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

项目总结/摘要 肢体发育受到基因调控网络的严格调控，基因调控网络由一个复杂的 调控元件、转录因子和表观遗传修饰物。每个组件都是相互连接的， 在发育时间轴的任何一点上的失调都可能导致先天性肢体缺陷， 两千个新生儿。调节小鼠和斑马鱼早期肢体发育和生长的一个因素是 PRDM1，尽管机制未知。揭示这一点变得越来越重要，因为我们 最近发现了三个人类家庭与分裂手/足畸形（SHFM）的未知遗传 病原学，但潜在的致病性PRDM1变体。PRDM1是一种多功能转录因子，能够 通过在锌指结构域直接与DNA结合或通过招募染色质修饰剂来激活和抑制 它的SET结构域来调节转录。PRDM1的DNA结合结构域是缺失的或突变的， 斑马鱼突变体和SHFM家族。因此，我假设通过锌的直接DNA结合 PRDM1的指状结构域对于维持AER所需的基因表达的激活是必需的 肢体发育过程中的活动。拟议的研究将在三个重点目标中检验这一假设。第一 aim将使用条件拯救实验来确定Prdm1a（斑马鱼）的功能活性结构域 直系同源物）。修改后的prdm1a的构造（其中删除了不同的域）将 注射到缺失的斑马鱼突变体中。第二个目标是确定Prdm1a在DNA结合过程中的位置。 胸鳍芽发育。第三个目标是测试每个SHFM PRDM1变体在斑马鱼中的致病性 随后通过CRISPR-Cas9诱变和同源定向修复。我会分析胸鳍芽 每个突变体的发育和DNA结合。总之，这项研究的结果将改善我们的 了解肢体发育的许多途径之一。它还将提供洞察如何 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的结果， 表型严重性基于给定的变体。

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.