted for Tet3 expression in conditioned DRG neurons at 1 dpi, higher when injury occurred at ZT12-15 (Fig. 8b, c).This also applied to 5hmC responsiveness to PL, with augmented 5hmC induction by PL at ZT12-15 than at ZT0-3 (Fig. 8d).We also examined transcriptional rhythmicity of the enzymes involved in DNA (de)methylation, and only Tet3 and Gadd45g displayed diurnal changes (Fig. S13a, b), both containing promoter binding motifs for circadian clock TFs (Fig. S13c).Gadd45g is implicated in locus-specific DNA demethylation by serving as an adapter for BER factors 76 ; its higher expression level at ZT0-3 thus aligns with lower 5hmC accumulation at this ZT phase.
We further examined rhythmicity of Tet3-5hmC expression in DRG neurons by performing IF at 4-hour intervals along the ZT cycle, which revealed zenith of Tet3 levels at ZT20 and 5hmC at ZT12, while nadir of Tet3 expression at ZT0 and 5hmC at ZT4 and ZT20 (Fig. S14a).Interestingly, Bmal1 protein levels as assessed by IF appeared higher at ZT12 than ZT0, opposite that of Bmal1 transcriptional oscillation, echoing reported lag time between Bmal1 mRNA and protein levels 77,78 (Fig. S14b).In this context, it is noteworthy that Bmal1 protein level does not equate to Bmal1 DNA binding or transcriptional activity as Bmal1 activity is additionally controlled by regulators of the feedback loops such as Per and Cry 47,77,79 .Indeed, highest Bmal1 activity has been reported when Bmal1 protein level is at its lowest 79 .Of note, in whole DRG lysates (from either male or female mice), WB did not reveal significant changes of Bmal1 protein levels between ZT0-3 and ZT12-15, although we detected a slight shift to higher molecular weight at ZT12-15 (Fig. S14c), likely reflecting post-translational modifications 79,80 .We also performed WB on DRG tissue lysates collected at ZT0-3 from Bmal1 cKO or control mice, which revealed only a slight but not statistically significant reduction of Bmal1 in mutant samples, likely due to neuronal-specific cKO that spared glial cells (Fig. S14d).

Time-of-day effect on axon regrowth potential in vitro and in vivo
Having revealed diurnal oscillation of Bmal1 and Tet3-5hmC in DRG neurons (Fig. 8e), we next examined a potential time-of-day effect on axon growth potential by comparing primary DRG neurons collected at ZT0-3 and ZT12-15 (Fig. 9a).While the initial phase of axon elongation at 12 h after plating did not show differences, by 22 h, DRG neurons collected at ZT12-15 extended longer neurites than those from ZT0-3 (Fig. 9b).This applied for DRG neurons from both female and male mice, indicating a sex-independent diurnal effect on axon growth (Fig. 9c).We further conducted neurite outgrowth assay on primary DRG neurons isolated every 6 h, wherein the ones isolated at ZT12 extended the longest neurites while those isolated at ZT0 extended the shortest neurites (Fig. S16a).
We next conducted in vivo nerve regeneration studies.In congruence with the neurite outgrowth results, axonal regrowth appeared more robust for PL occurring at ZT12-15 than at ZT0-3, with SCG10 + axon length reaching twice as long at 1 dpi (Fig. 9d).Altogether, axonal injury at the onset of the active phase of animals appears to be associated with more robust axon regrowth.

Bmal1 cKO mimics the conditioning lesion in augmenting axon growth potential at different ZT
Our above studies showing that PL triggers Bmal1 downregulation and that Bmal1 deletion leads to enhanced axon regeneration were conducted mostly at ZT0-3.This prompted us to ask if these results would be affected by different ZTs (Fig. 10a).We found that Bmal1 downregulation at 1 dpi was more pronounced for PL at ZT12-15 than ZT0-3 (Fig. 10a, b).Consistently, conditioned DRGs with a prior PL at ZT12-15 extended longer axons than the ZT0-3 group (Fig. 10c), indicating a time-of-day influence on the conditioning lesion effect, more pronounced at the onset of active phase of animals.
Perhaps reflecting light entrainment or "masking", a response to light overriding the absence of circadian clock 34,52,82 , DRG neurons from Bmal1 cKO mice (maintained in regular light-dark (LD) cycle) continued to show time-of-day effect on neurite outgrowth, with longer neurites at ZT12 than ZT0 (Fig. S16b).Likewise, neurite outgrowth advantage of Bmal1 cKO also appeared more pronounced for the ZT12-15 group than ZT0-3 (Fig. 10d).Resonating with the above results, when we examined in vivo axon regrowth after sciatic nerve crush injury at different ZTs, the axon promoting effect from Bmal1 ablation was evident for both ZT cohorts, but more pronounced for the ZT12-15 cohort, with maximal axon length reaching 4.2 ± 0.39 mm at 3 dpi for ZT12-15 vs. 3.4 ± 0.84 mm for ZT0-3 (Fig. 10e, f).
Since the conditioning lesion operates partly through CREB activation 83,84 and p-CREB S133 expression displays diurnal changes in uninjured DRG neurons (see Fig. S15c), we next examined p-CREB S133 levels in axotomized DRG neurons, which showed higher levels at ZT12-15 than ZT0-3 for both 1 dpi and 3 dpi (Fig. S16c, d).Aligned with highly elevated p-CREB S133 in Bmal1 cKO DRGs (see Fig. 6f), treatment with db-cAMP, a cell permeable synthetic analog of cAMP which activates CREB, did not further enhance neurite outgrowth (Fig. S16e).Hence, while DRG neurons display diurnal changes of axon growth capacity, attenuating Bmal1 by either conditioning lesion or genetic ablation can further augment axon regeneration even for ZT12-15 (Figs. 10g, S16f).
Lastly, as another indicator of the time-of-day effect, we compared the DEGs profiled in conditioned DRG with PL performed at ZT0-3 or at undefined ZT time 14,85 (Fig. S17a).This revealed 815 shared genes with similar directionality of expression (Pearson correlation coefficient of 0.89) and featuring Circadian Rhythm, Axonal guidance, cAMP-mediated pathway, CREB, HIF, and Xenobiotic metabolism, as well as immune signaling (Fig. S17b, c; Supplementary Data 13).But notably, there was a large fraction of nonoverlapping DEGs: 73% of the DEGs from this study and 58% of the published study 14,85 .

