A Truncated Form of the Human CAF-1 p150 Subunit Impairs the Maintenance of Transcriptional Gene Silencing in Mammalian Cells

Chromatin assembly factor 1 (CAF-1) is a protein complex formed of three subunits, p150, p60, and p48, conserved from the yeast Saccharomyces cerevisiae to humans, which can promote nucleosome assembly onto newly replicated DNA. In S. cerevisiae, deletion of the genes encoding any of the three CAF-1 subunits (cacΔ mutants), although nonlethal, results in a silencing defect of genes packaged into heterochromatin. Here we report on a mammalian cell model that we devised to monitor gene silencing and its reversal in a quantitative manner. This model relies on the use of a cell line stably transfected with a reporter gene in a silenced state. Reversal of reporter gene silencing was achieved upon treatment of the cells with 5-azacytidine, which resulted in the demethylation of the reporter gene copies. We show that expression of a cDNA for the human p150 CAF-1 subunit harboring 5′ truncations, but not that of a cDNA encoding the full-length p150 CAF-1 subunit, increases by more than 500-fold the frequency at which transcriptional silencing of the reporter gene copies is reversed in these cells. Reversal of gene silencing is dependent upon expression of a truncated protein, possibly acting as a dominant negative mutant of the wild-type CAF-1, is associated with alterations in chromatin structure as measured by an endonuclease sensitivity assay and is not associated with detectable changes in the methylation status of the silenced genes. These results suggest that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals, despite the lack of DNA methylation in yeast chromatin.

染色质组装因子1（Chromatin assembly factor 1，CAF-1）是由p150、p60和p48三个亚基构成的蛋白质复合物，从酿酒酵母（Saccharomyces cerevisiae）到人类均具有进化保守性，可介导核小体（nucleosome）在新复制DNA上的组装。在酿酒酵母中，敲除编码CAF-1三个亚基中任意一个的基因（即cacΔ突变体）虽不会引发细胞致死，但会使包装于异染色质（heterochromatin）中的基因出现沉默缺陷。本研究构建了一种可定量监测基因沉默及其逆转过程的哺乳动物细胞模型，该模型依托于一株稳定转染了报告基因且该基因处于沉默状态的细胞系。用5-氮胞苷（5-azacytidine）处理细胞可逆转报告基因的沉默，该过程会导致报告基因拷贝发生去甲基化。我们发现，表达带有5'端截短的人类p150 CAF-1亚基的互补DNA（complementary DNA，cDNA），而非编码全长p150 CAF-1亚基的cDNA，可使该细胞系中报告基因拷贝的转录沉默被逆转的频率提升500倍以上。基因沉默的逆转依赖于截短蛋白的表达，该截短蛋白可能作为野生型CAF-1的显性负突变体发挥作用，其伴随有通过核酸内切酶敏感性实验检测到的染色质结构改变，且未检测到沉默基因甲基化状态的可观测变化。尽管酿酒酵母的染色质不存在DNA甲基化，但上述结果表明，CAF-1在基因表达的表观遗传调控中的功能在酵母与哺乳动物之间是保守的。