ANALYSIS OF THE FUNCTIONAL-ROLE OF THE POLYCOMB CHROMO DOMAIN IN DROSOPHILA-MELANOGASTER
ANALYSIS OF THE FUNCTIONAL-ROLE OF THE POLYCOMB CHROMO DOMAIN IN DROSOPHILA-MELANOGASTER
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DOI：
10.1101/gad.6.7.1241
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发表时间：
1992-07-01
影响因子：
10.5
通讯作者：
PARO, R
中科院分区：
文献类型：
--
作者：
MESSMER, S;FRANKE, A;PARO, R
染色质结构域被认为是Polycomb （Pc）和hp1（位置效应杂色基因Su(var)205的抑制因子编码的异染色质相关蛋白）之间的同源蛋白基序。Polycomb （Pc）是Pc组基因的成员，编码同源基因的转录抑制因子。结合先前的遗传学研究，这种分子相似性支持了产生异染色质和抑制同质基因的共同机制的建议。整个动物和植物界的色素结构域的进化保护意味着该蛋白基序具有重要的功能作用。我们利用转基因细胞系和果蝇组织培养细胞的瞬时表达实验来研究Pc色域的功能作用。野生型Pc蛋白在SL2细胞中内源性表达，存在于大的免疫可见复合物中。将突变的Pc蛋白表达为Pc- β -半乳糖苷酶融合蛋白，利用间接免疫荧光法检测其在组织培养细胞和转基因幼虫多烯染色体上的核分布。我们发现Pc蛋白的羧基端截断不影响融合蛋白的染色体结合。然而，仅影响染色质结构域的突变，包括体外产生的缺失，以及点突变，会取消染色体结合。我们的结果首次证明了染色质结构域对Pc的功能是重要的，并且它是Pc蛋白与染色质结合所绝对需要的。融合蛋白突变形式产生的一些核模式进一步表明，染色质结构域可能参与包装机制，这对于在异染色质或异染色质样复合体内压缩染色体蛋白至关重要。
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.