Kaller Markus, Euteneuer Ursula, Nellen Wolfgang
Kassel University, FB 18, Abt. Genetik, Heinrich-Plett-Strasse 40, 34132 Kassel, Germany.
Eukaryot Cell. 2006 Mar;5(3):530-43. doi: 10.1128/EC.5.3.530-543.2006.
Heterochromatin protein 1 (HP1) is a well-characterized heterochromatin component conserved from fission yeast to humans. We identified three HP1-like genes (hcpA, hcpB, and hcpC) in the Dictyostelium discoideum genome. Two of these (hcpA and hcpB) are expressed, and the proteins colocalized as green fluorescent protein (GFP) fusion proteins in one major cluster at the nuclear periphery that was also characterized by histone H3 lysine 9 dimethylation, a histone modification so far not described for Dictyostelium. The data strongly suggest that this cluster represents the centromeres. Both single-knockout strains displayed only subtle phenotypes, suggesting that both isoforms have largely overlapping functions. In contrast, disruption of both isoforms appeared to be lethal. Furthermore, overexpression of a C-terminally truncated form of HcpA resulted in phenotypically distinct growth defects that were characterized by a strong decrease in cell viability. Although genetic evidence implies functional redundancy, overexpression of GFP-HcpA, but not GFP-HcpB, caused growth defects that were accompanied by an increase in the frequency of atypic anaphase bridges. Our data indicate that Dictyostelium discoideum cells are sensitive to changes in HcpA and HcpB protein levels and that the two isoforms display different in vivo and in vitro affinities for each other. Since the RNA interference (RNAi) machinery is frequently involved in chromatin remodeling, we analyzed if knockouts of RNAi components influenced the localization of H3K9 dimethylation and HP1 isoforms in Dictyostelium. Interestingly, heterochromatin organization appeared to be independent of functional RNAi.
异染色质蛋白1（HP1）是一种从裂殖酵母到人类都保守的、特征明确的异染色质成分。我们在盘基网柄菌基因组中鉴定出三个类HP1基因（hcpA、hcpB和hcpC）。其中两个（hcpA和hcpB）表达，并且这些蛋白质作为绿色荧光蛋白（GFP）融合蛋白在核周边的一个主要簇中共定位，该簇也以组蛋白H3赖氨酸9二甲基化特征，这是迄今为止盘基网柄菌中未描述的一种组蛋白修饰。数据强烈表明该簇代表着丝粒。两个单敲除菌株仅表现出细微的表型，表明这两种异构体在很大程度上具有重叠功能。相比之下，两种异构体的破坏似乎是致死的。此外，HcpA的C末端截短形式的过表达导致了表型上不同的生长缺陷，其特征是细胞活力大幅下降。尽管遗传证据暗示功能冗余，但GFP-HcpA而非GFP-HcpB的过表达导致了生长缺陷，并伴随着非典型后期桥频率的增加。我们的数据表明盘基网柄菌细胞对HcpA和HcpB蛋白水平的变化敏感，并且这两种异构体在体内和体外对彼此表现出不同的亲和力。由于RNA干扰（RNAi）机制经常参与染色质重塑，我们分析了RNAi成分的敲除是否会影响盘基网柄菌中H3K9二甲基化和HP1异构体的定位。有趣的是，异染色质组织似乎独立于功能性RNAi。
