Regenerative Medicine Program, Ottawa Hospital Research Institute, Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6.
J Proteome Res. 2012 Aug 3;11(8):4326-37. doi: 10.1021/pr3004369. Epub 2012 Jul 3.
Mutations in PHF6 are the cause of Börjeson-Forssman-Lehman syndrome (BFLS), an X-linked intellectual disability (XLID) disorder, and both T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The PHF6 gene encodes a protein with two plant homeodomain (PHD)-like zinc finger domains. As many PHD-like domains function to target chromatin remodelers to post-translationally modified histones, this suggests a role for PHF6 in chromatin regulation. However, PHD domains are usually found in association with a catalytic domain, a feature that is lacking in PHF6. This distinct domain structure and the minimal information on its cellular function prompted us to perform a proteomic screen to identify PHF6 binding partners. We expressed recombinant Flag-tagged PHF6 in HEK 293T cells for coimmunoprecipitation, and analyzed the purified products by mass spectrometry. We identified proteins involved in ribosome biogenesis, RNA splicing, and chromatin regulation, consistent with PHF6 localization to both the nucleoplasm and nucleolus. Notably, PHF6 copurified with multiple constituents of the nucleosome remodeling and deacetylation (NuRD) complex, including CHD4, HDAC1, and RBBP4. We demonstrate that this PHF6-NuRD complex is not present in the nucleolus but is restricted to the nucleoplasm. The association with NuRD represents the first known interaction for PHF6 and implicates it in chromatin regulation.
PHF6 基因突变是 Börjeson-Forssman-Lehman 综合征（BFLS）的病因，BFLS 是一种 X 连锁智力障碍（XLID）疾病，同时也是 T 细胞急性淋巴细胞白血病（T-ALL）和急性髓系白血病（AML）的病因。PHF6 基因编码一种具有两个植物同源结构域（PHD）样锌指结构域的蛋白质。由于许多 PHD 样结构域的功能是将染色质重塑剂靶向到翻译后修饰的组蛋白上，这表明 PHF6 在染色质调节中发挥作用。然而，PHD 结构域通常与催化结构域相关联，而 PHF6 缺乏这一特征。这种独特的结构域结构和对其细胞功能的有限信息促使我们进行蛋白质组筛选以鉴定 PHF6 的结合伙伴。我们在 HEK 293T 细胞中表达重组 Flag 标记的 PHF6 进行共免疫沉淀，并通过质谱分析纯化产物。我们鉴定出参与核糖体生物发生、RNA 剪接和染色质调节的蛋白质，这与 PHF6 定位于核质和核仁相一致。值得注意的是，PHF6 与核小体重塑和去乙酰化（NuRD）复合物的多个组成部分共沉淀，包括 CHD4、HDAC1 和 RBBP4。我们证明这种 PHF6-NuRD 复合物不存在于核仁中，而是局限于核质中。与 NuRD 的关联是 PHF6 的第一个已知相互作用，并暗示其参与染色质调节。
