Circadian clock regulator Bmal1 gates axon regeneration via Tet3-5hmC mediated epigenetics
Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves epigenetic reconfigurations that rewire gene regulatory circuits to establish regenerative gene program. However, the mechanisms and transcriptional regulators remain poorly understood. Here, we conducted an unbiased survey of DNA differentially hydroxymethylated regions (DhMRs) in DRG after peripheral lesion, which identified enriched binding motif for Bmal1, a transcription factor and a central regulator of the circadian clock. Through applying conditional deletion of Bmal1, in vitro and in vivo models of axon regrowth, and transcriptomic profiling, we showed that Bmal1 inhibits axon regeneration in part through Tet3-dependent manner. Mechanistically, Bmal1 functions as a gatekeeper of neuroepigenetic injury responses by limiting Tet3 expression and restricting 5hmC modifications. Notably, Bmal1-regulated genes after axotomy not only concern axon guidance and axon regrowth, but also stress responses, energy homeostasis, and neuroinflammation. Furthermore, we uncovered diurnal oscillation of Tet3 and 5hmC in DRG neurons, and this epigenetic rhythm corresponded to time-of-day effect on axon growth potential. Collectively, our studies showed that Bmal1 deletion mimics the conditioning lesion in lifting epigenetic barriers to enhance axon regeneration. Overall design: For conditional deletion of Bmal1, 19 weeks-old Bmal1 cKO (Bmal1f/f Thy1-CRE-ERT2/EYFP) or control (Bmal1f/f or Bmal1 f/+) mice were injected with Tamoxifen at a dose of 100 mg/kg by intraperitoneal injection once a day for 5 consecutive days. Thoracic and lumbar DRGs were isolated 5 weeks post-tamoxifen treatment ZT2-4 with each mouse representing 1 sample.   Enriched DRG neuronal cultures were prepared and seeded on PLO/Laminin coated plates at ZT8.   RNA was collected at 28 hr post-seeding using QIAGEN RNeasy plus mini kit.  Next-generation sequencing was conducted on the Illumina NovaSeq platform.