Messmer S, Franke A, Paro R
Center for Molecular Biology (ZMBH), University of Heidelberg, Germany.
Genes Dev. 1992 Jul;6(7):1241-54. doi: 10.1101/gad.6.7.1241.
The chromo domain was identified as a homologous protein motif between Polycomb (Pc)--a member of the Pc-group genes encoding transcriptional repressors of the homeotic genes--and HP1--a heterochromatin-associated protein encoded by the suppressor of position effect variegation gene Su(var)205. Together with previous genetic studies, this molecular similarity supports the suggestion of a common mechanism used for generating heterochromatin and for repressing homeotic genes. The evolutionary conservation of the chromo domain throughout the animal and plant kingdoms implies an important functional role for this protein motif. We have used transgenic lines as well as transient expression assays employing Drosophila tissue culture cells to study the functional role of the Pc chromo domain. Wild-type Pc protein is endogenously expressed in SL2 cells and is found in large immunologically visible complexes. Mutated Pc proteins were expressed as Pc-beta-galactosidase fusion proteins, and their nuclear distribution was examined by indirect immunofluorescence in tissue culture cells and on polytene chromosomes of transgenic larvae. We show that carboxy-terminal truncations of the Pc protein do not affect chromosomal binding of the fusion protein. However, mutations affecting only the chromo domain including in vitro generated deletions, as well as point mutations, abolish chromosomal binding. Our results demonstrate for the first time that the chromo domain is important for the function of Pc and that it is absolutely required for binding of Pc protein to chromatin. Some of the nuclear patterns generated by the mutated forms of the fusion proteins suggest, furthermore, that the chromo domain could be involved in a packaging mechanism, essential for compacting chromosomal proteins within heterochromatin or heterochromatin-like complexes.
染色体结构域被鉴定为多梳蛋白（Pc）（一种编码同源异型基因转录抑制因子的Pc组基因成员）与HP1（一种由位置效应斑驳基因Su(var)205的抑制子编码的异染色质相关蛋白）之间的同源蛋白基序。结合先前的遗传学研究，这种分子相似性支持了一种用于产生异染色质和抑制同源异型基因的共同机制的观点。染色体结构域在整个动植物界的进化保守性意味着该蛋白基序具有重要的功能作用。我们利用转基因品系以及采用果蝇组织培养细胞的瞬时表达分析来研究Pc染色体结构域的功能作用。野生型Pc蛋白在SL2细胞中内源性表达，并存在于大型免疫可见复合物中。突变的Pc蛋白以Pc-β-半乳糖苷酶融合蛋白的形式表达，其核分布通过组织培养细胞和转基因幼虫多线染色体上的间接免疫荧光进行检测。我们发现，Pc蛋白的羧基末端截短不影响融合蛋白与染色体的结合。然而，仅影响染色体结构域的突变，包括体外产生的缺失以及点突变，会消除与染色体的结合。我们的结果首次证明，染色体结构域对Pc的功能很重要，并且是Pc蛋白与染色质结合所绝对必需的。此外，融合蛋白突变形式产生的一些核模式表明，染色体结构域可能参与一种包装机制，这种机制对于在异染色质或异染色质样复合物中压实染色体蛋白至关重要。