Discussion
Axon regeneration requires epigenetic reconfigurations to rewire the gene regulatory circuits.Here, we identified Bmal1 as a regulator axon regeneration: Bmal1 deletion led to enhanced axon regeneration, in part through augmenting Tet3-5hmC induction in response to axotomy.We also revealed diurnal oscillation of Tet3-5hmC in DRG and a time-of-day effect on axon growth potential.
Peripheral axotomy of DRG induces Tet3 expression to adjust epigenetic states in favor of axon regeneration 17 .Our unbiased survey of the PL-specific DhMRs revealed an enrichment of Bmal1 binding motif, thus implicating a link of Bmal1 with epigenetic regulation of axon regeneration.Interestingly, our data supported an antagonistic relationship between Bmal1 and Tet3 in gating axon regenerative responses: (i) rapid downregulation of Bmal1 after conditioning lesion of DRG, coinciding with Tet3-5hmC upregulation; (ii) augmented Tet3 upregulation and 5hmC gains with Bmal1 ablation.Tet3 may be under the direct control of Bmal1-Clock or other molecular oscillators (Tet3 promoter contains binding motifs for Clock and Bhlhe41).Tet3 recruitment to PL-DhMRs may also involve Bmal1 or other bHLH-PAS TFs.Alternatively, a recent study revealed that the Tet3 catalytic domain displays an intrinsic sequence preference for E-box motifs to establish DNA (de)methylation patterns; 86 thus, by occupying the E-box motifs, Bmal1 may sterically restrict access of Tet3 to these sites.The potential dual mechanism of limiting Tet3 expression and controlling Tet3 access to E-box motifs may ensure swift and robust gene regulation by the Bmal1 circadian machinery in response to axotomy.While our data demonstrated a transcriptional as well as a functional link of Bmal1 and Tet3 (as the growth advantage of Bmal1-deficient DRG neurons requires Tet3), future analysis of a possible proteinprotein interaction is needed.
By enhancing Tet3 expression and global 5hmC gains, Bmal1 deletion likely exerts a wide-spread influence on the DNA methylome and thereby transcriptional output.This may also underlie how Bmal1 operates as a pioneer-like TF 38,39 in the setting of axon regeneration.A pioneer-like function of Bmal1 for Tet3-5hmC epigenetic reconfigurations also fits with our findings that Bmal1 deletion alone seems insufficient to trigger large-scale pro-growth gene programs, but it primes DRGs for regenerative responses.The signal that triggers RAGs induction upon axotomy awaits future study, but may involve axonal retrograde transport of injury signals back to soma 87 .
While global 5hmC gains were observed with Bmal1 deletion, on the level of individual DhMRs and associated genes, either 5hmC gain or loss may occur, depending on the step along the DNA demethylation cascade 88,89 .Of note, although DNA methylation primarily occurs near TSS and generally leads to gene silencing 90 , 5hmC modifications typically occur at introns and intergenic regions rather than at promoters and CpG islands 17 , and may be associated with neuronal enhancer regions 91,92 .In congruence, CpG island DNA methylation analyses revealed only minor changes after PL vs. CL of DRG 15 .
How Bmal1 is downregulated after PL requires further study.In this regard, it is notable that Per1 is upregulated after PL; since Per1 is an immediate early gene 93 , it may participate in further suppressing Bmal1 transcriptional activity after PL.The numerous feedback loops of the circadian clock preclude a clear demarcation of the hierarchy of Bmal1 and Tet3 regulation at this point.Indeed, both Tet3 and Bmal1 genes contain PL-DhMRs, suggesting auto-regulatory loops and sensitivity to DNA methylation changes.
Our transcriptional profiling revealed a large repertoire of Bmal1regulated genes in conditioned DRG, with partial overlap with PL-DhMR associated genes.Hence, Bmal1 operates through Tet3dependent and -independent mechanisms in response to axotomy.It is noteworthy that the Bmal1 regulon not only concerns axon growth/ axon guidance, but also immune signaling, stress responses, lipid metabolism, and energy homeostasis, thereby pointing to additional pathways with potential impact on axon regeneration.Intriguingly, 46% of the Bmal1 regulon in injured DRG neurons encoded proteins located at plasma membrane or extracellular space, supporting nonautonomous interactions.Also of interest is whether Bmal1 activity in glial cells influences regeneration; in non-CNS, the beneficial effects of Bmal1 deletion for tissue repair have been attributed to immune or vascular changes [94][95][96][97] .
Circadian rhythm is governed not only by the central clock located in the suprachiasmatic nucleus, but also by interactions with peripheral clocks, which exist in every organ system including DRGs 81,98 .In our studies, we utilized the SLICK-H Thy1-CreER T2 line for Bmal1 cKO, which may potentially ablate Bmal1 in the suprachiasmatic nucleus, affecting the central clock.However, earlier studies using constitutive deletion of Bmal1 showed that behavioral rhythms and activity are maintained in knockout mice entrained by light:dark cycle 34,52,82 .As our mouse cohorts were kept in regular light:dark cycle, we expect that perturbation of the central clock is not a major factor for the observed phenotypes of axon regeneration in Bmal1 cKO animals.Bmal1 notably also has circadian-independent functions, e.g., in aging and tissue homeostasis 99 .
The circadian clock affects a wide range of systems, including metabolism, inflammation, and stress responses 100 .Our study uncovered Tet3-5hmC rhythmicity in connection with a time-of-day effect on axon regrowth potential.In echo, recent studies revealed changes of cytosine modifications in a circadian manner in mouse liver and lung, particularly at E-box elements of circadian transcripts in gene body region 101,102 .Tet3-5hmC diurnal changes in DRG neurons may be controlled not just by peripheral circadian clock, but also reflect light entrainment, sleep cycle, or neuronal activity.In the current study, the Bma1 cKO mice were maintained in regular light:dark cycle, thus the continued time-of-day differences of axon growth potential of DRG neurons from Bmal1 cKO mice might reflect "masking" or that the glial circadian clock remained intact in the cKO mice.To tease out whether the Tet3-5hmC epigenetic oscillation or axon growth potential is truly circadian or a result of light entrainment or neuronal activity requires further studies in animals kept in constant darkness.In addition, increasing neuronal activity by electrical stimulation or by exercise can improve regeneration 103 ; since exercise affects circadian rhythms and alters the amplitude of the clock 104 , its regenerative effect may partly operate through circadian modulation.It is also worth future exploration if other epigenetic mechanisms such as histone modifications display diurnal rhythmicity.Since conditioned DRG neurons extended longer axons than Bmal1 cKO neurons, Bmal1 circadian rhythm is only one factor at play for axon regeneration.
From an evolutionary point-of-view, faster initiation of the regenerative response in active phase may reflect an anticipation by neurons for heightened regenerative need during the active phase.Alternatively, gene expression program at the active phase may simply coincide with a large set of genes controlling axon regeneration.Since most rodent experiments are conducted during daytime with lights on (resting phase of rodents), our study underscores the importance of experimental setups in a circadian-informed manner 105,106 .
In sum, our study identifies a role of the circadian oscillator Bmal1 in the regulation of neural repair and provides a proof of principle that suppression of circadian TF such as Bmal1 can foster a sustained regenerative injury response.As a growing arsenal of pharmacological agents targeting circadian pathways are available 107 , the understanding of the connection between Bmal1 and Tet3/5hmC epigenetics, as well as regenerative and stress responses, provides a starting point to explore strategies to enhance axon regeneration.

