Replication-independent chromatin loading of Dnmt1 during G2 and M phases - PubMed (original) (raw)

Replication-independent chromatin loading of Dnmt1 during G2 and M phases

Hariharan P Easwaran et al. EMBO Rep. 2004 Dec.

Abstract

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation.

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Figures

Figure 1

Figure 1

Dnmt1 shows cell-cycle-dependent association with chromatin. Live-cell imaging of C2C12 cells expressing full-length Dnmt1 fusion to GFP (GMT) and RFP–ligase. Selected frames from this time series are shown. At the time imaging was started (0 h), this cell was in mids phase. The arrowheads indicate preferential association of GMT with heterochromatic regions during late S and G2. (The images from mitosis and G1 in this assembly are from a different cell.) Scale bar, 5 μm.

Figure 2

Figure 2

Mapping of Dnmt1 domains responsible for replication foci association. DMAP corresponds to the DMAP1 transcriptional repressor interacting domain (Rountree et al, 2000). For other domains, see text. *Association takes place not only in S phase (see Fig 3).

Figure 3

Figure 3

TS mediates association of Dnmt1 with chromatin, preferentially constitutive heterochromatin, which is independent of H3K9 trimethylation. (A) Live-cell imaging of C2C12 cells expressing F-TS–GFP and RFP–ligase. Selected frames from this time series are shown. After 3.5 h from start of imaging, the cell has entered late S phase (deciphered from the pattern of RFP–ligase) and the large F-TS–GFP foci (arrows) show colocalization with RFP–ligase. (B) C2C12 cells expressing GFP-tagged Dnmt1 lacking TS (GMTΔTS) analysed at the different cell cycle stages (for strategy, see supplementary Fig 1 online. (C) TS preferentially associates with constitutive heterochromatin and not with facultative heterochromatin. C2C12 cell expressing TS–GFP was immunostained with anti-trimethylated H3K27 antibody labelling the inactive X chromosome and DNA was visualized with TOPRO-3. These C2C12 cells are polyploid and have two inactive X chromosomes (H.P. Easwaran et al, unpublished observations). (D) PMEFs from wild-type and Suvar39h1/2 double-null mice expressing TS–GFP and FLAG-tagged HP1β were stained with anti-FLAG antibody and centromeric heterochromatin was visualized with Hoechst 33258. Scale bars, 5 μm.

Figure 4

Figure 4

Recovery of GFP-tagged Dnmt1 after photobleaching indicates de novo loading of Dnmt1 onto chromatin throughout G2. (A) FRAP series of a typical G2 nucleus expressing GMT (green) and RFP–PCNA (red). An area containing a heterochromatic focus was bleached (inset). Scale bar, 5 μm. (B) Recovery curve of the FRAP experiment shown in (A). Half-time of recovery is 11 s. (C) FRAP data of five nuclei were averaged. Mean curve (solid line) and standard deviation (dotted lines) are shown. Half-time of recovery of the mean curve is 11.7 s.

Figure 5

Figure 5

Summary of the role of PBD and TS in controlling the dynamic association of Dnmt1 with chromatin during the cell cycle. PBD directs association of Dnmt1 with RF throughout the S phase and TS mediates association with chromatin, preferentially heterochromatin, during G2 and M.

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