Genome Wide Search for Transcriptional Motifs in Central Nervous System Regeneration

Mentor 1

Dr. Ava J Udvadia

Location

Union Wisconsin Room

Start Date

28-4-2017 1:30 PM

End Date

28-4-2017 4:00 PM

Description

Adult mammals are unable to repair spinal cord, brain, and other CNS tissues. Although mature mammals cannot undergo regeneration of their CNS, some lower vertebrates can regenerate as adults. The differential response to CNS injury is due in part to the induction of regeneration-associated genes in lower vertebrates that remain inactive in mature mammals. In order to understand how regeneration-associated genes are regulated during successful CNS regeneration, we are studying gene regulatory networks that are activated after optic nerve injury in the model organism, zebrafish. Specifically, we are using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to identify regeneration-associated gene promoters and enhancers. This technique uses transposase to introduce PCR primers into regions of accessible chromatin to find potentially "active" promoter and enhancer binding sites. In order to perform this technique, we had to first optimize protocols for isolating regenerating retinal ganglion cells from GFP-expressing transgenic zebrafish retinas at different stages post optic nerve injury. My research focused on optimizing retinal tissue dissociation and isolation of cells by fluorescence activated cell sorting to enable this approach. As a result of this work we determined the number of zebrafish retinas required and the specific conditions for enzymatic and mechanical tissues disruption that has resulted in the successful construction of ATAC-seq libraries at 0-, 4-, and 7-days post injury. We anticipate that sequencing and bioinformatics analyses of these libraries, in conjunction with previously obtained RNA-seq data, will yield important insight into the critical regulatory factors enabling activation of regeneration-associated genes.

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Apr 28th, 1:30 PM Apr 28th, 4:00 PM

Genome Wide Search for Transcriptional Motifs in Central Nervous System Regeneration

Union Wisconsin Room

Adult mammals are unable to repair spinal cord, brain, and other CNS tissues. Although mature mammals cannot undergo regeneration of their CNS, some lower vertebrates can regenerate as adults. The differential response to CNS injury is due in part to the induction of regeneration-associated genes in lower vertebrates that remain inactive in mature mammals. In order to understand how regeneration-associated genes are regulated during successful CNS regeneration, we are studying gene regulatory networks that are activated after optic nerve injury in the model organism, zebrafish. Specifically, we are using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to identify regeneration-associated gene promoters and enhancers. This technique uses transposase to introduce PCR primers into regions of accessible chromatin to find potentially "active" promoter and enhancer binding sites. In order to perform this technique, we had to first optimize protocols for isolating regenerating retinal ganglion cells from GFP-expressing transgenic zebrafish retinas at different stages post optic nerve injury. My research focused on optimizing retinal tissue dissociation and isolation of cells by fluorescence activated cell sorting to enable this approach. As a result of this work we determined the number of zebrafish retinas required and the specific conditions for enzymatic and mechanical tissues disruption that has resulted in the successful construction of ATAC-seq libraries at 0-, 4-, and 7-days post injury. We anticipate that sequencing and bioinformatics analyses of these libraries, in conjunction with previously obtained RNA-seq data, will yield important insight into the critical regulatory factors enabling activation of regeneration-associated genes.