Mouse strains
Animal procedures were conducted according to the protocol (IPROTO202200000184) approved by the Institutional Animal Care and Use Committee (IACUC) at Mount Sinai.C57BL/6J (JAX stock #000664), Tg(Thy1-cre/ERT2,-EYFP)HGfng (also known as SLICK-H; stock #012708) 54 , B6.Cg-Tg(Nes-cre)1Kln/J (stock #003771) 108 , and Bmal1 fl/fl (stock #007668) 109 mice were from The Jackson Laboratory.All mice were bred onto C57BL/6J genetic background for at least three generations.Animals were housed under 12:12 h light:dark cycle with ad libitum access to food and water and were habituated to the facility for at least 2 weeks prior to conducting experiments.Tamoxifen (Sigma) dissolved in corn oil (Sigma) was administered at a dose of 100 mg/kg by intraperitoneal injection once a day for 5 consecutive days.Experiments were conducted 2 weeks after tamoxifen injections and within the first 3 h of light-on in animal facility (ZT0-3) unless otherwise specified.

Sciatic nerve lesion injury
Sciatic nerve crush injury was performed on anesthetized mice with isoflurane under aseptic conditions.After shaving the leg fur and sterile prepping, a skin incision was made on right mid-thigh region, and the fascia between the gluteus superficialis and biceps femoris muscles was gently opened to expose the sciatic nerve, which was then carefully freed from surrounding connective tissue.Crush injury was performed approximately 5 mm distal to the bifurcation point of the sural nerve, applying pressure with ultra-fine hemostats (FST, #13020-12) for 15 seconds.Wound area was closed with two reflex 9 mm clips (Braintree Scientific, EZC-2PK).Sciatic nerve transection injury was performed with 3 mm Vannas spring scissors (FST, #15000-00).

DRG isolation procedure
Dissections were performed under a Nikon SMZ645 stereo microscope.Mice were euthanized by carbon dioxide inhalation followed by cervical dislocation.Mice were laid on the dorsal side and immobilized on a dissection pad.Skin covering the ventral thorax and abdomen was removed together with internal organs to fully expose the ventral spinal column using surgical scissors (Fine Science Tools, #14054-13), supported by tissue forceps (Fine Science Tools, #11021-12).Muscles covering ventral side of the spinal column were removed using Spring Scissors (Fine Science Tools, #15751-11) to expose the lumbosacral peripheral nerves.To expose the lumbosacral DRGs, the ventral vertebrae were removed using the aforementioned Spring scissors, aided by Octagon forceps (Fine Science Tools, #11042-08) with care taken not to sever nerves.DRGs were gently picked starting with the lower lumbar level (L6) using a Dumont #3 forceps (World Precision Instruments, #50037) and isolated by cutting connecting nerves with Vannas Spring scissors (Fine Science Tools, #15000-00).

