Dissection of cell cycle–dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling

Abstract

DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle–dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

Quantitative Profiling of Chromatome Dynamics Reveals a Novel Role for HP1BP3 in Hypoxia-induced Oncogenesis

“…S3B and S3D), indicating that hypoxia stress substantially reduced their binding to chromatin. As chromatin organizing proteins and molecular platforms such as the condensin complex are closely associated with cell cycle regulation, it is possible that reduced chromatin binding by proteins in clusters a and c could be attributed to cell cycle disruption due to hypoxia stress (26,27). Consistent with these proteomic data, our cell cycle analysis of cancer cells that had been subjected to hypoxia revealed a corresponding cell cycle arrest in G 0 -G 1 phase (supplemental Figs.…”

RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

“…We considered that the main site of demethylation might occur in Satellite 2 repeat sequences (SAT2) for 3 reasons. First, the RFTS domain mediates association of DNMT1 to pericentromeric heterochromatin to maintain dense methylation 47,48 and SAT2 is the most abundant repeat in the region. 49 Second, SAT2-specific hypomethylation has been found in DNMT1-null cells 50,51 and in patients with RFTS-mutated DNMT1.…”

Bayesian mixed-effects model for the analysis of a series of FRAP images

“…In this paper, we use FRAP data sets of GFP-Dnmt1 expressed in mouse C2C12 myoblast cells (Schneider et al, 2013), which were obtained from multiple cell nuclei and can therefore be utilized to illustrate how our nonlinear regression model can be used to fit all available data at once. DNA methylation at position 5 of cytosines within CpG dinocleotide sequences is an important biochemical process for the stable epigenetic gene silencing in vertebrates (Bird, 2002, Spada et al, 2006.…”

“…To date, for the analysis of data from FRAP experiments for the same molecule on multiple similar cell nuclei with a mathematical model, either the data of each recovery curve is analyzed separately, and the results for all cell nuclei are regarded together (e.g., Schneider et al, 2013), or the data of the different cell nuclei are pooled, averaged and then analyzed (e.g., McNally, 2008). In the second case, the recovery curves of the cell nuclei are averaged to obtain a smooth curve that can then be analyzed by the same mathematical model that is usually used for the analysis of one recovery curve.…”

“…See Sprague and McNally (2005) for more information on FRAP experiments. In this paper, we concentrate on half-nucleus FRAP [as opposed to circle FRAP or strip FRAP (Sprague et al, 2004;Mueller et al, 2008)], which should cover representative fractions of heterogeneously distributed binding sites in all cell cycle stages (Schneider et al, 2013). An example for such data is given in Figure 1.…”