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. 2012 Oct;86(19):10524–10532. doi: 10.1128/JVI.01077-12

Fig 4.

Fig 4

Long-range interactions detected between the Mml regions and the 5′ c-myb region, including the promoter. Interactions between the 5′ c-myb region and regions up to −100 kb upstream were determined by 3C-qPCR. A HindIII fragment, including the c-myb promoter, was used as the bait. (A) The 3C-qPCR assay was performed on differentiated (M1 plus IL-6) and undifferentiated M1 cells, and the data show the cross-linking frequency between the upstream regions and the bait fragment. The locations of HindIII fragments are indicated below the graph. Mml1, Mml2, and Mml3 regions are marked by red arrows. Data are normalized to the ERCC3 internal cross-linking control (means and standard errors of the means [SEM]; n = 3). (B) Sequences of retrovirus integration sites in tumor cell lines. Virus-chromosomal DNA junction sequences were determined by a shotgun cloning method as described previously (34). Genomic DNA was digested with MseI and ligated with an adaptor (the sequence is available upon request). PCR was carried out using a viral LTR primer and an adaptor primer to amplify the junction sequences, followed by TA cloning and sequencing. Virus integration site sequences in tumor cell lines are shown as indicated. Red vertical arrows mark the virus integration sites. Black horizontal arrows indicate the orientation of retrovirus sequences inserted into the genome. A green solid rectangle with an arrow depicts the c-myb gene and its transcriptional orientation. (C) Long-range interactions detected between the 5′ c-myb region and Mml regions in tumor cells. 3C-qPCR assays were performed at the same time in M1 cells and tumor cell lines containing a provirus in one of the Mml regions. The upstream HindIII fragments examined in these experiments are indicated. Integration sites in tumor cell lines are marked by red arrows. The locations of integrated proviruses in cell lines 30C-18 (Mml1), 30-2-9 (Mml2), and 30-2-7 (Mml3) are depicted, and the genomic sequences at the virus-cell junctions are presented in panel B. The Mml3 location was previously determined by Southern blot analysis. Data are normalized to the ERCC3 internal cross-linking frequency control (means and SEM; n = 3). (D) 3C-qPCR assay of NIH 3T3 cells. Data are normalized to the ERCC3 control (means and SEM; n = 3).