Department of Human Genetics, University of Chicago, Howard Hughes Medical Institute, Chicago, IL 60637, USA.
Cell Res. 2012 May;22(5):848-58. doi: 10.1038/cr.2011.190. Epub 2011 Nov 29.
The progressive restriction of cell fate during lineage differentiation is a poorly understood phenomenon despite its ubiquity in multicellular organisms. We recently used a cell fusion assay to define a mode of epigenetic silencing that we termed "occlusion", wherein affected genes are silenced by cis-acting chromatin mechanisms irrespective of whether trans-acting transcriptional activators are present. We hypothesized that occlusion of lineage-inappropriate genes could contribute to cell fate restriction. Here, we test this hypothesis by introducing bacterial artificial chromosomes (BACs), which are devoid of chromatin modifications necessary for occlusion, into mouse fibroblasts. We found that BAC transgenes corresponding to occluded endogenous genes are expressed in most cases, whereas BAC transgenes corresponding to silent but non-occluded endogenous genes are not expressed. This indicates that the cellular milieu in trans supports the expression of most occluded genes in fibroblasts, and that the silent state of these genes is solely the consequence of occlusion in cis. For the BAC corresponding to the occluded myogenic master regulator Myf5, expression of the Myf5 transgene on the BAC triggered fibroblasts to acquire a muscle-like phenotype. These results provide compelling evidence for a critical role of gene occlusion in cell fate restriction.
尽管细胞命运在谱系分化过程中的逐渐限制在多细胞生物中普遍存在，但这一现象仍未得到充分理解。我们最近使用细胞融合测定法来定义一种我们称之为“阻断”的表观遗传沉默模式，其中受影响的基因通过顺式作用染色质机制沉默，而不管是否存在反式作用转录激活剂。我们假设谱系不当基因的阻断可能有助于细胞命运的限制。在这里，我们通过将缺乏阻断所需的染色质修饰的细菌人工染色体 (BAC) 引入小鼠成纤维细胞中来检验这一假设。我们发现，在大多数情况下，与被阻断的内源性基因相对应的 BAC 转基因是表达的，而与沉默但未被阻断的内源性基因相对应的 BAC 转基因则不表达。这表明在转染体中细胞环境支持大多数阻断基因在成纤维细胞中的表达，并且这些基因的沉默状态仅仅是顺式阻断的结果。对于与被阻断的肌源性主调控因子 Myf5 相对应的 BAC，BAC 上 Myf5 转基因的表达使成纤维细胞获得了类似于肌肉的表型。这些结果为基因阻断在细胞命运限制中的关键作用提供了有力证据。