Lentiviral production and DRG transduction experiments
HEK293T producer cells were subjected to multiple passaging steps and then seeded at density of 1 × 10 6 in a T75 flask containing 15 ml of complete media, DMEM (Invitrogen, #10569044), 1× NEAA (Invitrogen, #11140-050), and 10% FBS.Two days later, 3.8 × 10 6 cells were seeded in PLO coated 10 cm plate.Next day, cells were washed with DPBS and incubated with 10 ml OPTI-MEM (Invitrogen, #31985070).Transfection mix containing 1.3 pmol psPAX2 (Addgene #12260), 0.72 pmol pMD.2G (Addgene #12259), and 1.64 pmol pEGIP or Bmal1-OE plasmids and PEI (1:3 µg DNA to PEI ratio) was added to cells for 6 h.Cells were washed twice with DPBS and incubated in complete media.Media containing the virus was collected at 48 h, 72 h, and 96 h posttransfection.To concentrate virus, media was spun at 500 g for 5 min, filtered through 0.45 micron PES filter (Thermofisher, #725-2545), and spun at 24,000 rpm for 2 h using SW41 Beckman rotor.Media was aspirated carefully and pellet containing virus was suspended in 100 μl serum free DMEM per 10 ml initial volume.Virus was stored at 4 °C and used within 1 week of preparation.
For viral transduction of adult DRG neurons, neurons freshly isolated from thoracic and lumbar DRGs of one mouse were suspended in 1 ml of 2x complete DRG media without antibiotic/antimycotic.30 μl virus was mixed with 970 μl Neurobasal-A media and added to the 1 ml neuronal suspension.The resulting 2 ml neuron-virus mix was added to a 6-well plate precoated with PLO/laminin, spun down at 900 rpm for 90 min at room-temperature, and incubated at 37 °C overnight.Next day, media was changed to 1x complete DRG media with antibiotic and antimycotic.For neurite outgrowth analysis, transduced DRG neurons were washed once with DPBS, trypsinized for 3 min at 37 °C, and resuspended in DRG media by gentle pipetting.Neurons were split from one 6-well plate to six wells of a 24-well plate with PLO/laminin precoated coverslips for analysis.

DRG explant culture
DRGs were isolated from adult mice and placed individually in DMEM/ solution on ice, washed twice with ice-cold HBSS-HEPES buffer, and incubated in 9% collagenase (Worthington, #LS004196) for 30 min at 37 °C.Subsequently, collagenase was removed and DRGs were washed twice with HBSS-HEPES.DRGs were picked and placed individually on laminin-coated coverslips in 0.5 ml medium containing DMEM highglucose with Glutamax (Gibco, #10569-044), 10% fetal bovine serum, and 1× non-essential amino acids.A centrifugation step at 500 × g for 5 min was used to promote adherence.Explants were cultured at 37 °C for 4 days followed by fixation with 4% paraformaldehyde containing 4% sucrose for 20 min for immunofluorescence analysis.

Generation of induced neurons and replating assay
H9 human embryonic stem (hES) cells were obtained from the stem cell core at Mount Sinai, maintained and passaged in mTeSR1 media (Stem Cell Technologies, #85850) using aggregate passaging with ReLeSR (Stem Cell Technologies).0.4% of antibiotic-antimycotic agent was added to all media used in these studies (Gibco, 15240062).The study was approved by the Embryonic Stem Cell Research Oversight Committee (ESCRO) at Icahn School of Medicine at Mount Sinai.

Immunofluorescence imaging
For immunofluorescence cytochemistry, cultured DRG neurons or induced neurons derived from hES cells were washed once with PBS and fixed in 4% paraformaldehyde containing 4% sucrose in PBS for 15 min at room temperature.NPC fixation was performed in 4% formalin in PBS for 10 min at room temperature.After three 5 min washes with PBS, cells were incubated in blocking buffering (5% donkey serum, 0.3% Triton X-100 in PBS) for 45 min.Primary antibody incubation was performed overnight at 4 °C in antibody dilution buffer (1% BSA, 0.3% Triton X-100 in PBS), followed by 3×5 min washes with PBS.Secondary antibody incubation was performed together with DAPI (Invitrogen, D1306) at room temperature for 1 h, followed by 3x 5 min washes with PBS.Glass coverslips were mounted with Fluoromount-G (SouthernBiotech).
For immunofluorescence histochemistry, dissected DRGs or sciatic nerves were fixed in 4% paraformaldehyde at 4 °C, cryopreserved in 30% sucrose for 24 h at 4 °C, and embedded in Tissue-plus OCT compound (Fisher Healthcare, 4585).12 µm thick sections were prepared on a cryostat.For immunofluorescence for the 5hmC mark, DRG sections were permeabilized with 0.5% Triton-X for 30 min, washed twice with PBS for 5 min, and incubated with 100 µg/ml RNase A (Thermo Scientific, EN0531) in PBS at 37 °C.After two 5 min washes in PBS, DRG sections were denatured in 2 N HCl for 30 min and then neutralized with 100 mM Tris pH8.5 for 30 min.Sections were then washed three times for 5 min with PBS, blocked for 1 h, and immunostained as described above.
For wholemount immunofluorescence of paw pads, hind paw skin (ipsilateral to injury) was dissected, cleaned from fat and connective tissue, washed three times for 5 min with PBS, and post-fixed in 4% PFA at 4 °C.Tissue was rinsed thrice for 5 min in PBS and washed ten times for 30 min each with PBS containing 0.3% Triton X-100 (0.3% PBST).Primary antibody incubation was performed in blocking buffer (0.3% PBST containing 5% goat serum and 20% DMSO) at room-temperature for 5 days with gentle shaking.Tissue was washed ten times for 30 min each with 0.3% PBST and incubated with secondary antibodies in blocking buffer at room-temperature for 3 days with gentle shaking.After washing ten times for 30 min with 0.3% PBST, tissue was dehydrated in 50% methanol for 5 min, 100% methanol for 20 min, and finally cleared in benzyl alcohol: benzyl benzoate mix (1:2 ratio) overnight at room temperature.Tissue was imaged in clearance solution with a Zeiss LSM 780 confocal microscope.
Immunofluorescence images were captured with a Zeiss Axio Imager.A2 microscope with AxioCam MRm camera.For β-catenin imaging and whole mount nerve reinnervation imaging, maximal intensity projections of z-stacks that were captured with a Zeiss LSM 710 confocal microscope were used.Brightfield imaging was performed using an Olympus CKX53 microscope.

