Regulator of chromatin condensation 1 (RCC1) is the only known guanine nucleotide-exchange factor for the Ran GTPase and has pivotal roles in nucleo-cytoplasmic transport, mitosis, and nuclear-envelope assembly 1 . RCC1 associates dynamically with chromatin through binding to histones H2A and/or H2B in a Ran-regulated manner 2,3 . Here, we report that, unexpectedly, the amino-terminal serine or proline residue of RCC1 is uniquely methylated on its α-amino group. Methylation requires removal of the initiating methionine, and the presence of proline and lysine at positions 3 and 4, respectively. Methylation-defective mutants of RCC1 bind less effectively than wild-type protein to chromatin during mitosis, which causes spindle-pole defects. We propose a bimodal attachment mechanism for RCC1 in which the tail promotes stable RCC1 association with chromatin through DNA binding in an α-N-methylation-dependent manner. These data provide the first known function for N-terminal protein methylation.RCC1 has a propeller-like structure, one face of which binds to Ran 4 , whereas the other face binds chromatin. A flexible N-terminal tail contains a nuclear localization signal (NLS) 5,6 . Although the propeller seems rigid, Ran allosterically modulates RCC1 association with chromatin 3 , and the RCC1 D182A mutant, which does not detectably bind Ran, shows reduced chromatin association 7,8 . Removal of the N-terminal tail also leads to exclusion of RCC1 from chromosomes 7 , whereas phosphorylation on Ser 2 and Ser 11 in human RCC1 reportedly permits stable association with mitotic chromosomes 8,9 . These phosphorylations have been proposed to reduce the affinity of the NLS for its nuclear transport receptor, Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ 4 Correspondence should be addressed to I.G.M. (igm9c@virginia.edu).Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONST.C. made the RCC1 mutant proteins, identified the methyltransferase activity and its recognition motif, and performed the assays to identify its function. T.L.M. and J.S. performed the mass spectrometry. C.E.S.-T. made the antibodies against methylated RCC1 and performed the experiments with them. D.F.H. directed the mass spectrometry. I.G.M. directed the biochemical and cell biological studies. T.C. and I.G.M. wrote the paper. All authors discussed results and contributed to the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript importin-α3, which, when bound to RCC1, interferes with chromatin attachment 9 . However, Ser 2 is found only in simians and is a proline residue in other mammalian species.The N-terminus of RCC1 resembles histone tails, which also contain NLSs, and which are phosphorylated, acetylated and methylated in complex patterns that comprise a histone code 10,11 . We asked whether ...