Identification of the genetic pathways that give rise to the chicken rod-free zone and human fovea using epigenomic profiling

使用表观基因组分析鉴定产生无鸡棒区和人类中央凹的遗传途径

• 批准号: 9248366
• 负责人: Brian P Hafler
• 金额: $ 27.69万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9248366
• 关键词: ATAC-seq; Age; Age related macular degeneration; Anatomy; Area; Binding; Binding Sites; Biological Assay; Biological Models; CRISPR/Cas technology; Candidate Disease Gene; Cell Count; Cells; Chickens; Chromatin; Clinical; Cone; Conserved Sequence; Data; Development; Electrophoretic Mobility Shift Assay; Electroporation; Embryo; Environment; Future; Gene Expression; Generations; Genes; Genetic; Goals; Human; Human Development; Immunohistochemistry; In Situ Hybridization; Inherited; Institutes; Investigation; Journals; K-Series Research Career Programs; Knowledge; Laboratories; Lead; Macular degeneration; Medical; Mentors; Methods; Molecular; Ophthalmology; Optic vesicle; Pathway Analysis; Pathway interactions; Patients; Pattern; Peripheral; Photoreceptors; Phylogenetic Analysis; Process; Quality of life; Regulation; Replacement Therapy; Reporter; Reporter Genes; Research; Research Personnel; Research Proposals; Retina; Retinal; Retinal Cone; Retinal Diseases; Scientist; Site; Spottings; Structure; Testing; Thyroid Hormones; Tissues; Training; Training Programs; Transcript; Transcription Initiation Site; Transposase; Vision; Visual Acuity; Work; base; candidate identification; career; career development; design; epigenomics; experience; fovea centralis; improved; in vivo; insight; interest; macular dystrophy; medical schools; new technology; professor; programs; public health relevance; retinal progenitor cell; retinal rods; skills; transcription factor; transcriptome; transcriptome sequencing

 DESCRIPTION (provided by applicant): This mentored clinical scientist research career development award is a training program designed to help the candidate achieve his long-term career goal to have an academic career with a focus on the retina. For the current training and future career, Brian Hafler intends to perform research focused on understanding the molecular mechanisms underlying development of the human fovea and translational applications to retinal disease. The fovea is responsible for high acuity central vision, yet the genes that establish this region are unknown. This research proposal will focus on identifying the molecular pathways that generate this region using the chicken as a model system, as it has a central rod-free spot, much like the human fovea. In this research proposal, Dr. Hafler will use new technologies like ATAC-seq to perform open chromatin profiling of cells in the developing chicken rod-free zone. ATAC-seq data will be compared with RNA-seq data, which is in the process of being generated in the Cepko lab to prioritize candidate genes of interest. Immunohistochemistry and in situ hybridization will be performed to assess whether the transcripts associated with regions of open chromatin specifically localize to the rod-free central zone in the chicken embryonic retina. Candidate cis- regulatory modules located in regions of open chromatin will be aligned and assessed for conservation with species that contain a fovea. The most promising sequences will be cloned into reporter constructs and tested for activity in the developing chicken retina using electroporation assays. Transcription factors that bind to the cis-regulatory modules will be identified using electrophoretic mobility shift assays. Immunohistochemistry and in situ hybridization on embryonic human retinas will be used to examine if the transcription factors of interest are conserved during human retinal development. Completion of this work would not only give insight into how the fovea is generated during human development, it will also help with the development of cell replacement therapies for patients with age-related macular degeneration. The candidate has an excellent mentor, Dr. Constance Cepko, who is a leader in the field and is committed to the scientific development and execution of the project. She is the Bullard Professor of Genetics and Ophthalmology at Harvard Medical School and a Howard Hughes Medical Institute Investigator. The environment at Harvard Medical School has a strong and established research program related to retinal development. They will provide necessary facilities for career enhancement to become an independent investigator. There are numerous courses offered, seminars, journal clubs, and presentations with opportunities for intellectual interactions with other research scientists. The experience, knowledge, and skills gained through the research plan and career development activities will carry the candidate forward towards a career as an independent clinician-scientist.

 描述（由申请人提供）：该受指导的临床科学家研究职业发展奖是一项培训计划，旨在帮助候选人实现其长期职业目标，即在当前的培训和未来的职业生涯中从事以视网膜为重点的学术职业。 Brian Hafler 打算进行研究，重点是了解人类中央凹发育的分子机制以及视网膜疾病的转化应用中央凹负责高敏锐度中央视力，但建立该区域的基因尚不清楚。该提案将重点关注使用鸡作为模型系统来确定生成该区域的分子途径，因为它有一个无杆中心点，很像人类的中央凹。在这项研究提案中，Hafler 博士将使用 ATAC 等新技术。 -seq 对正在发育的鸡无杆区细胞进行开放染色质分析，ATAC-seq 数据将与 Cepko 实验室正在生成的 RNA-seq 数据进行比较，以优先考虑感兴趣的候选基因。将进行免疫组织化学和原位杂交来评估与开放染色质区域相关的转录物是否特异性定位于无杆中央 位于鸡胚胎视网膜开放染色质区域的候选顺式调控模块将与含有中央凹的物种进行比对和保守性评估，最有希望的序列将被克隆到报告构建体中并测试发育中鸡的活性。使用电穿孔测定的视网膜，将使用电泳迁移率变化测定和原位杂交人视网膜来鉴定结合顺式调节模块的转录因子。用于检查感兴趣的转录因子在人类视网膜发育过程中是否保守。完成这项工作不仅可以深入了解人类发育过程中中央凹是如何产生的，还有助于开发针对患有视网膜病变的患者的细胞替代疗法。该候选人有一位出色的导师 Constance Cepko 博士，她是该领域的领导者，致力于该项目的科学开发和执行，她是哈佛大学遗传学和眼科教授。医学院和霍华德休斯医学研究所研究员 哈佛医学院拥有与视网膜发育相关的强大且成熟的研究项目，他们将为成为一名独立研究者提供必要的设施。通过研究计划和职业发展活动获得的经验、知识和技能将推动候选人走向独立临床医生科学家的职业生涯。