Transcriptional Regulatory Complexes in Epidermal Differentiation

表皮分化中的转录调控复合物

• 批准号: 9223483
• 负责人: Vanessa LopezPajares
• 金额: $ 12.89万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-08 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9223483
• 关键词: ATAC-seq; Address; Affinity Chromatography; Architecture; Area; Binding Sites; Biochemistry; Bioinformatics; Biology; Biotinylation; CRISPR screen; CRISPR/Cas technology; Cell Cycle; Cells; ChIP-seq; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Computer Analysis; Coupled; DNA; Data; Development; Differentiated Gene; Disease; Elements; Enhancers; Epidermis; Epithelial; Equilibrium; Factor Analysis; Faculty; Foundations; Future; Gene Activation; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genome; Genomics; Goals; Homeostasis; Human; Knock-out; Knowledge; Lead; Leadership; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mentors; Mentorship; Molecular Biology; Molecular Profiling; PRDM1 gene; Play; Process; Proteins; Proteomics; Psoriasis; Recruitment Activity; Regulation; Regulator Genes; Regulatory Element; Research; Research Personnel; Role; Scientist; Shapes; Site; Skin; Solid; Stem cells; Testing; Time; Tissue Model; Tissues; Training; Transcriptional Regulation; Universities; Untranslated RNA; base; career; chronic wound; design; epigenome; epigenomics; gene repression; genetic regulatory protein; genome editing; genome-wide; interest; keratinocyte; keratinocyte differentiation; member; novel; overexpression; progenitor; programs; promoter; protein complex; self-renewal; skin disorder; transcription factor; treatment strategy

Transcriptional Regulatory Complexes in Epidermal Differentiation Project Summary My long-term career goal is to lead a productive academic group at a research university, advancing the field of epithelial biology, and serving as a mentor to young scientists. My scientific interests lie in understanding the complex biology behind transcriptional regulatory networks mediating epidermal homeostasis. As I build a solid research program in this area, my immediate goals are to obtain mentorship in new areas, including epigenomics, bioinformatics, and proteomics, which will greatly enhance my current expertise in genetics, biochemistry and molecular biology. In addition to the valuable expertise provided by my mentor, six additional faculty members at Stanford University with expertise in these areas have agreed to serve as collaborators for the proposed studies. Furthermore, I will acquire additional knowledge through limited didactic courses, and participation in leadership and management seminars offered at Stanford will further prepare me for my transition to independence. Altogether, these efforts will allow me to expand my current research to provide a platform for establishing a new research group and aid in my transition to independent investigator. The goal of this K01 proposal is to investigate the transcriptional regulatory complexes involved in epidermal differentiation. I have recently identified the MAF/MAFB transcription factors (TFs) as critical regulators of epidermal homeostasis mediating progenitor cell cycle exit and terminal differentiation. These TFs are themselves regulated by long non-coding RNAs and mediate downstream activation of important epidermal TFs in cooperation with p63, the master regulator of epidermis. Furthermore, our analysis revealed MAF/MAFB localized to putative enhancers at the genomic level. Due to their ability to both suppress progenitor genes and activate terminal differentiation genes, we believe MAF/MAFB exist simultaneously in activating and repressive complexes. Thus, the aims are to 1) Characterize the epigenomic effects of MAF/MAFB during epidermal differentiation and, 2) Functionally characterize the MAF/MAFB protein interactome to understand how MAF/MAFB function in activating and repressive complexes. We will begin by determining the epigenomic effects of MAF/MAFB in Aim I. The contribution of MAF/MAFB to chromatin accessibility and genome architecture will be addressed in the context of MAF/MAFB CRISPR-mediated knockout keratinocytes. We will use ATAC-seq to identify changes in chromatin accessibility comparing MAF/MAFB loss to control differentiated keratinocytes. To assess changes in genome shape, we will interrogate promoter:DNA contacts using capture Hi-C in control vs MAF/MAFB knockout keratinocytes that have undergone differentiation. These unbiased approaches will provide genome-wide data for characterizing the epigenome during epidermal differentiation, assessing the impact of MAF/MAFB loss, and identifying critical genomic regulatory elements required for differentiation. Furthermore, characterization of regulatory elements will allow for subsequent identification of additional factors, or potential co-regulators, involved in MAF/MAFB mediated gene regulation. We will first test candidate co-regulators already identified through FOCIS analysis of MAF/MAFB bound loci and then incorporate results from epigenomic studies to identify and characterize putative co-regulators. In Aim II, we will focus on functionally characterizing the MAF/MAFB protein interactome. Because of their ability to suppress progenitor genes and activate differentiation genes, we hypothesized that MAF/MAFB exist simultaneously in activating and repressive complexes to mediate gene expression. We will first characterize the MAF/MAFB interactome using tandem affinity purification and a proximal protein biotinylation (BioID) approach coupled with mass spectrometry to identify proteins associating with MAF/MAFB during epidermal differentiation. These approaches will identify stable and transient MAF/MAFB interactors. To functionally characterize candidate MAF/MAFB co-regulators, we have developed a CRISPR/Cas9 screen to identify factors required for terminal differentiation. Candidates that arise from this screen will be validated by gene expression profiling and in tissue models. Taken together, this aim will serve to functionally characterize MAF/MAFB activating and repressive complexes operating during epidermal differentiation. Over the course of the training period, I plan to have expanded my expertise into the areas of epigenomics, bioinformatics, and proteomics, all while characterizing the complexity of epidermal transcriptional regulation. The findings arising from this proposal will inform the skin biology community of critical DNA regulatory elements required for human epidermal differentiation, how MAF/MAFB participates in functional regulation of these elements, and what additional factors are critical for epidermal differentiation. These findings may form the foundation of future treatment strategies for disorders characterized by disrupted epidermal homeostasis.

