Nucleic Acids Research, 2007, Vol. 35, No. 20 6709 Figure 6. Distinct pre-RC proteins are absent in terminally diﬀerentiated myotubes. (A) Myogenic diﬀerentiation of C2C12 cells. Phase contrast microscopy showing cultures containing terminally diﬀerentiated myotubes by day 5 in diﬀerentiation medium (DM) (upper panel). Cell cycle proﬁles of proliferating (54.4% G1 phase, 34.4% S phase, 11.2% G2 phase) and diﬀerentiated (95.6% G1 phase, 2.4% S phase, 1.9% G2 phase) C2C12 cells (middle panel). BrdU incorporation: representive areas examined by indirect immunoﬂuorescence microscopy demonstrate the cessation of DNA synthesis in diﬀerentiated C2C12 cells (lower panel). (B) Immunoblot analyses of pre-RC proteins during diﬀerentiation of C2C12 cells. Nuclear (NE) and soluble (SE) extracts were prepared from proliferating (p), conﬂuent (c) and diﬀerentiating C2C12 cells at the times indicated in diﬀerentiation medium (DM). Same amounts (20 mg) of extracts were dissolved by SDS-PAGE and immunoblot analyses were performed using antibodies raised against pre-RC proteins. GM: growth medium. (C) ChIP analyses of pre-RC components interacting with fragment B in the OBR region in proliferating C2C12 myoblasts or terminally diﬀerentiated C2C12 myotubes after ﬁve days in DM. ChIP analysis was performed as described above with the antibodies indicated. Representative results of three experiments are shown. terminally diﬀerentiated myotubes at all, whereas ORC2 was detectable, albeit at lower levels than in proliferating myoblasts. These data demonstrate that the loss of proliferation capacity of mammalian cells during diﬀer- entiation is associated with down-regulation of constitu- ents of the pre-RC. In vivo occupancy of the OBR region with pre-RC proteins in terminally differentiated myotubes We performed ChIP analyses comparing chromatin of proliferating cells to that of diﬀerentiated cells to elucidate if members of the pre-RC still occupy the OBR region in non-replicating cells. Formaldehyde cross-linked nuclear proteins of proliferating and diﬀerentiated C2C12 cells after 5 days in DM were prepared. Chromatin was immunoprecipitated with antibodies raised against ORC1, -2, -4 and -5 and was analyzed with primer pairs speciﬁc to the pre-RC assembly region (fragment B) and to the control region in the 5.8 S rRNA gene (fragment D). Preimmune serum served as control. Densitometric values of PCR results are shown in Supplementary Figure 2. All investigated proteins are bound to OBR sequences in asynchronously growing cells (Figure 6C, upper panel). When cells exit the cell cycle during diﬀerentiation, ORC1 is released from chromatin (Figure 6C, lower panel). ORC2, -4 and -5 remain bound to the OBR region in terminally diﬀerentiated cells. The control region in the 5.8 S rRNA gene showed no interaction with any of these proteins. These results probably indicate that chromatin associa- tion of ORC1 is regulated during transition from proliferation to terminal diﬀerentiation. DISCUSSION In the current work, we demonstrated that a pre-RC-binding site is located in a region positioned from 2519 to 2152 upstream the transcription start site of RNA polymerase I within the OBR of the murine rDNA gene cluster. This pre-RC-binding site has been veriﬁed by three independent approaches. First, we showed in vivo by ChIP experiments a cell cycle-dependent association of pre-RC proteins with this sequence in murine FM3A cells. Second, we demonstrated in vitro by EMSA a sequence-speciﬁc and ATP-stimulated bind- ing of protein complexes containing ORC proteins to this DNA region. Deletion mutagenesis revealed that this binding site is located within 119 bp at 2365 to 2247 and that a repeated element, CTCGGGAGA, is crucial for the assembly of pre-RC proteins. Third, we showed by ChIP experiments in terminally diﬀerentiated murine C2C12 cells that this binding site still exists, but that only a subset of pre-RC proteins is bound at this region in diﬀerentiated myotubes.