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

项目摘要/摘要 逆转嵌合体(Rm)是一种罕见但自然的现象，细胞携带致病突变。 与突变被正常等位基因自发取代的细胞共存。鱼鳞病伴发 五彩纸屑(IWC)是一种罕见的皮肤病，通常表现为皮肤RM。IWC患者出生时就患有 皮肤红斑，鳞状，在儿童时期，他们开始形成组织学上正常的五彩纸屑样斑块 看起来像皮肤，其中突变已经丢失。随着时间的推移，这些补丁的大小和数量都会增加， 最终变成数千人，其中每一人都是一个独立的致病逆转事件 突变。此外，这些无性系的扩张表明，存在着相反的选择压力 突变的克隆和突变的细胞，这暗示着细胞竞争。我们实验室最近发现 IWC是由角蛋白1(KRT1)或角蛋白10(KRT10)尾部区域的显性负突变引起的 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 DNA损伤，并导致拷贝中性杂合性丢失。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，突变体之间的竞争 而回复细胞还没有被很好地理解。为了实现WT或K10 IWC角质形成细胞的可视化 和它们的核，我将K10IWC/K14-CreERT/mTmG与K14-CreERT/H2 BmCherry小鼠(mCherry核)杂交。 创造K10WT(m番茄)或K10IWC(MGFP)角质形成细胞，并观察到mCherry核。至 询问细胞间竞争的机制，我将使用这个新的三重荧光IWC 小鼠体内进行K10IWC镶嵌表皮的IVLI及体外免疫荧光检测 确定导致野生型角质形成细胞相对于突变型角质形成细胞具有克隆优势的细胞行为。这就做 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，以确定 影响竞争的特定调解人。第二，为了探索回归的机制，我们还 开发了一种利用杂合Tk1基因敲除(Tk1/-)来量化逆转速率的体外系统 小鼠角质形成细胞。在这个系统中，表达至少一个功能拷贝的tk酶的细胞 三氟代胸苷(TFT)是一种有毒的代谢物，在TFT介质中生长时会死亡。细胞只有在以下情况下才会形成克隆 Tk基因发生重组，导致tk酶的丢失。我发现，更高的 IWC突变体KRT10和KRT的表达导致克隆形成，但导致逆转的步骤如下 仍不清楚。为此，我将进行CRISPR击倒筛选与我们的体外Tk1/-系统配对，其中 将允许高通量、不偏不倚地评估所有的小鼠基因，这些基因介导了 IWC突变角蛋白表达引起的逆转。

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.