表皮分化中的转录调节复合物 项目摘要 我的长期职业目标是领导一所研究大学的富有成效的学术小组，促进 上皮生物学领域，并成为年轻科学家的导师。我的科学利益在于 了解介导表皮的转录调节网络背后的复杂生物学 稳态。当我在该领域构建一个可靠的研究计划时，我的直接目标是获得指导 新领域，包括表观基因组学，生物信息学和蛋白质组学，这将极大地增强我的当前 遗传学，生物化学和分子生物学方面的专业知识。除了我提供的宝贵专业知识 导师，斯坦福大学的另外六名教职员工在这些领域具有专业知识 作为拟议研究的合作者。此外，我将通过 有限的教学课程，并参加斯坦福大学提供的领导力和管理研讨会 进一步为我的过渡到独立做好准备。总之，这些努力将使我扩大我的 当前的研究提供了一个平台来建立一个新的研究小组，并帮助我过渡到 独立研究者。 该K01提案的目的是调查涉及的转录调节复合物 表皮分化。我最近将MAF/MAFB转录因子（TF）确定为关键 表皮稳态的调节剂介导祖细胞周期出口和终末分化。这些TF 本身受到长期非编码RNA的调节，并介导了重要表皮的下游激活 TFS与表皮总体调节器P63合作。此外，我们的分析揭示了MAF/MAFB 位于基因组水平的推定增强子。由于它们能够抑制祖细胞基因和 激活末端分化基因，我们认为MAF/MAFB同时存在于激活和抑制作用中 复合物。因此，目的是1）表征表皮期间MAF/MAFB的表观基因组效应 分化和2）在功能上表征MAF/MAFB蛋白质相互作用，以了解如何 MAF/MAFB在激活和抑制性复合物中功能。 我们将首先确定MAF/MAFB在AIM I中的表观基因组作用。 MAF/MAFB到染色质的可及性和基因组结构将在MAF/MAFB的背景下解决 CRISPR介导的敲除角质形成细胞。我们将使用ATAC-SEQ识别染色质的变化 可访问性比较MAF/MAFB损失与控制分化的角质形成细胞。评估基因组的变化 形状，我们将询问启动子：使用Capture Hi-C中的DNA触点在控制中与MAF/MAFB敲除 具有分化的角质形成细胞。这些公正的方法将提供全基因组数据 为了表征表皮分化过程中的表观基因组，评估MAF/MAFB损失的影响 并确定分化所需的关键基因组调节元素。此外，表征 监管要素的范围将允许随后识别其他因素或潜在共同调节因子 参与MAF/MAFB介导的基因调节。我们将首先测试已确定的候选共同调节器 通过对MAF/MAFB结合基因座的焦点分析，然后将表观基因组研究的结果纳入 识别并表征推定的共同调节器。 在AIM II中，我们将专注于功能表征MAF/MAFB蛋白质相互作用组。因为他们 能够抑制祖细胞基因并激活分化基因的能力，我们假设存在MAF/MAFB 同时激活和抑制复合物介导基因表达。我们将首先描述 MAF/MAFB使用串联亲和力纯化和近端蛋白生物素化（Bioid）相互作用 方法与质谱法结合以识别表皮期间与MAF/MAFB相关的蛋白质 分化。这些方法将确定稳定且瞬态的MAF/MAFB交互作用。在功能上 候选MAF/MAFB共同调节器的特征，我们开发了CRISPR/CAS9屏幕以识别 终端分化所需的因素。该屏幕引起的候选人将通过基因验证 表达分析和组织模型。综上所述，这个目标将在功能上表征 MAF/MAFB在表皮分化过程中活化和抑制性复合物。 在整个培训期间，我计划将我的专业知识扩展到表观基因组学领域， 生物信息学和蛋白质组学，同时表征表皮转录调节的复杂性。 该提案产生的发现将为皮肤生物学社区提供关键DNA调节的信息 人类表皮分化所需的要素，MAF/MAFB如何参与功能调节 这些要素，以及哪些其他因素对于表皮分化至关重要。这些发现可能形成 未来的治疗策略的基础是以表皮稳态的破坏为特征。