Understanding the role of PRDM1 in limb development and split hand/foot malformation.

了解 PRDM1 在肢体发育和手/足裂畸形中的作用。

• 批准号: 10220869
• 负责人: Brittany Truong
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220869
• 关键词: Affect; Apical Ectodermal Ridge; Binding; Biological; CRISPR/Cas technology; Cells; Chromatin; Chromatin Remodeling Factor; Complex; Congenital Abnormality; DNA; DNA Binding; DNA Binding Domain; Data; Defect; Development; Developmental Biology; Digit structure; Disease; Disease Outcome; Ectoderm; Embryo; Epigenetic Process; Ethylnitrosourea; Etiology; Event; Family; Fibroblast Growth Factor; Future; Gene Activation; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Predisposition to Disease; Genetic Transcription; Goals; Growth; Hand; Human; Laboratories; Lead; Limb Development; Limb structure; Mesoderm; Mus; Mutagenesis; Mutate; Mutation; Newborn Infant; Orthologous Gene; PRDM1 gene; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pectoral; Phenotype; Play; Polydactyly; Population; Positioning Attribute; Process; Regulator Genes; Regulatory Element; Repression; Role; SET Domain; Severities; Signal Transduction; Testing; TimeLine; Tissues; Tretinoin; Variant; Work; Zebrafish; Zinc Fingers; base; causal variant; cell motility; conditional knockout; disease phenotype; exome sequencing; experimental study; foot; improved; in vivo; insight; knock-down; malformation; morphogens; mutant; negative affect; novel; null mutation; recruit; repaired; transcription factor; transcriptome sequencing; variant of unknown significance

PROJECT SUMMARY/ABSTRACT Limb development is strictly regulated by gene regulatory networks governed by a complex network of regulatory elements, transcription factors, and epigenetic modifiers. Each component is interconnected, and misregulation at any point in the developmental timeline can lead to congenital limb defects, which affect 1 in 2,000 newborns. One factor that regulates early limb initiation and outgrowth in both mice and zebrafish is PRDM1, though the mechanism is unknown. Uncovering this has become increasingly more important as we have recently identified three human families with split hand/foot malformation (SHFM) of unknown genetic etiology but potentially pathogenic PRDM1 variants. PRDM1 is a versatile transcription factor, capable of both activation and repression by binding directly to DNA at its zinc finger domain or by recruiting chromatin modifiers to its SET domain to regulate transcription . The DNA-binding domain of PRDM1 is either absent or mutated in zebrafish mutants and in SHFM families. Therefore, I hypothesize that direct DNA binding through the zinc finger domain of PRDM1 is necessary for activation of gene expression required for maintaining AER activity during limb development. The proposed study will test this hypothesis in three focused aims. The first aim will use a conditional rescue experiment to determine the functionally active domain of Prdm1a (zebrafish ortholog) during limb development. Constructs of modified prdm1a, in which different domains are deleted, will be injected into null zebrafish mutants. The second aim will determine where Prdm1a binds to DNA during pectoral fin bud development. The third aim will test the pathogenicity of each SHFM PRDM1 variant in zebrafish following mutagenesis by CRISPR-Cas9 and homology directed repair. I will analyze pectoral fin bud development and DNA binding in each mutant. Together, the results from this study will improve our understanding of one of the many pathways involved in limb development. It will also provide insight into how mutations in PRDM1 can lead to congenital limb disorders and help us better predict disease outcomes or phenotype severity based on the given variant.

项目摘要/摘要 肢体发育受到基因调控网络的严格控制，基因调控网络由复杂的 调节元件、转录因子和表观遗传修饰物。每个组件相互连接，并且 发育时间线上任何时间点的调节不当都会导致先天性肢体缺陷，影响1/4。 2000个新生儿。调节小鼠和斑马鱼早期肢体起始和生长的一个因素是 PRDM1，但机制尚不清楚。发现这一点变得越来越重要，因为我们 我最近确认了三个患有未知基因的手足分裂畸形(SHFM)的人类家族 病原学，但可能致病的PRDM1变异。PRDM1是一种多功能转录因子，既能 通过直接结合DNA的锌指结构域或通过招募染色质修饰物来激活和抑制 到其设定的结构域来调节转录。PRDM1的DNA结合区缺失或突变 斑马鱼突变体和SHFM家族。因此，我假设通过锌直接与DNA结合 PRDM1的指状结构域是激活维持AER所需的基因表达所必需的 肢体发育过程中的活动。这项拟议的研究将在三个重点目标上检验这一假设。第一 AIM将使用条件救援实验来确定Prdm1a(斑马鱼)的功能活性区域 在肢体发育过程中。其中删除了不同结构域的修改的prdm1a的结构将 被注射到无效的斑马鱼突变体中。第二个目标将确定Prdm1a在什么地方与DNA结合 胸鳍芽发育。第三个目标是测试斑马鱼中每种SHFM PRDM1变种的致病性 用CRISPR-Cas9和同源定向修复进行诱变。我来分析一下胸鳍的花蕾 每个突变体的发育和DNA结合。总而言之，这项研究的结果将改善我们的 了解肢体发育的众多途径中的一条。它还将提供有关如何 PRDM1的突变可以导致先天性肢体疾病，并帮助我们更好地预测疾病结局或 基于给定变种的表型严重程度。