Direct Observation of Cell-Cycle-Dependent Interactions between CTCF and Chromatin

Abstract

The three-dimensional arrangement of chromatin encodes regulatory traits important for nuclear processes such as transcription and replication. Chromatin topology is in part mediated by the architectural protein CCCTC-binding factor (CTCF) that binds to the boundaries of topologically associating domains. Whereas sites of CTCF interactions are well characterized, little is known on how long CTCF binds to chromatin and how binding evolves during the cell cycle. We monitored CTCF-chromatin interactions by live cell single molecule tracking in different phases of the cell cycle. In G1-, S-, and G2-phases, a majority of CTCF molecules was bound transiently (∼0.2 s) to chromatin, whereas minor fractions were bound dynamically (∼4 s) or stably (>15 min). During mitosis, CTCF was mostly excluded from chromatin. Our data suggest that CTCF scans DNA in search for two different subsets of specific target sites and provide information on the timescales over which topologically associating domains might be restructured. During S-phase, dynamic and stable interactions decreased considerably compared to G1-phase, but were resumed in G2-phase, indicating that specific interactions need to be dissolved for replication to proceed.

Figure 1

Comparison of residence times and frequencies of SiR-Halo-CTCF-chromatin interactions between different cell cycle phases. (A) Shown here are chromatin residence times of SiR-Halo-CTCF in G1-, S-, and G2-phases. The symbol area is proportional to the fraction of molecules exhibiting the corresponding binding time. Error bars denote SD. (B) Shown here are relative frequencies of SiR-Halo-CTCF molecules exhibiting transient (light blue), dynamic (blue), and stable (dark blue) interactions with chromatin in S- and G2-phases compared to G1-phase. (Left inset) Shown here are fractions of molecules bound to chromatin (dark red) and of molecules exhibiting effective diffusion (red and light red). (Right inset) Shown here are fractions of molecules exhibiting transient (light blue), dynamic (blue), and stable (dark blue) interactions with chromatin in G1-, S-, and G2-phases. Error bars denote SD. To see this figure in color, go online.

Figure 2

Shown here is a model of cell-cycle-dependent CTCF-chromatin interactions. To see this figure in color, go online.