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

点击翻译按钮获取中文摘要

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.