Dynein Light Chain 1 (DYNLT1) Interacts with Normal and Oncogenic Nucleoporins

The chimeric oncoprotein NUP98-HOXA9 results from the t(7;11)(p15;p15) chromosomal translocation and is associated with acute myeloid leukemia. It causes aberrant gene regulation and leukemic transformation through mechanisms that are not fully understood. NUP98-HOXA9 consists of an N-terminal portion of the nucleoporin NUP98 that contains many FG repeats fused to the DNA-binding homeodomain of HOXA9. We used a Cytotrap yeast two-hybrid assay to identify proteins that interact with NUP98-HOXA9. We identified Dynein Light Chain 1 (DYNLT1), an integral 14 KDa protein subunit of the large microtubule-based cytoplasmic dynein complex, as an interaction partner of NUP98-HOXA9. Binding was confirmed by in vitro pull down and co-immunoprecipitation assays and the FG repeat region of NUP98-HOXA9 was shown to be essential for the interaction. RNAi-mediated knockdown of DYNLT1 resulted in reduction of the ability of NUP98-HOXA9 to activate transcription and also inhibited the ability of NUP98-HOXA9 to induce proliferation of primary human hematopoietic CD34+ cells. DYNLT1 also showed a strong interaction with wild-type NUP98 and other nucleoporins containing FG repeats. Immunofluorescence analysis showed that DYNLT1 localizes primarily to the nuclear periphery, where it co-localizes with the nuclear pore complex, and to the cytoplasm. Deletion studies showed that the interactions of the nucleoporins with DYNLT1 are dependent predominantly on the C-terminal half of the DYNLT1. These data show for the first time that DYNLT1 interacts with nucleoporins and plays a role in the dysregulation of gene expression and induction of hematopoietic cell proliferation by the leukemogenic nucleoporin fusion, NUP98-HOXA9.嵌合癌蛋白NUP98-HOXA9由t(7;11)(p15;p15)染色体易位产生，与急性髓系白血病相关。其通过尚未完全阐明的机制引发异常基因调控与白血病转化。NUP98-HOXA9由含有大量FG重复序列的核孔蛋白NUP98的N端部分，与HOXA9的DNA结合同源结构域融合而成。本研究采用Cytotrap酵母双杂交实验，筛选可与NUP98-HOXA9相互作用的蛋白，最终鉴定出动力蛋白轻链1（Dynein Light Chain 1, DYNLT1）——一种分子量为14kDa的大型基于微管的胞质动力蛋白复合物的固有亚基——作为NUP98-HOXA9的互作伴侣。通过体外下拉实验与免疫共沉淀实验验证了二者的结合，并证实NUP98-HOXA9的FG重复区域是该相互作用的必需结构。对DYNLT1进行RNA干扰介导的敲低，可削弱NUP98-HOXA9的转录激活能力，同时抑制其诱导原代人造血CD34+细胞增殖的能力。DYNLT1还可与野生型NUP98及其他含有FG重复序列的核孔蛋白发生较强相互作用。免疫荧光分析显示，DYNLT1主要定位于核周区域（与核孔复合物共定位）及细胞质中。缺失实验表明，核孔蛋白与DYNLT1的相互作用主要依赖于DYNLT1的C端半区。本研究首次证实DYNLT1可与核孔蛋白结合，并在致白血病核孔蛋白融合蛋白NUP98-HOXA9引发的基因表达失调与造血细胞增殖诱导过程中发挥作用。