Deciphering the Mechanism of Revertant Mosaicism and Cellular Competition in Ichthyosis with Confetti

用五彩纸屑破译鱼鳞病中恢复嵌合和细胞竞争的机制

基本信息

批准号：
10315913
负责人：
DIANA ALEXANDRA YANEZ
金额：
$ 3.09万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-16 至 2023-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10315913
关键词：
Affect Alleles Apoptosis Area Biological Assay Cell Nucleus Cells Characteristics Childhood Clonal Expansion Clustered Regularly Interspaced Short Palindromic Repeats Complex Cutaneous Cytoplasm Cytoskeleton DNA DNA Damage DNA Repair Data Disease Dominant-Negative Mutation Enzymes Epidermis Event Fluorescence Frequencies Genes Genetic Genetic Recombination Growth and Development function Histologic Human Ichthyosis en confetti Image Immunofluorescence Immunologic In Vitro Individual Intermediate Filament Gene Intermediate Filament Proteins Ker10 protein Keratin Knock-out Lead Loss of Heterozygosity Maintenance Malignant Neoplasms Measures Mediating Mediator of activation protein Molecular Mosaicism Mus Mutation Natural Selections Nuclear Oncogenic Pathway interactions Patients Play Process Role Skin Spottings Suggestion System Tail Therapeutic Time Trifluridine Visualization cell behavior cell type disease-causing mutation gene therapy genome integrity genome-wide high throughput screening homologous recombination human disease in vitro Assay in vivo keratinocyte knock-down mutant novel pressure protein complex response single-cell RNA sequencing skin disorder skin patch tool two-photon

项目摘要

PROJECT SUMMARY / ABSTRACT Revertant mosaicism (RM) is a rare but naturally phenomenon wherein cells carrying disease-causing mutations coexist with cells in which the mutation has been spontaneously replaced with a normal allele. Ichthyosis with confetti (IWC) is a rare skin disorder that commonly displays cutaneous RM. Patients with IWC are born with erythematous scaly skin, and during childhood they start to develop confetti-like patches of histologically normal appearing skin, in which the mutation has been lost. These patches increase in size and number over time, eventually becoming thousands, each of which is an independent event of reversion of the disease-causing mutation. Moreover, the expansion of these clones suggests the existence of opposing selective pressures on the revertant clones and mutant cells, which is suggestive of cellular competition. Our lab recently discovered that IWC is caused by dominant negative mutations in the tail domains of the keratin 1 (KRT1) or 10 (KRT10) intermediate filament genes which normally contribute to the stability of the cytoskeleton and reside in the cytoplasm. These mutations result in nuclear mislocalization of affected keratins, which lead to an increase in DNA damage and result in copy-neutral loss of heterozygosity. The molecular mechanisms regulating reversion and the steps leading to recombination are largely unknown, and competition between mutant and revertant cells is not well understood. In order to achieve visualization of WT or K10 IWC keratinocytes and their nuclei, I crossed K10IWC/K14-CreERT/mTmG with K14-CreERT/H2BmCherry mouse (mCherry nucleus) to create K10 WT (mTomato) or K10 IWC (mGFP) keratinocytes with readily observable mCherry nuclei. To interrogate the mechanism underlying intercellular competition, I will use this novel triple-fluorescence IWC mouse to perform IVLI of mosaic K10IWC epidermis in vivo and to perform immunofluorescence assays in vitro to determine the cellular behaviors that result in clonal advantage of wild type over mutant keratinocytes. I will also perform single cell RNA sequencing of mutant and wild type keratinocytes from K10IWC mice to identify specific mediators affecting competition. Second, to explore the mechanisms of reversion, we have also developed an in vitro system to quantify the rate of reversion employing heterozygous Tk1 knockout (Tk1+/-) murine keratinocytes. In this system, cells expressing at least a functional copy of the tk enzyme, which turns trifluorothymidine (TFT) into a toxic metabolite, die when grown in TFT-medium. Cells only form colonies, if recombination at the tk locus occurs resulting in the loss of the tk enzyme. I have found that higher rates of colony formation result from expression of IWC mutant KRT10 and KRT but the steps leading to reversion are still unclear. To do this, I will perform a CRISPR knockdown screen paired with our in vitro Tk1+/- system, which will allow for a high-throughput, unbiased assessment of all murine genes that mediate the altered rate of reversion resulting from IWC mutant keratin expression.

项目概要/摘要回复嵌合体 (RM) 是一种罕见但自然的现象，其中细胞携带致病突变与突变已自发被正常等位基因取代的细胞共存。鱼鳞病五彩纸屑 (IWC) 是一种罕见的皮肤病，通常表现为皮肤 RM。 IWC 患者出生时患有红斑鳞状皮肤，在儿童时期开始出现组织学正常的五彩纸屑状斑块出现突变已丢失的皮肤。这些斑块的大小和数量随着时间的推移而增加，最终变成数千个，每个都是致病逆转的独立事件突变。此外，这些克隆的扩张表明存在对立的选择压力回复克隆和突变细胞，这表明存在细胞竞争。我们的实验室最近发现 IWC 是由角蛋白 1 (KRT1) 或 10 (KRT10) 尾部结构域的显性失活突变引起的中间丝基因通常有助于细胞骨架的稳定性并驻留在细胞质。这些突变导致受影响角蛋白的核错误定位，从而导致角蛋白增加 DNA 损伤并导致复制中性杂合性丢失。调控的分子机制回复和导致重组的步骤在很大程度上是未知的，并且突变体之间的竞争回复细胞尚不清楚。为了实现 WT 或 K10 IWC 角质形成细胞的可视化及其细胞核，我将 K10IWC/K14-CreERT/mTmG 与 K14-CreERT/H2BmCherry 小鼠（mCherry 细胞核）杂交创建具有易于观察的 mCherry 细胞核的 K10 WT (mTomato) 或 K10 IWC (mGFP) 角质形成细胞。到探究细胞间竞争的机制，我将使用这款新型三荧光 IWC 小鼠体内进行镶嵌 K10IWC 表皮的 IVLI 并进行体外免疫荧光测定确定导致野生型相对于突变型角质形成细胞的克隆优势的细胞行为。我会还对 K10IWC 小鼠的突变型和野生型角质形成细胞进行单细胞 RNA 测序，以鉴定影响竞争的特定中介因素。其次，为了探索回归机制，我们还开发了一种体外系统来量化采用杂合 Tk1 敲除 (Tk1+/-) 的回复率鼠角质形成细胞。在这个系统中，细胞至少表达 tk 酶的功能性副本，该酶将三氟胸苷 (TFT) 转化为有毒代谢物，在 TFT 培养基中生长时死亡。细胞仅形成集落，如果 tk 基因座发生重组，导致 tk 酶丢失。我发现更高的比率集落形成是 IWC 突变体 KRT10 和 KRT 表达的结果，但导致回复的步骤是仍不清楚。为此，我将与我们的体外 Tk1+/- 系统配对进行 CRISPR 敲除筛选，这将允许对介导改变率改变的所有小鼠基因进行高通量、公正的评估 IWC 突变角蛋白表达导致的逆转。