Determinants of Context Specific Transcription Factor Function

项目摘要： 发育中的胚胎依赖于严格的时间和数量水平上的基因表达。 没有适当的协调，受精卵遭受严重的后果，如生长问题，甚至胚胎 终止早期胚胎基因调控是由母体沉积的转录因子进行的， 能够找到它们的同源顺式序列，并以巨大的功效激活合子基因组， 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- 在整个基因组中的特定功能，即使共享相似的顺式元件序列结构。我的目标是定义 转录因子和它们的顺式序列之间的关系，通过询问 DNA序列和基因组背景对早期胚胎转录因子功能的影响？我将用 一个优秀的遗传模型系统果蝇，以研究特定的功能， 母体沉积转录因子MSL蛋白质的染色质连接衔接子（CLAMP）。夹具 靶向胚胎基因组中富含GA的顺式元件，但取决于 基因组背景例如，CLAMP启动男性X染色体以进行剂量补偿，调节 启动子全基因组，并促进保守的组蛋白基因座体（HLB）的形成， 复制依赖性组蛋白基因的发育和细胞周期表达。CLAMP定位发生 在胚胎中，当CLAMP耗尽时，胚胎在合子基因组被激活之前终止。虽然 CLAMP在所有三种情况下都靶向相似的顺式元件，但它招募非常不同的基因座特异性转录 因素我假设CLAMP输入DNA序列信息和基因组背景来执行其功能。 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果蝇品系， 用来自不同基因组来源的CLAMP募集GA富集元件替换天然顺式元件。我会 评估这些顺式元件如何影响早期胚胎HLB形成、CLAMP结合和组蛋白基因 转录以确定来自不同来源的相似顺式元件如何影响CLAMP功能。在目标2中，我将 进行（A）体外凝胶位移测定和（B）体内实验，其中改变CLAMP募集的长度， 顺式元件并决定结合亲和力和转录输出。在这个目标下，我将决定 顺式元件对转录因子功能的亲和力。总的来说，提出的目标将定义顺式元件DNA如何 序列和基因组环境影响早期胚胎中的环境特异性转录因子功能。

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.