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Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation.

Zellner E, Herrmann T, Schulz C, Grummt F - Nucleic Acids Res. (2007)

Bottom Line: Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus.During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses.ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biocenter at the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.

ABSTRACT
Eukaryotic DNA replication initiates at origins of replication by the assembly of the highly conserved pre-replicative complex (pre-RC). However, exact sequences for pre-RC binding still remain unknown. By chromatin immunoprecipitation we identified in vivo a pre-RC-binding site within the origin of bidirectional replication in the murine rDNA locus. At this sequence, ORC1, -2, -4 and -5 are bound in G1 phase and at the G1/S transition. During S phase, ORC1 is released. An ATP-dependent and site-specific assembly of the pre-RC at origin DNA was demonstrated in vitro using partially purified murine pre-RC proteins in electrophoretic mobility shift assays. By deletion experiments the sequence required for pre-RC binding was confined to 119 bp. Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus. During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses. ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

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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 µg) 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.
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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 µg) 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.

Mentions: To address this issue experimentally, we analyzed protein levels of pre-RC components during myogenic differentiation and determined whether components of the pre-RC are attached to the OBR in terminally differentiated myotubes. For that we used murine C2C12 cells, which can be induced to terminal differentiation into myotubes by exposure to low mitogen medium for several days. Differentiation was assessed by morphological examination. After 5 days in differentiation medium (DM), predominantly elongated myotubes were present in the culture (Figure 6A, upper panel). The percentage of S phase cells considerably decreased during myogenic differentiation (34.4% in proliferating versus 2.4% in differentiated cells) concomitantly with a significant increase in the percentage of myotubes in the G0/G1 phase of the cell cycle (54.4% in proliferating versus 95.6% in differentiated cells, middle panel). The cessation of DNA replication in myotubes could further be verified by BrdU incorporation. After five days in differentiation medium only two persisting myoblasts were replicating in the examined area (lower panel).Figure 6.


Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation.

Zellner E, Herrmann T, Schulz C, Grummt F - Nucleic Acids Res. (2007)

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 µg) 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.
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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 µg) 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.
Mentions: To address this issue experimentally, we analyzed protein levels of pre-RC components during myogenic differentiation and determined whether components of the pre-RC are attached to the OBR in terminally differentiated myotubes. For that we used murine C2C12 cells, which can be induced to terminal differentiation into myotubes by exposure to low mitogen medium for several days. Differentiation was assessed by morphological examination. After 5 days in differentiation medium (DM), predominantly elongated myotubes were present in the culture (Figure 6A, upper panel). The percentage of S phase cells considerably decreased during myogenic differentiation (34.4% in proliferating versus 2.4% in differentiated cells) concomitantly with a significant increase in the percentage of myotubes in the G0/G1 phase of the cell cycle (54.4% in proliferating versus 95.6% in differentiated cells, middle panel). The cessation of DNA replication in myotubes could further be verified by BrdU incorporation. After five days in differentiation medium only two persisting myoblasts were replicating in the examined area (lower panel).Figure 6.

Bottom Line: Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus.During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses.ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biocenter at the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.

ABSTRACT
Eukaryotic DNA replication initiates at origins of replication by the assembly of the highly conserved pre-replicative complex (pre-RC). However, exact sequences for pre-RC binding still remain unknown. By chromatin immunoprecipitation we identified in vivo a pre-RC-binding site within the origin of bidirectional replication in the murine rDNA locus. At this sequence, ORC1, -2, -4 and -5 are bound in G1 phase and at the G1/S transition. During S phase, ORC1 is released. An ATP-dependent and site-specific assembly of the pre-RC at origin DNA was demonstrated in vitro using partially purified murine pre-RC proteins in electrophoretic mobility shift assays. By deletion experiments the sequence required for pre-RC binding was confined to 119 bp. Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus. During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses. ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

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