| Accession | GMS-169-1
URL copied to the clipboard |
| Title | Replication-dependent histone labeling dissects the physical properties of euchromatin/heterochromatin in living human cells |
| Submit date | 2025-05-08 10:47:41 |
| Last update date | 2025-05-08 12:12:17 |
| Contact | Kazuhiro MAESHIMA kmaeshim AT nig.ac.jp National Institute of Genetics & SOKENDAI |
| Sharing | |
| Total file size | 135.47 MB |
| Keywords | Cell cycle Mesoscale Chromatin motion Interphase S phase Euchromatin Heterochromatin HeLa RPE-1 DNA replication |
| Experiment type | Single-nucleosome imaging microscopy Live-cell imaging Cell lines |
| Summary | Chromatin is organized in the cell with distinct genome functions, from euchromatin to heterochromatin. Understanding physical differences between them is crucial, yet specific labeling methods in living cells remain limited. We developed replication-dependent histone (Repli-Histo) labeling to mark nucleosomes in euchromatin and heterochromatin based on DNA replication timing. This approach enables investigation of local nucleosome motion across the four chromatin classes with distinct labeling patterns, classes IA and IB (euchromatin) and classes II and III (heterochromatin), in living human cells. More euchromatic regions (earlier-replicated) show greater nucleosome motion, while more heterochromatic regions (later-replicated) exhibit lesser motion. The motion profile in each chromatin class persists throughout interphase. Two-point MSD analysis from dual-color single-nucleosome imaging shows that euchromatin behaves more like a liquid and heterochromatin acts more like a gel. |
| Citation(s) | Minami K, Nakazato K, Ide S, Kaizu K, Higashi K, Tamura S, Toyoda A, Takahashi K, Kurokawa K, Maeshima K. Replication-dependent histone labeling dissects the physical properties of euchromatin/heterochromatin in living human cells. Sci Adv. 2025 Mar 28;11(13):eadu8400. doi: 10.1126/sciadv.adu8400. Epub 2025 Mar 28. PMID: 40153514; PMCID: PMC11952110. https://doi.org/10.1126/sciadv.adu8400 |
| Organism | Human |
| Cell (Tissue) | HeLa and RPE-1 |
| Protocol | For replication-dependent histone (Repli-Histo) labeling, asynchronous cells expressing H3.2-HaloTag were incubated with 10 µM 7-bromo-1-heptanol (7BRO) for 1 h, then washed three times. The cells were then exposed to HaloTag TMR/JF646 ligands for 30 min, subsequently washed three times before imaging. The labeled patterns (classes IA/IB/II/III) were categorized based on previous studies. G1-synchronized HeLa cells were obtained by labeling asynchronous cells with Repli-Histo, chasing for 7 h, then treating with 400 µM L-mimosine for 17 h. CDC45-Halo in HeLa cells was labeled with 5 nM HaloTag JF646 ligand for 30 min, followed by overnight labeling with 10 nM HaloTag TMR ligand. S-phase cells with chromatin-bound CDC45-Halo foci were observed. Single nucleosomes or CDC45 were observed using a Nikon Ti2 with oblique illumination, imaging at 50 ms/frame with an ORCA-Fusion BT. Dual-color imaging was performed with a beam splitter W-VIEW GEMINI. |
| Data processing | For single-molecule movement analysis, the displacement and mean square displacement (MSD) of the fluorescent dots were calculated based on their trajectory. To analyze the relative movements of two nucleosomes labeled with two colors (TMR and JF646), we picked closely located (< 150 nm) pairs of TMR and JF646 trajectories from each image movie. The MSD between them (two-point MSD) was calculated based on the relative distance between the nucleosome dot pairs. [see https://doi.org/10.1126/sciadv.adu8400 for more information] |
| [902] sciadv.adu8400_movie_s05.mp4