RNA isolation and qRT-PCR analysis
DRGs were isolated and immediately placed in RNAlater stabilizing solution (Thermo Scientific, AM7024).Total RNA isolation was performed using the RNeasy plus micro kit (Qiagen, 74034).Briefly, DRGs were completely lysed in 80 µl of RLT buffer supplemented with βmercaptoethanol using RNase-free pestles (Fisher, #12-141-368).Subsequently, additional RLT buffer was added to a final volume of 350 µl and samples were vigorously pipetted before proceeding with RNA isolation.RNA isolation from H9 hES cells, NPCs, and neurons was performed using standard TRIzol extraction (Invitrogen, #15596026).Reverse transcription was performed using Superscript III reverse transcriptase (Invitrogen, #18080051).Real-time qRT-PCR was performed using ABI Prism 7900HT Sequence Detection instrument and SYBR green master mix (QuantaBio, #95073-012).Gene specific primers (Supplementary Data 14) were purchased from Integrated DNA Technologies.Melting curves were performed for each run to ensure specificity of the product and the housekeeping genes Hprt and GAPDH were used for normalization of mouse and human samples, respectively.Relative gene expression was analyzed according to the ΔΔCt method.For conditioned DRG analysis, ipsilateral L4-L6 DRGs were pooled from 2 mice for each sample.

Co-immunoprecipitation assay and Western blot analysis
Co-immunoprecipitation was performed based on protocols from the Longworth laboratory 115 .Neuro-2a cells were cultured in low glucose DMEM (Gibco, 11885084) containing 10% FBS.On the first day, Neuro-2a cells were seeded at a density of 8.5 × 10 6 cells in a T75 flask for each immunoprecipitation sample.On day3, cells were washed once with PBS and serum-starved for 24 hrs.For each sample, lysis was performed in 1 ml of ice-cold high salt lysis buffer containing 300 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 0.1% Triton, 10% glycerol, 1 mM DTT, 1× complete mini protease inhibitors (Sigma, 11836170001) for 1 h at 4 °C.Lysate was cleared by centrifugation at 13,000 g for 15 min at 4 °C.50 ml of protein G Dynabeads (Thermo scientific, 10003D) were used for each immunoprecipitation sample.Beads were washed two times with blocking buffer (0.5% BSA, 0.2% sodium azide in PBS) and blocked for 30 min at room-temperature under shaking.Anti-Tet3 antibody (Active motif, #61743, 1:100) or IgG control (Millipore, NI04-100UG) was conjugated to the beads in blocking buffer for 4 h at roomtemperature.Beads were washed twice with blocking buffer and lysate was applied and incubated overnight at 4 °C.Next day, beads were washed four times 5 min each with ice-cold 1× PBS at 4 °C, then suspended in 60 ml of 2× NuPAGE LDS buffer (Invitrogen, NP0007) with β-mercaptoethanol.For DRG analysis, extraction was performed in RIPA buffer containing 1× complete mini protease inhibitors (Sigma, 11836170001) and 1× PhosSTOP phosphatase inhibitor (Sigma, 4906845001) by homogenization on ice with pestles and rotating at 4 °C for 30 min.Subsequently, lysate was centrifuged at 13,000 × g for 10 min at 4 °C, boiled in 1× NuPAGE LDS sample buffer, and used for analysis.Western blot detection was performed using anti-Tet3 (Active motif, #61743, 1:1,000), anti-Bmal1 (Novus, NB100-2288, 1:1000), or anti-β-actin (Sigma, A1978, 1:10,000) antibodies as previously described 116 and membranes were scanned with LI-COR Odyssey Classic system.

Image analysis
Image analysis was performed with the Fiji package of ImageJ 117 .Prior to importing the Tiff image into ImageJ, the global scale was set according to the magnification used.For measurement of neurite length, a simple neurite tracer (SNT) tool was used to determine the length of longest neurite for each DRG neuron 118 .Explant cultures were analyzed with Neurite J 1.1 plugin as previously described 119 .For analysis of axon regrowth in injured nerve, micrographs of nerves were stitched together using Microsoft Paint.Images were then imported into ImageJ and a rectangle was drawn to outline the length of the nerve.The SCG10 immunointensity profile along the length of the nerve was determined using the Plot Profile tool.Finally, the intensity profile plot values were normalized to the highest intensity value, representing the beginning of the crush site.
Micrographs of footpads were analyzed with maximum intensity projections of z-stack images.PGP9.5 or NFH positive areas were measured by thresholding and using the Analyze Particle (with limit to threshold) summary tool.Total footpad area was measured by manual tracing and using the 'Measurement' tool.
5hmC, Tet3, pCREB S133 , and ATF3 immunofluorescence intensities in DRG neurons were analyzed by thresholding and using the Analyze Particle tool by setting the minimal particle size limit to 50 µm and the circularity value to 0.8-1 to exclude staining of the smaller satellite cells.Quantification of the expression of macrophage markers was performed on two randomly selected 100 mm 2 areas of DRG images, using the Analyze Particle tool.For all analyses, thresholding values were kept constant between groups.Analysis of β-catenin localization was performed by measuring the average IF intensity of randomly selected areas of membrane or cytoplasm and calculating the ratio of membrane to cytoplasm localization.

RNA-seq analysis
RNA samples of DRG tissue and dissociated neurons were collected with the QIAGEN RNeasy plus mini kit and sent to Psomagen, Inc for next-generation sequencing on the Illumina NovaSeq platform.The NGS-Data-Charmer pipeline was used for preprocessing, QC, and alignment of the FASTQ files (https://github.com/shenlab-sinai/NGS-Data-Charmer).In brief, adapter trimming was achieved by using the Trim-Galore tool 120 (v0.6.5),followed by alignment of trimmed reads to the mouse mm10 genome assembly using Bowtie2 121 (v2.4.1), and duplicated read pairs were further removed using the 'rmdup' module of SAMtools 122 (v1.10).featureCounts was used to obtain a gene expression matrix, using the parameters "--fraction -t gene" on the GENCODE annotation (vM25).Genes were filtered to remove lowly expressed genes, defined as fewer than 5 samples showing a minimum read count of 2 reads, prior to performing differential analysis with DESeq2.

