Roukens M Guy, Alloul-Ramdhani Mariam, Vertegaal Alfred C O, Anvarian Zeinab, Balog Crina I A, Deelder André M, Hensbergen Paul J, Baker David A
Signaling and Transcription Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Mol Cell Biol. 2008 Apr;28(7):2342-57. doi: 10.1128/MCB.01159-07. Epub 2008 Jan 22.
Cell proliferation and differentiation are governed by a finely controlled balance between repression and activation of gene expression. The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue Yan, play pivotal roles in cell fate determination although the precise mechanisms by which repression of gene expression by these factors is achieved are not clearly defined. Here, we report the identification and characterization of the primary site of sumoylation of Tel, lysine 11 (K11), which is highly conserved in vertebrates (except Danio rerio). We demonstrate that in cells PIAS3 binds to Tel and stimulates sumoylation of K11 in the nucleus. Both Tel monomers and oligomers are efficiently sumoylated on K11 in vitro; but in cells only Tel oligomers are found conjugated with SUMO, whereas sumoylation of Tel monomers is transitory and appears to sensitize them for proteasomal degradation. Mechanistically, sumoylation of K11 inhibits repression of gene expression by full-length Tel. In accordance with this observation, we found that sumoylation impedes Tel association with DNA. By contrast, a Tel isoform lacking K11 (TelM43) is strongly repressive. This isoform results from translation from an alternative initiation codon (M43) that is common to all Tel proteins that also contain the K11 sumoylation consensus site. We find that PIAS3 may have a dual, context-dependent influence on Tel; it mediates Tel sumoylation, but it also augments Tel's repressive function in a sumoylation-independent fashion. Our data support a model that suggests that PIAS-mediated sumoylation of K11 and the emergence of TelM43 in early vertebrates are linked and that this serves to refine spatiotemporal control of gene expression by Tel by establishing a pool of Tel molecules that are available either to be recycled to reinforce repression of gene expression or are degraded in a regulated fashion.
细胞增殖和分化受基因表达抑制与激活之间精确控制的平衡所调控。脊椎动物Ets转录抑制因子Tel（ETV6）及其无脊椎动物同源物Yan在细胞命运决定中发挥关键作用，尽管这些因子实现基因表达抑制的确切机制尚不清楚。在此，我们报告了Tel的主要SUMO化位点赖氨酸11（K11）的鉴定和表征，该位点在脊椎动物中高度保守（除斑马鱼外）。我们证明在细胞中PIAS3与Tel结合并刺激K11在细胞核中的SUMO化。Tel单体和寡聚体在体外均能有效地在K11上发生SUMO化；但在细胞中，仅发现Tel寡聚体与SUMO结合，而Tel单体的SUMO化是短暂的，似乎使其对蛋白酶体降解敏感。从机制上讲，K11的SUMO化抑制全长Tel对基因表达的抑制。根据这一观察结果，我们发现SUMO化阻碍Tel与DNA的结合。相比之下，缺乏K11的Tel异构体（TelM43）具有强烈的抑制作用。这种异构体来自于一个替代起始密码子（M43）的翻译，该起始密码子在所有也包含K11 SUMO化共有位点的Tel蛋白中都很常见。我们发现PIAS3可能对Tel有双重的、依赖于上下文的影响；它介导Tel的SUMO化，但也以不依赖SUMO化的方式增强Tel的抑制功能。我们的数据支持一个模型，该模型表明PIAS介导的K11 SUMO化与早期脊椎动物中TelM43的出现有关，并且这通过建立一组Tel分子来完善Tel对基因表达的时空控制，这些Tel分子要么可被循环利用以加强对基因表达的抑制，要么以受调控的方式被降解。
