Moreover, unbiased analysis of potential genome interactions using Hi-C clearly shows that intra-chromosomal interactions within CTs are at least 2 orders of magnitude more frequent than inter-chromosomal interactions ; this level is consistent with the potential for chromatin mixing described herein. Hence, while the formation of extensive interaction networks within mammalian cells appears to conflict with the idea that individual CTs are spatially self-contained , dynamic changes at the interaction interfaces of neighboring CTs can be sufficient to allow the formation of widespread gene interactions while preserving CTs as higher-order chromatin structures. As a growing body of evidence supports the formation of cell type specific ��interactomes�� during cell differentiation , it is important to understand how different patterns of gene expression correlate with the formation of interaction networks and how these interactions define spatial and temporal changes in genome structure and function within individual cells. HeLa cells were grown in the presence of different dTTP analogues to label sites of DNA synthesis, as described in detail by Maya-Mendoza et al. . The following precursors were used: AlexaFluor488-dUTP ; Cy3-dUTP; biotin-dUTP and bromo-deoxyuridine . AF488-dUTP and Cy3-dUTP were visualized either in living cells using time-lapse light microscopy or by confocal microscopy after fixation using routine procedures. For fixation, cells growing on glass coverslips were rinsed briefly in PBS . These fixation conditions preserved the structure of chromatin domains present in living cells and no changes in structure of the chromatin foci was seen under the imaging conditions used. Fixed cells were washed 36in PBS, Nutlin-3 treated with 0.5% Triton 6100 in PBS, rinsed 36in PBS, incubated with 5 mg/ml Hoechst 33258 for 10 min, rinsed 36 in PBS and mounted with either Vectashield or Prolong mounting media. Alternatively, DNA foci were labeled by indirect immuno-fluorescence . Where secondary fluorescent antibodies were replaced by Qdots the following changes were applied: 1) permeabilization was altered to 1% Triton 6100 for 10 min; 2) Qdots were applied to coverslips in 24 well plates and incubation performed for 15 h at 4uC with shaking ; fixation, primary antibody incubation and washes were as for routine immuno-labeling. We note that in our hands the performance of Qdots was very variable from batch to batch, with some batches giving high background staining. Qdots were from Invitrogen: streptavidin LY2109761 conjugated Qdot-525 was used to detect biotin labeled CTs and secondary anti-rat antibody conjugated with Qdot-605 to detect BrdU. TSA was purchased from Sigma. Western blotting was performed as described using appropriate antibodies , as shown. For confocal imaging, samples were examined using a Zeiss LSM510META confocal microscope following well-established imaging protocols .