Figure 5.

c-Myc associates with the rDNA IGS region in a growth-dependent fashion and is physically associated with the gene looping structures that are found in growing cells. (A) Quantitative real-time PCR ChIP assays to detect association of c-Myc with rDNA in starved HeLa cells before (−serum) and after addition of serum (+serum) or serum and 10058-F4 (+serum, +10058-F4). The upper panel shows a human rDNA repeat marked with locations of E boxes (canonical and non-canonical) and primer pairs for qPCR. Binding values are expressed relative to levels detected after parallel ChIP reactions with IgG from non-immunized rabbit. Error bars show standard deviation about the mean (n ≥ 4). (B) c-Myc is physically associated with rDNA-IGS-mediated gene looping structures in growing HeLa cells. The re-ligated rDNA chromatin, immunoprecipitated by a specific antibody (Myc) or agarose beads as a negative control (N), is amplified by using primer pairs throughout the rDNA IGS region (sequences of primer sets are shown in Supplementary Table S6). The expected migration of the ligation product is shown by the positive control lane (C). (C) A schematic diagram describing the main conclusions of the work. Hypomethylated (potentially active) rDNA genes associate with nucleolar matrix via the IGS in a growth- and Myc-dependent manner. Matrix association appears to be the cause of the growth-related gene looping structures that characterize the rDNA in growing cells. Myc is associated with the growth-stimulated gene looping structures via interaction with the rDNA IGS.