Synthetic HoxA to dissect transcriptional regulatory logic - TRFR

解析转录调控逻辑的合成 HoxA - TRFR

• 批准号: 10891949
• 负责人: Esteban Orlando Mazzoni
• 金额: $ 62.52万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10891949
• 关键词: 20 year old; Achievement; Address; Alleles; Animals; Anterior; Behavior; Bilateral; Binding; Binding Sites; Biological Process; Biology; Boundary Elements; CRISPR/Cas technology; Carpet; Cell Lineage; Cells; ChIP-seq; Chromatin; Code; Complement; Complex; Computers; DNA; Data; Defect; Development; Disease; Distal; Elements; Embryo; Engineering; Epigenetic Process; Gene Cluster; Gene Expression; Gene Expression Regulation; Gene Structure; Genes; Genetic; Genetic Transcription; Genome; Genomic approach; Genomics; Homeobox Genes; Human; In Vitro; Knock-out; Light; Location; Logic; Methods; Modeling; Motor Neurons; Mus; Mutate; Neighborhoods; Neuronal Differentiation; Output; Pattern; Phenotype; Rattus; Regulation; Regulatory Element; Rodent; Signal Transduction; Site; System; TFRC gene; Technology; Testing; Transcriptional Regulation; Variant; Work; Writing; Yeasts; cost; design; embryonic stem cell; experimental study; genetic variant; genomic locus; in vivo; mutant; novel; synthetic construct; tool; transcription factor; tumorigenesis

Project Summary The “big picture” of this proposal is that using our newfound ability to build big synthetic DNAs (1 Mb scale) at will and with great efficiency and precision, we propose a new paradigm for “systems genetics” of gene regulation in the context of a unique master developmental regulator, the HoxA cluster. We use yeast to make hundreds of mammalian gene loci variants rapidly, with high precision and cos-effective. Leveraging this technology, we have already assembled and deliver 134kb and 170 kb long constructs containing the entire rat HoxA cluster, as well as the mouse counterparts. We can deliver precision-engineered large HoxA constructs to either an ectopic location at the Hprt1 locus, and working on delivering to the allelic location as well. Using these powerful new tools, we describe how we can deliver the rat and mouse HoxA clusters to Hprt1 in mouse ES cells. Using the heterologous rat HoxA clusters allows us to internally compare the Hox loci from both species in the same cell using a range of chromatin “omics-based” readouts. The HoxA cluster is extremely highly conserved at the DNA level; we will take advantage of the rat HoxA, since it is a rodent gene it is likely to complement function in the mouse, but is densely carpeted with genomic variants (one per 10 bp on average across HoxA). These experiments will be done in a data-rich system using in vitro differentiation of mES cells to motor neurons. These experiments can be done in the context of already generated HoxA+/+, +/–, or –/– ES cells. The aims include asking the question Do Hox clusters require their genomic context containing long- distance regulatory elements for the establishment of chromatin boundaries and initial gene expression? (Aim 1), attempting functional complementation with our ectopic synthetic loci in HoA-/- knockout background (Aim 2) and asking Is it possible to establish novel regulatory domains within Hox clusters? (Aim 3).

项目总结

项目成果

ISSCR Education Committee syllabus and learning guide for enhancing stem cell literacy.

• DOI: 10.1016/j.stemcr.2022.12.004
• 发表时间: 2023-02-14
• 期刊: STEM CELL REPORTS
• 影响因子: 5.9
• 作者: Perlin, Julie R.;Anderson, William J.;Bartfeld, Sina;Couturier, Anna;de Soysa, Yvanka;Hawley, R. Scott;Hu, Ping;Loh, Yuin-Han;Mandal, Lolitika;Master, Zubin;Muotri, Alysson R.;Piddini, Eugenia;Polo, Jose M.;Mazzoni, Esteban O.
• 通讯作者: Mazzoni, Esteban O.

Capybara: A computational tool to measure cell identity and fate transitions.

• DOI: 10.1016/j.stem.2022.03.001
• 发表时间: 2022-04-07
• 期刊: CELL STEM CELL
• 影响因子: 23.9
• 作者: Kong, Wenjun;C. Fu, Yuheng;Holloway, Emily M.;Garipler, Gorkem;Yang, Xue;Mazzoni, Esteban O.;A. Morris, Samantha
• 通讯作者: A. Morris, Samantha