Atoh7 cis regulation and gene regulatory network analysis during retinal ganglion cell development

视网膜神经节细胞发育过程中Atoh7顺式调控及基因调控网络分析

• 批准号: 10662505
• 负责人: Joel B Miesfeld
• 金额: $ 24.9万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662505
• 关键词: ATAC-seq; ATOH7 gene; Adult; Alleles; Architecture; BHLH Protein; Bacterial Artificial Chromosomes; Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Models; Blindness; Brain; CRISPR screen; California; Cell Differentiation process; Cell Transplantation; Cell division; Cells; Chromatin; Chromatin Structure; Community Developments; Competence; Complex; DNA; DNA Sequence; Data; Data Analyses; Development; Disease; Elements; Enhancers; Environment; Eye; Flow Cytometry; Generations; Genetic Enhancer Element; Genetic Models; Genetic Transcription; Glaucoma; Goals; Human; Image; In Vitro; Individual; KDM1A gene; Laboratory mice; Learning; Lysine; Mentors; Messenger RNA; Methods; Modeling; Muller' s cell; Multipotent Stem Cells; Mus; Neuroglia; Neurons; Nucleic Acid Regulatory Sequences; Optic Nerve; Organoids; Pathogenicity; Pathway Analysis; Patients; Pattern; Population; Regulation; Regulatory Element; Reporter; Reporter Genes; Repression; Research; Research Personnel; Retina; Retinal Ganglion Cells; Role; Scientist; Solid; Stereotyping; Structure; System; Techniques; Testing; Therapeutic; Time; Tissues; Tracer; Transgenes; Transgenic Mice; Transgenic Organisms; Universities; Untranslated RNA; Vision; Work; Xenopus; Zebrafish; autosome; career development; cell fate specification; cell type; chromatin immunoprecipitation; cofactor; competence factor; gene regulatory network; gene repression; genetic corepressor; histogenesis; improved; induced pluripotent stem cell; malformation; mutant; neural; neurogenesis; notch protein; postmitotic; prevent; progenitor; promoter; research and development; retinal progenitor cell; stem cells; success; tool; transcription factor; transcriptome; transcriptome sequencing

Project Summary/Abstract Retinal ganglion cells (RGCs) connect the eyes to the brain. They are essential for vertebrate vision and pathogenic targets in glaucoma. One therapeutic goal of vision scientists is to fully understand the factors required for RGC development, so these cells can be generated in vitro. The proneural basic helix-loop-helix (bHLH) protein ATOH7 is expressed transiently in a subpopulation of early retinal progenitor cells, which give rise to the 7 major cell types of the retina but is only essential as a competence factor for RGC genesis. Loss of ATOH7 causes optic nerve aplasia and severe secondary retinovascular malformations. Cre-lox lineage data show only 55% of RGCs descend from Atoh7+ progenitors. What factors control genesis of the other 45% of RGCs? Why do only some Atoh7+ cells become RGCs? In humans with nonsyndromic congenital retinal nonattachment (NCRNA), a remote 5’ conserved enhancer for ATOH7 is deleted, preventing development of RGCs and leading to total blindness. This DNA segment is obviously vital, but its exact role is unknown. In transgene reporter mice, this ‘shadow’ enhancer (SE) appears to be wholly redundant with the ‘primary’ (promoter-adjacent) enhancer (PE), despite is requirement in human NCRNA. In preliminary studies, we observed that Atoh7 SE deletion mice retain optic nerves. How do these dual enhancer elements coordinately regulate the rapid onset and offset of Atoh7 expression? Here, we propose to investigate functional differences between the human NCRNA and mouse SE deletion, to determine how specific DNA sequences control the level, timing and pattern of ATOH7 expression, to analyze ATOH7 transcriptional repression, and to identify cofactors influencing ATOH7+ cell fate decisions during RGC genesis. First, we will apply a multi-species approach to test the necessity and sufficiency of each ATOH7 regulatory element and determine precisely how each component contributes to the dynamic tissue and cellular expression pattern. Second, we will investigate mechanisms of ATOH7 transcriptional repression via Notch effector RPBJ and Kdm1a, using a high-throughput zebrafish screen, transgenic reporters and RNAseq. Third, we will use single-cell and pooled ATACseq and RNAseq methods to profile retinal progenitors in detail as they progress through stages of Atoh7 expression. These data will illuminate mechanisms controlling ATOH7 transcription, the onset of retinal neurogenesis and RGC fate specification; the action of binary enhancers generally; and the potential generation of RGCs in vitro for cell transplantation. My work toward these goals will be aided by the strong research and career development community at the University of California, Davis and my established team of mentors. Together, the proposed research and environment will provide a solid platform for my continued career development as a vision scientist – learning new techniques and model systems, and interacting with a wide variety of scientists (short term goals), which will pave the way for me to become an independent academic researcher probing gene regulatory networks that control ATOH7, RGC fate and retinal histogenesis (long term goals).

项目总结/文摘