Date of Completion
Feliks (Ephraim) Trakhtenberg, Royce Mohan, Paola Bargagna-Mohan
Field of Study
Master of Science
Retinal ganglion cells (RGCs) are projection neurons of the eye, which process and pass visual information collected in the eyes to the brain. However, epigenetic regulation of RGC fate specification remains poorly understood, in large part due to the technical challenges associated with purifying RGCs, which comprise only 1% of all retinal cells, and performing ChIP-seq profiling on a small number of cells. To overcome these limitations, we have purified RGCs from multiple mice by immunoppanning for Thy1, a surface marker on RGCs, and analyzed pulled chromatin by ChIP-seq for histone 3 acetylated at K27 (H3K27ac), which is an epigenetic marker of active (i.e. accessible) enhancers. We also utilized recently developed ChIP-seq Kit for processing small cell number samples and ultra low chromatin input (truChIP Ultra Low Cell Chromatin Shearing Kit, Covaris; Zymo-Spin ChIP Kit, Zymo Research; and Accel-NGS 2S Plus DNA Library Kit, Swift Biosciences). Immunoprecipitation chromatin was sequenced and mapped to mouse reference genome (mm10), peaks were called using MACS2 (sheared RGC chromatin was used for background control), and peak annotation was performed using the HOMER software. We have identified enhancers that are active in RGCs and cross-referenced them to the mouse cell types, and some were novel and unique to RGCs. Next, using HOMER software, we performed DNA motif analysis in the identified enhancer regions and predicted the transcription factors to those we have identified as expressed in RGCs by mRNA-seq. These analyses enabled us to associate the transcription factors that are expressed in RGCs with the enhancers that are active in RGCs, and also to predict which of these transcription factors could bind to the same enhancers, and thus may cooperate with each other in regulating gene expression in RGCs. Taken together, our epigenetic profiling of RGCs predicted active enhancers, which are potentially unique to RGCs, as well as predicted the interactions of transcription factors and enhancers, which are involved in determining RGC-specific gene program.
Xing, Jian, "Epigenetic Profiling of Active Enhancers in Mouse Retinal Ganglion Cells" (2018). Master's Theses. 1268.
Feliks (Ephraim) Trakhtenberg