The developmental dynamics and disease potential of random monoallelic gene expression

X-chromosome inactivation (XCI) in females and allelic exclusion of olfactory receptor or immunoglobulin loci, represent classic examples of random monoallelic expression (RME). RME of some single copy genes has also been reported, but the in vivo relevance of this remains unclear. Here we identify several hundred RME genes in clonal neural progenitor cell lines derived from embryonic stem cells. RME occurs during differentiation and once established, the monoallelic state can be highly stable. We show that monoallelic expression also occurs in vivo, in the absence of DNA sequence polymorphism, similarly to XCI. Several of the genes identified play important roles in development and have also been implicated in human autosomal dominant disorders. We propose that monoallelic expression of such genes contributes to the fine-tuning of the developmental regulatory pathways they control and in the context of a mutation, RME can predispose to loss of function in a proportion of cells and thus contribute to disease. Overall design: The hybrid mouse ES cell lines F1-21.6 and F1-23 (129Sv-Cast/EiJ), previously described in (Luikenhuis et al., 2001) and (Jonkers et al., 2009), were grown on mitomycin C-inactivated MEFs in ES cell media containing 15% FBS (Gibco), 10-4 M b-mercaptoethanol (Sigma), 1000U/ml of leukaemia inhibitory factor (LIF, Chemicon). Mouse ES cells were differentiated into neural progenitor cells (NPC) as previously described (Conti et al., 2005; Splinter et al., 2011). Total RNAs were prepared from the mouse ES cell lines, 4 NPC clones derived from ES cell line F1-21.6 plus one replicate, and 4 NPC clones derived from ES cell line F1-23 plus one replicate. Library preparation after polyadenylated RNA purification was performed according to the standard Illumina instructions. Paired-end 100bp reads were generated using the HiSeq 2000 sequencing platform.

雌性个体的X染色体失活（X-chromosome inactivation, XCI）以及嗅觉受体或免疫球蛋白基因座的等位基因排斥，是随机单等位基因表达（random monoallelic expression, RME）的经典范例。已有研究报道部分单拷贝基因存在RME现象，但该现象在体内的相关意义至今尚不明确。本研究在胚胎干细胞（embryonic stem cell, ES）衍生的克隆神经前体细胞系中鉴定出数百个RME基因。RME发生于细胞分化过程中，且一旦建立单等位基因表达状态后，其状态可保持高度稳定。我们证实，单等位基因表达在体内同样存在，且不依赖DNA序列多态性，这一点与XCI类似。本研究鉴定出的诸多基因在发育过程中发挥重要作用，同时也与人类常染色体显性遗传病相关。我们提出，这类基因的单等位基因表达有助于微调其调控的发育调控通路；而当发生突变时，RME可使部分细胞出现功能丧失易感性，进而参与疾病发生。

总体实验设计：本研究使用此前已在Luikenhuis等人（2001）及Jonkers等人（2009）的研究中报道过的杂交小鼠ES细胞系F1-21.6与F1-23（129Sv-Cast/EiJ），将其培养于丝裂霉素C灭活的小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）饲养层上，所用ES细胞培养基包含15%胎牛血清（Gibco）、10^-4 M β-巯基乙醇（Sigma）以及1000U/ml白血病抑制因子（leukaemia inhibitory factor, LIF, Chemicon）。按照此前报道的方法（Conti等人，2005；Splinter等人，2011），将小鼠ES细胞诱导分化为神经前体细胞（neural progenitor cell, NPC）。分别从以下样本中提取总RNA：上述小鼠ES细胞系、源自ES细胞系F1-21.6的4个NPC克隆及1个重复样本、源自ES细胞系F1-23的4个NPC克隆及1个重复样本。在纯化聚腺苷酸化RNA后，按照Illumina标准流程进行文库构建。采用HiSeq 2000测序平台生成100bp双端测序读段。