Differential effects of heterochromatin protein 1 isoforms on mitotic chromosome distribution and growth in Dictyostelium discoideum
Differential effects of heterochromatin protein 1 isoforms on mitotic chromosome distribution and growth in Dictyostelium discoideum
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DOI：
10.1128/ec.5.3.530-543.2006
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发表时间：
2006-03-01
期刊：
影响因子：
--
通讯作者：
Nellen, W
中科院分区：
文献类型：
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作者：
Kaller, M;Euteneuer, U;Nellen, W
异染色质蛋白1 （HP1）是一种具有良好特征的异染色质成分，从裂变酵母到人类都是保守的。我们在盘状盘齿龙基因组中鉴定了3个hp1样基因（hcpA、hcpB和hcpC）。其中两种（hcpA和hcpB）被表达，并且这些蛋白作为绿色荧光蛋白（GFP）融合蛋白在核外围的一个主要簇中共定位，该簇也以组蛋白H3赖氨酸9二甲基化为特征，这种组蛋白修饰迄今尚未在盘基骨菌中描述过。这些数据有力地表明这个簇代表着着丝粒。两种单基因敲除菌株仅显示出微妙的表型，这表明两种亚型在很大程度上具有重叠的功能。相反，两种同工异构体的破坏似乎是致命的。此外，c端截断形式的HcpA过表达导致明显的生长缺陷，其特征是细胞活力强烈下降。虽然遗传证据表明功能冗余，但过表达GFP-HcpA而非GFP-HcpB会导致生长缺陷，并伴有非典型晚期桥的频率增加。我们的数据表明盘状盘基钢鞘细胞对HcpA和HcpB蛋白水平的变化很敏感，并且这两种异构体在体内和体外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，我们分析了敲除RNAi成分是否影响Dictyostelium中H3K9二甲基化和HP1亚型的定位。有趣的是，异染色质组织似乎独立于功能性RNAi。
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.