additionally aﬀected by surrounding DNA sequences, by chromatin structure or by epigenetic events. Initiation proteins in terminally differentiated cells As cells undergo terminal diﬀerentiation, they enter an irreversible cell cycle arrest and generally lose the capacity to go through DNA replication and cell division. In previous studies, it has been proposed that the absence of proteins involved in initiation of DNA replication in quiescent or diﬀerentiated cells seems to be crucial for the maintenance of the ‘out-of-cycle’ state (22). To investigate the fate of replication proteins upon cell cycle exit, we took advantage of the in vitro diﬀerentiation of murine C2C12 myoblast cells to myotubes. These cells undergo terminal diﬀerentiation when exposed to a low mitogen medium. Using immunoblot analysis we demonstrated that ORC1 and -2 were abundant in proliferating cells, but the levels of these proteins were reduced after cells underwent diﬀerentiation. The reduction of ORC1 and -2 levels during diﬀerentiation is in agreement with a previous report showing that ORC1 and -2 are lacking in terminally diﬀerentiated Xenopus erythrocytes (45). ORC1 was reduced notably faster than ORC2, which suggests that ORC1 might be the limiting component of the active murine pre-RC. This is aﬃrmed by the fact that during normal cell cycle transit the activity of ORC1, as the only ORC subunit, is regulated by ubiquitination. This modiﬁcation results either in degradation of human ORC1 (11,46–48) or in dissociation of ORC1 from chromatin in hamster cells (6). In a similar manner, the degradation of ORC1 in terminally diﬀerentiated cells might contribute to the maintenance of the postmitotic state. Contrary to studies showing that ORC2 levels remain unaﬀected in human diﬀerentiated or quiescent cells (22,49,50) we observed that levels of ORC2 were reduced, but did not completely disappear, during terminal diﬀerentiation of murine C2C12 cells. A slower migrating soluble variant of ORC2 was observed in the course of diﬀerentiation, probably a result of phosphor- ylation. In S. cerevisiae (51), S. pombe (52) and Xenopus laevis (53) ORC2 is phosphorylated by CDK/cyclin complexes. The phosphorylation might result in confor- mational changes within the ORC and thereby probably inhibits binding of additional factors and prevents re-replication (54–56). ChIP experiments revealed that ORC1 dissociated from the pre-RC-binding site during diﬀerentiation, whereas ORC2, -4 and -5 were still bound to the OBR. The loss of ORC1 from chromatin in terminally diﬀerentiated cells is in agreement with the fast reduction of ORC1 shown by immunoblotting and could explain the function of this protein as regulator of the initiation of DNA replication in proliferating cells. Since ORC2 was present in the nucleus of myotubes at a very low level, but was detected by ChIP experiments at the OBR, this suggests that a persisting fraction of ORC2 remains bound at origins previously used in proliferating cells. This observation could also imply that ORC2 is not yet fully degraded upon diﬀerentiation and that soluble ORC2 is reduced ﬁrst and chromatin-bound ORC2 later. Nucleic Acids Research, 2007, Vol. 35, No. 20 6711 ORC4 and -5 remain bound to chromatin in diﬀerentiated cells. Formerly, it was also demonstrated that Orc2, -3 and -5 can be detected in non-proliferating human cells (50). Alternatively, the persistence of ORC subunits in non-cycling cells could be due to the fact that despite their function in initiation of DNA replication these proteins play additional roles, for example in transcrip- tional silencing (39,57,58). Taken together, the reduction and the chromatin dissociation of several proteins involved in the events of initiation of DNA replication could be demonstrated during myogenic diﬀerentiation of C2C12 cells. The loss of the important regulatory initiation protein ORC1 in terminally diﬀerentiated cells might contribute to the establishment and maintenance of the ‘out-of-cycle’ state, as a part of a highly sophisticated and regulated network. Nevertheless, the function of pre-RC proteins remaining at the OBR in terminally diﬀerentiated C2C12 cells remains unknown. ACKNOWLEDGEMENTS We are grateful to E. Dinkl for technical help and to R. Friedel for the performance of cell cycle analyses. We thank A. Chari, B. Laggerbauer and S. Meister for comments and discussions. This work was supported by the Deutsche Forschungsgemeinschaft. Funding to pay the Open Access publication charges for this article was provided by by the University of Wuerzburg. Conﬂict of interest statement. None declared. 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