Determinants of Context Specific Transcription Factor Function

上下文特异性转录因子功能的决定因素

• 批准号: 10462505
• 负责人: Lauren Jessica Hodkinson
• 金额: $ 4.68万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462505
• 关键词: Address; Affinity; Bar Codes; Binding; Binding Proteins; Biological Models; Cell Cycle; Cell Nucleus; Cessation of life; Chromatin; Chromosomes; Crowding; DNA; DNA Sequence; DNA-Binding Proteins; Deposition; Development; Disease; Dosage Compensation (Genetics); Drosophila genus; Drosophila melanogaster; Electrophoretic Mobility Shift Assay; Elements; Embryo; Engineering; Environment; Failure; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Models; Genetic Transcription; Genome; Genomics; Goals; Growth; Histones; Human; Human Genome; In Vitro; Length; Link; Location; Measures; Modeling; Mutation; Nucleic Acid Regulatory Sequences; Nucleotides; Output; Process; Proteins; Quantitative Reverse Transcriptase PCR; Role; Site; Structure; System; Techniques; Testing; Transgenic Organisms; X Chromosome; dosage; experimental study; fly; genome-wide; in vivo; male; promoter; protein function; recruit; tool; transcription factor; zygote

Project Summary: The developing embryo relies on gene expression to be orchestrated on strict temporal and quantitative levels. Without proper coordination, the zygote suffers severe consequences such as growth issues and even embryo termination. Early embryonic gene regulation is carried out by maternally deposited transcription factors which are able to find their cognate cis sequences and activate the zygotic genome with tremendous efficacy despite the crowded environment of the nucleus. Once correctly targeted, transcription factors can perform context- specific functions across the genome even while sharing similar cis element sequence structure. I aim to define the relationship between transcription factors and their cis sequences by asking what are the contributions of DNA sequence and genomic context to transcription factor function in the early embryo? I will use the excellent genetic model system Drosophila melanogaster to investigate context-specific functions of the maternally deposited transcription factor Chromatin-Linked Adapter for MSL Proteins (CLAMP). CLAMP targets GA-rich cis elements in the embryonic genome, but performs several distinct functions depending on genomic context. For example, CLAMP primes the male X-chromosome for dosage compensation, regulates promoters genome-wide, and promotes formation of the conserved histone locus body (HLB), which regulates developmental and cell cycle expression of the replication-dependent histone genes. CLAMP targeting occurs in the embryo and when CLAMP is depleted, embryos terminate before the zygotic genome is activated. Although CLAMP targets similar cis elements in all three contexts, it recruits very different locus-specific transcription factors. I hypothesize that CLAMP inputs both DNA sequence information and genomic context to perform its context-specific functions. In Aim 1, I will engineer fly lines carrying a transgenic histone locus in which I have replaced the natural cis elements with CLAMP-recruiting GA-rich elements from different genomic origins. I will assess how these cis elements impact early embryo HLB formation, CLAMP binding, and histone gene transcription to determine how similar cis elements from different origins impact CLAMP function. In Aim 2, I will perform (A) in vitro gel-shift assays and (B) in vivo experiments, in which I vary the length of the CLAMP-recruiting cis elements and determine binding affinity and transcriptional output. In this aim, I will determine the contribution of cis element affinity to transcription factor function. Overall, the proposed aims will define how cis element DNA sequence and genomic context influence context-specific transcription factor function in the early embryo.

项目总结： 发育中的胚胎依赖于基因表达在严格的时间和数量水平上进行协调。 如果没有适当的协调，受精卵会遭受严重的后果，如生长问题，甚至胚胎。 终止。早期胚胎的基因调控是由母体存放的转录因子进行的 能够找到它们的同源顺式序列并以巨大的效率激活合子基因组 原子核的拥挤环境。一旦被正确定位，转录因子就可以执行上下文- 即使在拥有相似的顺式元件序列结构的情况下，也可以在基因组中实现特定的功能。我的目标是定义 转录因子与其顺式序列之间的关系 DNA序列和基因组背景对早期胚胎中转录因子功能的影响？我将使用 优秀的遗传模型系统黑腹果蝇研究特定背景下的功能 母系转录因子染色质连锁MSL蛋白接头(CLAP)。夹具 靶向胚胎基因组中富含GA的顺式元件，但执行几种不同的功能取决于 基因组背景。例如，夹具启动男性X染色体以进行剂量补偿，调节 全基因组范围内的启动子，并促进保守的组蛋白基因座体(HLb)的形成，HLb调节 依赖复制的组蛋白基因的发育和细胞周期表达。发生夹具定位 在胚胎中，当夹子被耗尽时，胚胎在合子基因组被激活之前终止。虽然 CLAMP在所有三种环境中都以相似的顺式元件为靶标，它招募了非常不同的位点特异性转录 各种因素。我假设CLAMP既输入DNA序列信息又输入基因组环境来执行其 特定于上下文的功能。在目标1中，我将设计携带转基因组蛋白基因座的飞行线路，我在其中有 用来自不同基因组来源的富含GA的夹子来取代天然的顺式元件。这就做 评估这些顺式元件如何影响早期胚胎HLB的形成、钳夹结合和组蛋白基因 转录以确定来自不同来源的相似的顺式元件如何影响钳制功能。在《目标2》中，我会 进行(A)体外凝胶移位分析和(B)体内实验，其中我改变钳子招募的长度 顺式元件，并确定结合亲和力和转录输出。在这个目标中，我将确定贡献 顺式元件对转录因子功能的亲和力。总体而言，拟议的目标将定义顺式DNA元素如何 序列和基因组背景影响早期胚胎中特定于背景的转录因子的功能。