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

项目总结/摘要 回复突变嵌合体（RM）是一种罕见但自然的现象，其中细胞携带致病突变， 与突变已被正常等位基因自发取代的细胞共存。鱼鳞病伴 五彩纸屑（IWC）是一种罕见的皮肤疾病，通常显示皮肤RM。IWC患者出生时 在儿童时期，他们开始发展出组织学上正常的五彩纸屑样斑块， 出现皮肤，其中突变已经丢失。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，每一个都是一个独立的致病逆转事件， 突变此外，这些克隆的扩张表明存在相反的选择压力， 回复突变克隆和突变细胞，这表明细胞竞争。我们的实验室最近发现 IWC是由角蛋白1（KRT 1）或10（KRT 10）尾部结构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的角蛋白的核错误定位，这导致角蛋白表达的增加。 DNA损伤并导致拷贝中性杂合性丢失。的分子调控机制 逆转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 和回复突变体细胞还不是很清楚。为了实现WT或K10 IWC角质形成细胞的可视化 将K10 IWC/K14-CreERT/mTmG与K14-CreERT/H2 BmCherry小鼠杂交（mCherry nucleus）， 以产生具有容易观察到的mCherry核的K10 WT（mTomato）或K10 IWC（mGFP）角质形成细胞。到 为了探究细胞间竞争的机制，我将使用这种新型的三重荧光IWC 小鼠体内进行嵌合K10 IWC表皮的IVLI，并在体外进行免疫荧光测定 以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的克隆优势的细胞行为。我会 还对来自K10 IWC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 影响竞争的具体媒介。第二，为了探索逆转机制，我们还 开发了一种体外系统，采用杂合Tk 1敲除（Tk 1 +/-） 小鼠角质形成细胞。在这个系统中，细胞表达至少一个功能性拷贝的tk酶， 三氟胸苷（TFT）转化为有毒代谢物，在TFT培养基中生长时死亡。细胞只形成菌落，如果 在TK基因座发生重组，导致TK酶的丢失。我发现， IWC突变体KRT 10和KRT的表达导致集落形成，但导致逆转的步骤是 目前仍不清楚.为了做到这一点，我将进行CRISPR敲除筛选与我们的体外Tk 1 +/-系统配对， 将允许高通量，无偏见的评估所有鼠基因介导的改变率， IWC突变角蛋白表达导致的逆转。

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.