6702 Nucleic Acids Research, 2007, Vol. 35, No. 20 In lower eukaryotes, origins can be deﬁned by rather short consensus elements (23). Metazoan origins, however, are less speciﬁed and can probably extend over thousands of base pairs. So far about two-dozen metazoan origins have been described, but they do not share any consensus sequences (24–26). Even so, sequence-speciﬁc and ATP- dependent binding of Drosophila melanogaster ORC has been observed at a control element in the chorion ampliﬁcation loci (27). Three described human origins, including the lamin B2 origin, the origin in the PRKDC/ MCM4 intergenic region, and that in the region upstream of the human TOP1 gene have been shown to interact with the ORC in vivo (5,28,29). However, it has also been shown that ORC exhibits sequence-independent DNA binding. For instance, puriﬁed human ORC binds preferentially to synthetic AT-rich polynucleotides, but does not discrimi- nate between natural DNA fragments with an origin and control fragments (30). Another work demonstrated that mammalian ORC binds to an extrachromosomally repli- cating plasmid in a sequence-independent manner (31). Even if it is commonly accepted that DNA replication initiates at speciﬁc sites, it is not clear whether speciﬁc consensus sequence elements are used to specify origins or whether higher order regulation, like chromatin organiza- tion, is more important. As an experimental system to explore the initiation of DNA replication we used murine rRNA genes. Approximately 400 tandemly repeated rDNA units, each 44 kb in size, are clustered in the nucleolar organizing regions of mouse cells. In the murine rRNA genes, one origin of bidirectional replication (OBR) was mapped by nascent strand length determination within a 3 kb region centered 1.6 kb upstream of the rDNA transcription start site of RNA polymerase I (32). In this report, we identiﬁed by chromatin immunopre- cipitation (ChIP) assays in vivo a pre-RC-binding site in the OBR of the murine rDNA cistron. At this site, ORC subunits 2, 4 and 5 are bound throughout the cell cycle whereas ORC1 assembles in a cell cycle-dependent manner. By electrophoretic mobility shift assays (EMSA) with partially puriﬁed nuclear extracts this binding site was conﬁrmed and conﬁned to a 119 bp DNA sequence. A protein complex containing ORC2 showed site-speciﬁc and ATP-stimulated DNA binding. Nucleotide substitutions in two 9 bp sequence elements within the 119 bp pre-RC assembly site reduced the formation of this DNA/protein complex signiﬁcantly. We further demonstrated by immunoblot analyses the loss of speciﬁc pre-RC proteins during myogenic diﬀerentia- tion of C2C12 cells. ChIP analyses revealed that ORC1 is lost from the pre-RC-binding site during terminal diﬀerentiation, but that a core complex of the pre-RC consisting of ORC2, -4, -5, still occupies the pre- RC-binding site in terminally diﬀerentiated myotubes. MATERIALS AND METHODS Cell culture and synchronization Murine FM3A cells were maintained at a density of 2 105 cells/ml in 1 RPMI-1640 medium (PAA, Austria), supplemented with 10% fetal calf serum at 378C under 5% CO2. Cells were synchronized in G1 phase by adding mevastatin (10 mM, ﬁnal), at the G1/S transition by adding mimosine (0.5 mM, ﬁnal) and in S phase by adding hydroxyurea (1 mM, ﬁnal) for at least 30 h. The synchronization in cell cycle was checked by propidium iodide staining of the cells followed by ﬂow cytometry using FACScan analysis. Analysis was carried out on a BD LSR System (Becton-Dickinson) and cell cycle phases were estimated using the MultiPlus software (Phoenix Flow Systems). Data are presented as histograms showing relative DNA content (x-axis) and cell number (y-axis). Mouse C2C12 myoblasts were maintained at subcon- ﬂuent densities in Dulbecco’s modiﬁed Eagle’s medium (PAA, Austria) supplemented with 10% fetal calf serum (growth medium, GM) at 378C under 10% CO2. To prepare diﬀerentiated multinucleated myotubes, myo- blasts were shifted to DMEM containing 2% horse serum (diﬀerentiation medium, DM) when the cells reached 70–80% conﬂuence. For the ﬁrst 12 h, the diﬀerentiation medium included 10 mg/ml insulin (Sigma), afterwards 10 mM cytosine b-D-arabinofurano- side (Ara-C, Sigma) was added to eliminate still replicat- ing non-diﬀerentiated cells. The media were changed every day. Antibodies Antibodies used in this work were generated against individual initiator proteins by immunization of rabbit (ORC2, ORC4, ORC5, ORC6) and sheep (ORC1) with recombinant proteins. Polyclonal antisera were aﬃnity puriﬁed on antigen columns. The speciﬁcity of antibodies has been described earlier (33). As a negative control in ChIP experiments corresponding to rabbit and sheep preimmune sera, respectively, were used. PCR primer pairs Fragment A: 50-GAA AGC AAA TCA CTA TGA AGA G-30 (2798f), 50-GCA CAG TTA GGC ACA GTT AGG- 30 (3136r); fragment B: 50-CCT AAC TGT GCC TAA CTG TGC-30 (3116f), 50-CGT TCC CGA AAC TTG TCA CC-30 (3483r); fragment C: 50-AAT GAG TGA GTG AAT GTG GCG-30 (3380f), 50-GTC CTC TCG GCC TCA GAT GTA-30 (3815r); fragment D: 50-CAC GTG TCT CGT TTC GTT CC-30 (12361f), 50-ACA ACC GCC CAC ACG TCT G-30 (12790r); 5."100: 50-TAC TTT CGT TTT TGG GTG CC-30 (3218f); 5."200: 50-GTA CGC TGC TCC GTC GTG-30 (3313f); 3."100: 50-CTC ACT CAT TCT GTC TCT CCG-30 (3389r); 3."200: 50-ACA AAC GGT CCC CAT CGT-30 (3287r); 119 bp: 50-ACG ATG GGG ACC GTT TGT-30 (3270f), 50-TCA CTC ATT CTG TCT CTC CGG-30 (3388r). Numbers in parenthesis indicate the location of oligonu- cleotides within the Mus musculus 45S pre rRNA gene (accession number: X82564). In vivo cross-linking and nuclear extract preparation DNA/protein cross-linking was performed by incubating FM3A (2 107 per condition) with 1% (v/v) formalde- hyde for 10 min at RT under gentle agitation. The reaction