Bioinformatics
Unbiased transcription factor (TF) binding motif enrichment analysis of DhMRs from DRGs after sciatic nerve lesion (previously reported in our study 17 ) was performed with the Homer package 123 , using the Perl script findMotifsGenome.pl.Background sequences were chosen at random from the mouse mm9 genome.Targeted enrichment analysis of motifs was performed with the Perl script annotatePeaks.pl.Chromatin accessibility of DhMRs and associated genes after nerve injury

Fig. 1 |
Fig. 1 | Bmal1 is linked to peripheral lesion of DRG and interacts with Tet3. a Graphical summary of genome-wide mapping of 5hmC reconfigurations after PL (peripheral lesion) vs. DCL (dorsal column lesion) or no injury, which identified 1,036 differentially hydroxymethylated regions (DhMRs) after PL 17 .Pie chart shows proportion of PL-DhMRs with 5hmC gain versus loss.b Top, normalized 5hmC signals after PL versus no injury across DhMRs 17 .Bottom, pie charts showing distribution of DhMRs in genomic regions.c Bubble plot showing enriched TF binding motifs in PL-specific DhMRs.Members of the bHLH-PAS family are labeled in blue and members of the bHLH family in brown.Circle size indicates DhMR numbers and color scale indicates significance of enrichment.d Bar graphs showing the number (y-axis) and percentage of PL-specific DhMRs that contain binding motifs for circadian-related or hypoxia-related transcription factors.e Left, diagram of the long and short isoforms of Tet3.ATG denotes start codon, CXXC indicates DNA binding domain.Right, qRT-PCR shows predominant expression of short isoform Tet3-S in uninjured DRGs.Data represent mean ± SEM. n = 4 independent studies, each with 6-9 pooled DRGs.f Model of potential Bmal1 and Tet3 interaction at DhMRs that may impact Tet3 targeting and 5hmC reconfigurations after peripheral lesion of DRG.g Ingenuity pathway analysis (IPA) highlighting the top enriched pathways for the 616 genes harboring PL-specific DhMRs with Bmal1 binding motif in response to axonal injury.Right-tailed Fisher's exact test.h Analysis of ATAC-seq signals from DRG after sham or PL at 1 dpi (raw data from Palmisano et al. 14 ) showed increased chromatin accessibility at PL-specific DhMRs with Bmal1 binding motif and associated genes near transcription start sites (TSS).TES, transcription end site.Welch's two-sample t-test.

Fig. 2 |
Fig. 2 | Peripheral lesion of DRG axons results in downregulation of Bmal1.a Schematic of core circadian clock components and feedback loops that control Bmal1 expression and activity.E-box, enhancer box; RRE, REV-ERB/ROR response element.b Left, experimental paradigm of ipsilateral L4-L6 DRGs analyzed by RNAseq at 1-day after peripheral lesion (PL at ZT2-4) or no injury.Top right, RNA-seq coverage tracks for differentially expressed circadian genes after PL at 1 dpi pooled from n = 3 samples per group.Bottom right, the expression levels of circadian clock genes as fragments-per-kilobase-per-million reads (FPKM).n = 3, each pooled from L4-L6 DRGs.Data represent mean ± SEM.Unpaired two-tailed Student's t-test.c Left, experimental paradigm of peripheral lesion (PL) and collection of ipsilateral L4-L6 DRGs for gene expression analysis.Right, qRT-PCR shows transcriptional changes of circadian clock genes in axotomized DRG at 1 dpi after PL as compared to uninjured DRG.n = 5-6 independent samples, each pooled from L4-L6 DRGs.Data represent mean ± SEM.Unpaired two-tailed Student's t-test.d Time-course analysis of Bmal1 transcription in L4-6 DRGs after PL by qRT-PCR.n = 3 independent DRG samples, pooled from two CD1 mice.Data represent mean ± SEM.Kruskal-Wallis with Dunn's multiple comparison's test.e Representative IF images and quantification of L5 DRGs show diminished nuclear signal of Bmal1 in axotomized DRG neurons (TUJ1 + ) at 1 dpi after PL.Violin plots of n = 100 uninjured and 183 axotomized neurons from 4 DRGs from 4 mice per group.Median is shown by black line.Mann-Whitney test.

Fig. 3 |
Fig. 3 | Bmal1 inhibition enhances axon outgrowth.a Top, experimental paradigm.Bottom, representative IF images and quantification of TUJ1 + area normalized to the number of soma per image, n = 39 images from 2 independent experiment.Data represent mean ± SEM, Mann-Whitney test.b Top, schematic of lentiviral vectors.Bottom, representative IF images and quantification of neurite length and percentage of neurite initiation of control (EGFP + ) or Bmal1 overexpression (mCherry + ) in Neuro-2a cells.n > 140 neurons for neurite length, and 60 images per group for percentage of neurite initiation.Data represent mean ± SEM.Mann-Whitney test.c Top left, diagram of pharmacological drugs stabilizing negative regulators of Bmal1 transcriptional activity.Top right, paradigm of neurite outgrowth assay of iNeurons.Bottom, representative IF images show longer neurite outgrowth from drug-treated iNeurons.Arrows point to neurites.Quantification: n = 600-900 neurons pooled from 3 independent cultures.Data represent mean ± SEM.Mann-Whitney test for KL001 vs. DMSO.Kruskal-Wallis with Dunn's multiple comparison test for PF-670462 vs. DMSO.d Experimental paradigm for tamoxifen administration to adult mice for neuron specific Bmal1 cKO.e IF images and quantification of neurite outgrowth of primary DRG neurons.Violin plots of n = 348 control, 305 Bmal1 cKO , and 148 conditioned neurons from n = 3 mice.Median is shown by black line.Kruskal-Wallis with Dunn's multiple comparison test.f IF images show that Bmal1 cKO could augment DRG conditioning lesion effect (with PL 3 days prior) in enhancing neurite outgrowth.Note early time point at 10 h after plating due to exuberant neurite outgrowth from the conditioning effect.Violin plots of n = 375 control and 626 Bmal1 cKO neurons collected from n = 3 mice per group.Data represent mean ± SEM.Two-way ANOVA followed by Bonferroni's multiple comparison test.g IF images of adult DRG explants show longer neurite outgrowth from Bmal1 cKO DRGs compared to control at 4 d of culture.Quantification of 8 images collected from n = 6 DRGs (L4-L6) from n = 2 mice per group.Two-way ANOVA followed by Bonferroni's multiple comparison test.

