The overall objective of this project is to identify combinatorial sets of transcription factors that can act in central nervous system (CNS) neurons to improve their regenerative abilities. To do so we will take advantage of a well- characterized transition in nervous system development, in which maturing neurons abruptly switch from an intrinsic state that allows rapid axon growth to one in which axon growth is slow or abortive. It is clear that this transition involves changes in gene expression, and we have had partial success in improving axon growth in older neurons by supplying pro-regenerative transcription factors. Here our central hypothesis is that in addition to transcriptional changes, underlying changes in chromatin structure also act to limit regenerative ability. It is well established in other fields that during cellular maturation, regions of chromatin can adopt a closed conformation that prevents access of transcription factors to specific genomic loci. Thus similar restriction events are likely to limit the expression of regeneration-associated genes in CNS neurons, and to prevent their re- expression even when pro-growth transcription factors are supplied. A special class of transcription factors, termed pioneer factors, can ameliorate this constraint by binding to closed chromatin and re-opening it, thus allowing subsequent waves of transcription factors to drive transcription. Our objective here is to use a newly developed technique called ATACseq to create genome-wide maps of chromatin accessibility in corticospinal tract neurons as they age and lose regenerative ability, and as they respond to axon injury. This will create a detailed picture of the relationship between regenerative ability and chromatin structure. Then, by examining regions of the genome that specifically open and close across time and in response to injury, we will use a bioinformatic approach to identify transcription factors that interact with these regions, with particular attention paid to potential pioneer factors. Finally we will perform a first-pass test of combined expression of predicted pioneers with known pro-regenerative factors in two cell culture models of axon growth. Ultimately these studies will fill a critical gap in knowledge in the field regarding chromatin accessibility and regenerative ability, and will use this information to identify transcription factors that act through a novel chromatin-based mechanism to improve axon growth.
To improve outcomes after nervous system trauma such as spinal cord injury, it is critical to devise effective methods of improving the growth of axons, the long processes that carry information. One limit to axon growth is the closed conformation of DNA that prevents activation of pro-growth genes. We will study the connection between DNA structure and axon growth, and identify new transcription factors that can alter DNA structure and promote nervous system repair.
