Regulation of 3D genome organisation and function in axonal regeneration

Injury in the central nervous system (CNS) such as spinal cord injury (SCI) results in severe neurological functional impairment and poor recovery due to failure in regeneration and re-connectivity of injured axons. To date, it is still unclear why nerves in the brain or spinal cord cannot regrow after injury. While it is known that many genes that could support the growth of nerves cannot be activated after injury, the reasons for this failure remain obscure. Recent accumulating evidence by others and us suggests that changes in the accessibility of genes that control the regeneration programme may facilitate repair and regeneration after spinal injury for example.
Understanding how the regeneration programme is regulated could be very important to improve regeneration of nerves and recovery of function in people affected by nerve and spinal injuries.
The regulation of gene expression is affected by the 3D structure of the chromatin that contains our genetic information. Dynamic 3D changes in chromatin are essential for proper embryonic development for example and have been recently implicated in cancer.
This proposal will investigate for the first time how chromatin state is affected by nerve injury and how this affects regenerative gene expression in models of nerve and spinal injury in mice. We will not only try to identify important mechanisms but we will also attempt to reshape chromatin structure to promote nerve regeneration.
Our studies will use sciatic and spinal nerve injury models in the mouse combined with state of the molecular techniques and bioinformatics analysis that will allow to clarify how gene expression and chromatin structure change after injury
In summary, our studies will provide a novel understanding of chromatin-dependent regulation of nerve regeneration and will also propose novel avenues to foster repair after nerve and spinal injury.

Following injury, peripheral nervous system axons regenerate while axons in the central nervous system fail to do so. The dorsal root ganglia (DRG) bipolar neurons allow investigating the molecular mechanisms of this differential regenerative ability: a single cell body extends one regenerative competent axon in the peripheral nerves and one regenerative incompetent branch into the spinal cord. We recently performed genome-wide gene expression and epigenetic studies (ATAC and ChIPSeq for histone PTMs) in the mouse DRG after peripheral nerve injury (SNA) vs central spinal dorsal column axotomy (DCA). These revealed that chromatin accessibility is modified via histone acetylation affecting the transcription programme facilitating regeneration following SNA but not after DCA. We also discovered enrichment in CCCTC binding factor (CTCF)-dependent transcriptional binding sites at promoters of regenerative genes in proximity of more accessible chromatin and that conditional neuronal deletion of CTCF leads to impaired axonal regeneration after sciatic nerve injury. Since CTCF plays an important role in 3D genome organisation, here we hypothesize that CTCF-mediated genome organization contributes to the injury-induced transcriptional programme and to axonal regeneration. We will investigate (i) CTCF-dependent and independent mechanisms of chromatin and transcriptional regulation following axonal injury by Circular Chromosome Conformation Capture sequencing (4Cseq), RNAseq and CTCF ChIPseq from DRG after spinal or sciatic injury (DCA vs SNA) comparing control versus CTCF-deleted DRG neurons; (ii) CTCF post-translational modifications following sciatic versus spinal injury that affect CTCF activity and DRG regenerative growth; (iii) the ability to enhance axonal regeneration following injury, by altering 3D genome conformation, via CRISPR-cas9 genome editing. In summary, these studies will provide a novel understanding of chromatin-dependent regulation of axonal regeneration.

Impact Summary

The focus of this project is to provide a novel understanding of chromatin-dependent regulation of nerve regeneration and to propose novel avenues to foster repair after nerve and spinal injury. Although it is difficult to anticipate immediate economic and societal benefits from this research, the results of this project should have an impact on the following groups;

Academics: As outlined in the academic beneficiaries section the proposed research will add to the still incomplete body of knowledge on the regenerative mechanisms of regenerative failure after nerve spinal cord injury (SCI) and will offer novel insight into the regulation of 3D chromatin changes in disease states. Provided that we identify key regulatory mechanisms that control chromatin contacts for axonal regeneration, this will prompt further research to build on this knowledge base eventually leading to clinical studies in patients. By disseminating our research findings through publications and attendance at conferences this should lead to further research and collaborations which may set the groundwork for novel avenues of nerve regeneration treatment in SCI patients; ultimately impacting the groups below.

Individuals with nerve and spinal cord injury: The prevalence for SCI is estimated to be approximately of 250,000 in the US and of 50,000 in the UK alone, affecting mainly young adults. To date, there is no effective treatment to improve disability in spinal cord patients, who rely only on long-term, time consuming and physically as well as psychologically demanding rehabilitation plans. Peripheral nerve injury affects millions of people worldwide leading to chronic disability without a cure. It is hoped that the investigation into how chromatin state is affected by nerve injury and how this affects regenerative gene expression in models of nerve and spinal injury in mice may eventually lead to novel avenues of nerve regeneration treatment in SCI patients. If successful this would improve patient quality of life and lead to wider societal and economic benefits. To ensure that these patients and their carers have the opportunity to benefit from this project, engagement with these groups and communication of our results is vital. This will be discussed further in the pathways to impact.

NHS and 3rd sector organisations: The health costs to the UK in limiting the disability in SCI and nerve injury patients is clearly substantial. The cost of loss of employment, chronic care facilities and treatment of chronic disease associated problems is a serious burden on the health system in general. Indeed, although we do not envisage that a therapy derived from our research will be available in the clinic within the time span of this research programme, we are hopeful that it will contribute to set the groundwork for targeted clinical trials in the near future which could lead to a reduction in the economic burden of this condition and a change in the treatment guidelines. This work might also inspire donations to support research in spinal cord and nerve regeneration research including via charities such as Wings for Life (WFL) and International Spinal Research Trust (ISRT).

Industry: While this research programme will not immediately attract industrial partnership, it does have the potential for industrial exploitation via the design of novel compounds to modify chromatin architecture for regenerative gene reprogramming. This will be discussed further in the pathways to impact.

The general public: This project should be of interest to the lay public and through various engagement activities that will be outlined in the pathways to impact, we will increase the public awareness and understanding of the proposed research. This should not only increase their knowledge of SCI research but could also provide essential feedback to shape the current project and future SCI research directions, thereby enhancing its associated impact.