Fig. 4 |
Fig. 4 | Neuronal deletion of Bmal1 accelerates axon regeneration after sciatic nerve injury.a Experimental paradigm of in vivo axon regeneration analysis at 1, 3, and 21 d after sciatic nerve injury.Representative IF images and quantifications of axon regeneration (SCG10 + ) at sciatic nerve crush site at 1 dpi (b, c) or 3 dpi (d, e).Note enhanced axon regeneration in Bmal1 cKO as compared to littermate controls.Dashed vertical lines denote lesion center defined by maximal SCG10 immunointensity.Right panels show enlarged images of boxed areas.Data represent mean ± SEM and unpaired two-tailed Student's t test for maximal axon length.Two-way ANOVA for regeneration index, measured by SCG10 immunointensity normalized to value at lesion center.n = 5-6 mice per genotype.f Diagram of analysis of innervation of footpad epidermis.Representative images and quantifications of whole mount IF staining for pan-neuronal marker PGP9.5 (g) or axon marker NF-H (h) show improved footpad epidermis innervation in Bmal1 cKO at 21 dpi.Micrographs are representative confocal scans of a 400 µm segment, spanning the top, middle, and lower portion of the epidermis.n = 3 control and 4 Bmal1 cKO mice per group.Data represent mean ± SEM.Unpaired two-tailed Student's t-test.i Working model for a role of Bmal1 as repressor of axon regeneration; removing this brake augments axon regeneration after peripheral nerve injury.

Fig. 5 |
Fig. 5 | Bmal1 affects Tet3-5hmC induction and RAGs expression after peripheral lesion of DRG.Representative IF images of Tet3 (a) and 5hmC (b) expression in lumbar DRGs at 3 dpi after PL (ZT12-15).Violin plots of n = 225 control and 250 Bmal1 cKO (Tet3) and n = 90 (5hmC) DRG neurons from n = 9 L4-6 DRGs of 3 mice per genotype.Median is shown by black line.Mann-Whitney test.c qRT-PCR of gene expression in lumbar DRGs with no injury (left) or PL at 1 dpi (right panel).Bmal1 cKO augmented RAGs expression only after PL. n = 3 independent studies at ZT0-3, each with 6 L4-6 DRGs pooled from 2 mice.Data represent mean ± SEM.Unpaired two-tailed Student's t-test.d IF images of NPY expression in L4-L6 DRGs at 3 dpi after PL. n = ~70 neurons from n = 9 L4-6 DRGs of 3 mice per genotype.Median is shown by black line.Mann-Whitney test.e, f IF images of primary DRG neurons at 28 h after plating show that siTet3 reversed the growth advantage of Bmal1 deletion.n = 70-124 DRG neurons pooled from 2 mice per condition.Median is shown by black line.Two-way ANOVA followed by Bonferroni's multiple comparison test.g Quantification of 5hmC immunointensity in individual DRG neurons.Violin plots of n = 150-170 DRG neurons (L4-L6) from 2 mice for each condition.Black lines represent median.Two-way ANOVA followed by Bonferroni's multiple comparison test.h IF images of neurite outgrowth and quantification.Violin plots of n = 115-145 DRG neurons (L4-6) from 2 mice for each condition.Median is shown by black line.Two-way ANOVA followed by Bonferroni's multiple comparison test.i Model of Bmal1 and DNA hydroxymethylation in conditioned DRG neurons.Bmal1 deletion augments Tet3 induction, but elevated ROS inhibits Tet3 activity.NACA alleviates ROS inhibition, leading to further 5hmC gains and axon regeneration.ROS Reactive oxygen species, NACA N-acetyl-cysteine amide.

Fig. 6 |
Fig. 6 | Tet3-5hmC regulon governs axon growth and immune response after peripheral lesion of DRG. a Top, Venn diagram showing the overlap of genes associated with PL-DhMRs (n = 1705) and differentially regulated genes (DEGs) at 1-day post sciatic nerve crush injury compared to no injury (RAGs), identified by RNA-seq.Bottom, volcano plot of the 346 overlapping PL RAGs, with labeled top DEGs.Welch's two-sample t-test.b Enrichr analysis showing top enriched ontologies of the 346 RAGs identified in (a).c IPA showing the top enriched canonical pathways of the 346 RAGs.Bar color indicates predicted activation (orange), and inhibition (blue) of pathways, and gray bars indicate insufficient evidence for prediction of activation state.Right-tailed Fisher's exact test.d IPA analysis for top upstream transcriptional regulators of the 346 RAGs.Bar color indicates predicted activation (orange) or inhibition (blue) of pathways.Right-tailed Fisher's exact test.e Representative IF images and quantifications show higher membrane association of β-catenin in axotomized lumbar Bmal1 cKO DRG neurons than in controls (TUJ1 + ) at

Fig. 7 |
Fig. 7 | Bmal1 regulon concerns stress responses and energy homeostasis during axon regeneration.a Top, schematic of experimental paradigm for RNAseq analysis on primary DRG neurons isolated from Bmal1 cKO or control mice at ZT2-4 and cultured for 28 h.Bottom, principal component analysis of RNA-seq data shows segregation of samples from Bmal1 cKO vs. control DRG neurons.b Heatmap of DEGs between Bmal1 cKO and control neurons, cutoff: abs (log 2 FC) > 0.38 and P < 0.01.c Pie chart of subcellular localization of n = 625 Bmal1regulated genes computed by IPA, cutoff: abs (log 2 FC) > 0.25 and P < 0.01.d IPA showing the top enriched canonical pathways of Bmal1 regulon, with color of bars indicating predicted activation (orange) or inhibition (blue) of pathways.Righttailed Fisher's exact test.e Top, Venn diagram showing the overlap between genes associated with PL-DhMRs (n = 1705) and Bmal1 regulated genes (n = 625).Bottom, volcano plot of the 85 overlapping genes, with top DEGs labeled.f IPA highlights top canonical pathways of 85 Bmal1-regulated genes containing PL-DhMRs.Bar color indicates predicted activation (orange) or inhibition (blue).Pathway associated genes are labeled.Note, predicted activation of CREB and repression of Wnt/ β-catenin signaling with Bmal1 cKO.Right-tailed Fisher's exact test.g.Enrichr result of top pathways enriched in 36 Bmal1 regulated genes with Bmal1-motif in DhMRs.

