Loll-Krippleber Raphaël, Feri Adeline, Nguyen Marie, Maufrais Corinne, Yansouni Jennifer, d'Enfert Christophe, Legrand Mélanie
Unité Biologie et Pathogénicité Fongiques, Département Mycologie, Institut Pasteur, Paris, France INRA USC2019, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Paris, France.
Plate-Forme de Cytométrie, Imagopole, Institut Pasteur, Paris, France.
Eukaryot Cell. 2015 Mar;14(3):311-22. doi: 10.1128/EC.00286-14. Epub 2015 Jan 16.
Loss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogen Candida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH in C. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched for C. albicans genes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability in C. albicans and more generally in eukaryotic cells.
杂合性缺失（LOH）在基因组动态变化中发挥着重要作用，尤其是在肿瘤发生过程中。在真菌病原体白色念珠菌中，LOH有助于获得抗真菌耐药性。为了研究调控白色念珠菌中LOH的机制，我们建立了一种新方法，该方法结合了携带蓝色荧光蛋白和绿色荧光蛋白标记的人工杂合位点以及流式细胞术，以在单细胞水平检测LOH事件。使用这种基于荧光的方法，我们证实了高温、甲磺酸甲酯处理以及Mec1 DNA损伤检查点激酶的失活会引发LOH频率的增加。利用这个系统，我们筛选了白色念珠菌中过表达会引发LOH增加的基因，并鉴定出四个候选基因，其中一些是已知的基因组动态变化调节因子，其人类同源物与癌症进展有关。因此，本文介绍的方法将有助于开展新的筛选，以鉴定对白色念珠菌乃至更广泛的真核细胞基因组稳定性至关重要的基因。
