Klocko Andrew D, Rountree Michael R, Grisafi Paula L, Hays Shan M, Adhvaryu Keyur K, Selker Eric U
Institute of Molecular Biology, University of Oregon, Eugene, Oregon, United States of America.
PLoS Genet. 2015 Mar 20;11(3):e1005083. doi: 10.1371/journal.pgen.1005083. eCollection 2015 Mar.
Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6(dim-3) allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.
异染色质及相关的基因沉默过程在发育、基因组防御和染色体功能中发挥作用。在许多物种中，组成型异染色质被赖氨酸9三甲基化的组蛋白H3（H3K9me3）和胞嘧啶甲基化修饰。在粗糙脉孢菌中，一个由五种蛋白质组成的复合物DCDC催化H3K9甲基化，进而指导DNA甲基化。在此，我们鉴定并表征了一个对DCDC功能至关重要的基因dim-3（甲基化缺陷-3），它编码核输入伴侣蛋白NUP-6（输入蛋白α）。dim-3中的关键突变导致NUP-6的一个ARM重复序列发生替换，并导致H3K9me3和DNA甲基化大量缺失。令人惊讶的是，在dim-3菌株中，所有参与组蛋白和DNA甲基化的已知蛋白质以及一种典型的转运底物的核运输似乎正常。DCDC成员之间的相互作用似乎也正常，但nup-6(dim-3)等位基因导致DCDC成员DIM-5和DIM-7从异染色质中错误定位，并且NUP-6dim-3自身也从核膜错误定位，至少在分生孢子中是这样。SAGA组蛋白乙酰转移酶复合物的成员GCN-5在dim-3中也表现出定位改变，这增加了NUP-6在核质运输后定位多个染色质复合物所必需的可能性。