Fig. 8 |
Fig. 8 | Diurnal expression changes of Tet3 and 5hmC in DRG neurons.a qRT-PCR reveals diurnal transcriptional changes of circadian clock genes in DRGs.Note the anti-phasic expression of Bmal1 and circadian genes of the translation/transcription feedback loops that control Bmal1 rhythmicity.n = 6 independent samples for Bmal1, Clock, Per1, Per2, Cry1 and n = 4 for Nr1d1 and Nr1d2, each sample pooled from n = 2 mice.Samples were collected on consecutive days at the indicated ZT.Data represent mean ± SEM.Unpaired two-tailed Student's t-test.b Representative IF images and quantifications show higher nuclear levels of Tet3 in uninjured DRG neurons at ZT12-15 than ZT0-3.Note the time-of-day effect persisted on Tet3 upregulation after PL, with higher level at 1 dpi when injury occurred at ZT12-15.n = 9 DRGs isolated from 3 mice per condition.Data represent mean ± SEM.Two-way ANOVA followed by Bonferroni's multiple comparison test.c qRT-PCR shows diurnal changes of Tet3 transcription in uninjured DRGs, higher at ZT12-15 than ZT0-3.PL resulted in a robust Tet3 induction for ZT0-3, and no further gain for ZT12-15 (which has a high baseline level of Tet3).n = 4 independent samples from 4 mice for each condition.Data represent mean ± SEM, Two-way ANOVA followed by Bonferroni's multiple comparison test.d Representative IF images and quantifications show diurnal changes of 5hmC in uninjured or axotomized DRG neurons (TUJ1 + ).Note more pronounced 5hmC elevation for PL at ZT12-15 than ZT0-3.n = 9 DRGs (L4-L6) isolated from 3 mice per condition.Median is shown by black line.Two-way ANOVA followed by Bonferroni's multiple comparison test.e.Schematic model of epigenetic rhythm of Tet3-5hmC in DRG neurons.

Fig. 9 |
Fig. 9 | Time-of-day effect on axon regeneration after conditioning lesion.a Experimental scheme of neurite outgrowth assay with primary DRG neurons isolated at ZT0-3 or ZT12-15.b Representative IF images and quantification show DRG neurons from ZT12-15 extended longer neurites than those from ZT0-3 at 22 h but not 12 h in vitro.Violin plots of n = 77 neurons, pooled from 3 independent cultures isolated from 3 mice per group.Median shown by black line.Kruskal-Wallis with Dunn's multiple comparison's test.c Quantifications show time-of-day effect on neurite outgrowth from DRG neurons isolated from both male and female mice.n = 250 neurons from 3 mice per group.Median shown by black line.Two-way ANOVA followed by Bonferroni's multiple comparison test.d Representative IF images and quantifications show faster axonal regeneration (SCG10 + ) at 1 d after sciatic nerve crush injury at ZT12-15 compared to ZT0-3.For regeneration index, Two-way ANOVA.n = 6 mice per group.For maximal axon length, data represent mean ± SEM.Unpaired two-tailed Student's t-test.

Fig. 10 |
Fig. 10 | Time-of-day impact on conditioning lesion effect for axonal regrowth.a Experimental scheme to evaluate time-of-day effect on the conditioning effect from PL. Mice were subjected to sciatic nerve lesion (PL) at ZT12-15 or ZT0-3, and DRGs were dissociated at 1 dpi for neurite outgrowth assay.b qRT-PCR shows that Bmal1 downregulation at 1 dpi was more pronounced when the sciatic nerve injury occurred at ZT12-15 than ZT0-3 (uninjured DRG data were also shown in Fig. 8a).Each sample pooled from 3 ipsilateral lumbar DRGs from n = 4 mice per group.Data represent mean ± SEM.Two-way ANOVA followed by Bonferroni's multiple comparison test.c Representative IF images and quantification of neurite outgrowth of DRG neurons 24 h after plating.A conditioning lesion was performed 1 day prior at either ZT12-15 or ZT0-3.Quantification of 149 neurons collected from n = 4 mice per group.Data represent mean ± SEM.Two-way ANOVA followed by Bonferroni's multiple comparison test.d Quantification of time-of day effect on neurite outgrowth from Bmal1 cKO or control DRG neurons.n = 115-135 DRG neurons for ZT0-3 and n = ~90 for ZT12-15 per group (ZT0-3 for control mice were shown in Fig. 5h).Data represent mean ± SEM.Two-way ANOVA followed by Bonferroni's multiple comparison test.Quantifications of in vivo axon regrowth show that Bmal1 cKO resulted in enhanced axon regeneration (SCG10 + ) at 3 dpi for ZT0-3 (e) and ZT12-15 (f) injury groups.Two-way ANOVA.Bar graphs of maximal axon length are shown on right.Data represent mean ± SEM.Unpaired two-tailed Student's t test.n = 5 mice per genotype.g Working model of how Bmal1 ablation mimics the conditioning lesion in lifting the circadian block, leading to enhanced axon growth capacity, in part through augmented Tet3-5hmC reconfiguration.