Project Summary
Axon regeneration after peripheral nerve (PN) injury is often incomplete. There is currently no effective
treatment beyond surgical reconstruction, which is only beneficial for a small percentage of cases.
Understanding the repair mechanisms is thus crucial. Here, we investigate a previously unknown function of
the core circadian regulator Bmal1 in gating neuroregenerative responses after PN injury. The study is based
on our new data that neuron-specific deletion of Bmal1 accelerates axon regeneration after PN injury. This
exciting finding was made in the context of a series of advances from our laboratory in deciphering
transcriptional networks that control neuronal intrinsic axon growth potential, i.e., how transcription factors
(TFs) cooperate with epigenetic factors to reshape the chromatin landscape for induction of regeneration-
associated genes (RAGs). Our most recent work has leveraged our genome-wide mapping of DNA
hydroxymethylation (5hmC) dynamics in regenerating dorsal root ganglia (DRG) neurons. Intriguingly, we
discovered enrichment of the Bmal1 binding motifs in genomic loci displaying 5hmC changes after PN injury,
suggesting an interaction of Bmal1 with the 5mC/5hmC converting enzyme Tet3. Indeed, Bmal1 cKO in mice
showed that the Bmal1-Tet3-5hmC axis regulates genes linked to axon growth, metabolism, and immune
interactions. Moreover, pilot data show for the first-time a diurnal epigenetic rhythm of Tet3 and 5hmC in DRG
neurons that is anti-phasic to Bmal1 transcriptional oscillation and corresponds to time-of-day differences in
regenerative responses. Here, we will test the central hypothesis that Bmal1 functions as an inhibitor of axon
regeneration and a gatekeeper of injury-trigged immune activation via regulation of 5hmC reprogramming. In
Aim 1, we will characterize Bmal1-gated regenerative gene programs and examine the effect of
pharmacological inhibition of Bmal1 transcription by SR9009, a potent Rev-Erb agonist with CNS penetration
capability. Mechanistically, we will test a “two-hit model” wherein Bmal1 deletion primes DRG neurons, but an
injury signal is required for RAGs induction. In Aim 2, we will characterize Tet3/5hmC epigenetic rhythmicity,
correlation with “neurite outgrowth clock”, and the underlying mechanisms by testing the working model that
Bmal1 controls Tet3 expression as well as Tet3 recruitment to target loci for 5hmC reprogramming. We will
then map short- and long-term impact of PN injury on Bmal1-Tet3-5hmC rhythmicity and whether these
rhythms return to normal upon axonal reconnection. In Aim 3, we pivot to in vivo study of the promoting effect
of Bmal1 inhibition on nerve repair, including motosensory functions. We will also address sustainability of the
effect, sex differences, and timed Bmal1 inhibition shortly after PN injury. In sum, our proposal has the
potential of connecting Bmal1 circadian pathway, Tet3/5hmC epigenetic reprogramming, injury-triggered
immune responses, and axon regeneration, thus advancing basic science of nerve regeneration and opening
translational paths.