Department of Molecular Biology, Faculty of Life Sciences, Ariel University, Ariel, Israel.
Ariel Center for Applied Cancer Research, Ariel University, Ariel, Israel.
Cell Prolif. 2022 Dec;55(12):e13348. doi: 10.1111/cpr.13348. Epub 2022 Nov 3.
SETDB1 is a methyltransferase responsible for the methylation of histone H3-lysine-9, which is mainly related to heterochromatin formation. SETDB1 is overexpressed in various cancer types and is associated with an aggressive phenotype. In agreement with its activity, it mainly exhibits a nuclear localization; however, in several cell types a cytoplasmic localization was reported. Here we looked for cytoplasmic functions of SETDB1.
SETDB1 association with microtubules was detected by immunofluorescence and co-sedimentation. Microtubule dynamics were analysed during recovery from nocodazole treatment and by tracking microtubule plus-ends in live cells. Live cell imaging was used to study mitotic kinetics and protein-protein interaction was identified by co-immunoprecipitation.
SETDB1 co-sedimented with microtubules and partially colocalized with microtubules. SETDB1 partial silencing led to faster polymerization and reduced rate of catastrophe events of microtubules in parallel to reduced proliferation rate and slower mitotic kinetics. Interestingly, over-expression of either wild-type or catalytic dead SETDB1 altered microtubule polymerization rate to the same extent, suggesting that SETDB1 may affect microtubule dynamics by a methylation-independent mechanism. Moreover, SETDB1 co-immunoprecipitated with HDAC6 and tubulin acetylation levels were increased upon silencing of SETDB1.
Taken together, our study suggests a model in which SETDB1 affects microtubule dynamics by interacting with both microtubules and HDAC6 to enhance tubulin deacetylation. Overall, our results suggest a novel cytoplasmic role for SETDB1 in the regulation of microtubule dynamics.
SETDB1 是一种组蛋白 H3-赖氨酸-9 的甲基转移酶，主要与异染色质形成有关。SETDB1 在各种癌症类型中过度表达，与侵袭性表型相关。与其活性一致，它主要表现为核定位；然而，在几种细胞类型中报道了细胞质定位。在这里，我们寻找 SETDB1 的细胞质功能。
通过免疫荧光和共沉淀检测 SETDB1 与微管的结合。在从诺考达唑处理中恢复期间和通过跟踪活细胞中的微管正端分析微管动力学。使用活细胞成像研究有丝分裂动力学，并通过共免疫沉淀鉴定蛋白质-蛋白质相互作用。
SETDB1 与微管共沉淀，并与微管部分共定位。SETDB1 的部分沉默导致微管的聚合更快， catastrophe 事件的速率降低，同时增殖率降低，有丝分裂动力学减慢。有趣的是，野生型或催化失活的 SETDB1 的过表达同样改变了微管聚合速率，表明 SETDB1 可能通过非甲基化机制影响微管动力学。此外，SETDB1 与 HDAC6 共免疫沉淀，并且沉默 SETDB1 后微管乙酰化水平增加。
总之，我们的研究表明了一个模型，其中 SETDB1 通过与微管和 HDAC6 相互作用影响微管动力学，从而增强微管去乙酰化。总体而言，我们的结果表明 SETDB1 在调节微管动力学方面具有新的细胞质作用。