A living HeLa cell with the euchromatin (class IA) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.37 MB MD5: 54ee66f82cedec3c2b6b4ea942911185 |
| [903] sciadv.adu8400_movie_s06.mp4
A living HeLa cell with the euchromatin (class IB) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.33 MB MD5: 993d3e400c30dcdaa701295abf989b90 |
| [904] sciadv.adu8400_movie_s07.mp4
A living HeLa cell with the heterochromatin (class II) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.24 MB MD5: 86b74d031bbd3e14d9b6a150c2956c02 |
| [905] sciadv.adu8400_movie_s08.mp4
A living HeLa cell with the heterochromatin (class III) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 5.76 MB MD5: c29c1cf7fe636ae4662ac6b93c536861 |
| [906] sciadv.adu8400_movie_s09.mp4
A living RPE-1 cell with the euchromatin (class IA) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.07 MB MD5: 7e4ed8dc2dbaa13530aab044a96f0119 |
| [907] sciadv.adu8400_movie_s10.mp4
A living RPE-1 cell with the euchromatin (class IB) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.31 MB MD5: f23539028c1e3055dfa66bb6b77b0a44 |
| [908] sciadv.adu8400_movie_s11.mp4
A living RPE-1 cell with the heterochromatin (class II) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.32 MB MD5: ff343bc323ccb524c242ce18158b10fd |
| [909] sciadv.adu8400_movie_s12.mp4
A living RPE-1 cell with the heterochromatin (class III) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.49 MB MD5: bd9a58b28ab06fd95b169e4a815da0e8 |
| [910] sciadv.adu8400_movie_s13.mp4
A G1-synchronized living HeLa cell with the euchromatin (class IA) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.13 MB MD5: 05b10e40e52eecbea8143d0dcc1e4069 |
| [911] sciadv.adu8400_movie_s14.mp4
A G1-synchronized living HeLa cell with the euchromatin (class IB) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.32 MB MD5: 03d74b10f17dcf95e4b5040d0746d32b |
| [912] sciadv.adu8400_movie_s15.mp4
A G1-synchronized living HeLa cell with the heterochromatin (class II) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.44 MB MD5: 7fdb1cc6ec71fd253a7964b05e59b2f4 |
| [913] sciadv.adu8400_movie_s16.mp4
A G1-synchronized living HeLa cell with the heterochromatin (class III) Repli-Histo labeling (left) and corresponding single nucleosomes (right). Scale bar: 5 μm. video/mp4 6.31 MB MD5: 95d7dcfb72c284af2b9b99ceac35f768 |
| [914] sciadv.adu8400_movie_s18.mp4
A living S-phase HeLa cell with the euchromatin (class IA)-bound CDC45-Halo foci (left) and corresponding single CDC45-Halo molecules (right). Scale bar: 5 μm. video/mp4 6.40 MB MD5: 05dffe0eb1109fad7b7be26856efca93 |
| [915] sciadv.adu8400_movie_s19.mp4
A living S-phase HeLa cell with the euchromatin (class IB)-bound CDC45-Halo foci (left) and corresponding single CDC45-Halo molecules (right). Scale bar: 5 μm. video/mp4 6.40 MB MD5: 5ea6d548892cdf4afac60599a83cdaf8 |
| [916] sciadv.adu8400_movie_s20.mp4
A living S-phase HeLa cell with the heterochromatin (class II)-bound CDC45-Halo foci (left) and corresponding single CDC45-Halo molecules (right). Scale bar: 5 μm. video/mp4 6.31 MB MD5: 215e43a6a46a06b4e137d00454a616ed |
| [917] sciadv.adu8400_movie_s21.mp4
A living S-phase HeLa cell with the heterochromatin (class III)-bound CDC45-Halo foci (left) and corresponding single CDC45-Halo molecules (right). Scale bar: 5 μm. video/mp4 6.43 MB MD5: 854d811b33ea4491f878d41039ac5a80 |
| [918] sciadv.adu8400_movie_s22.mp4
Dual-color single-nucleosome imaging with the euchromatin-specific (class IA) Repli-Histo labeling in a live HeLa cell. The two channels were recorded simultaneously. Scale bar: 5 μm. video/mp4 6.98 MB MD5: 314ab0b024137010b804b75f0e5e18a8 |
| [919] sciadv.adu8400_movie_s23.mp4
A magnified view of Movie [918], showing the closely labeled single-nucleosome pairs (yellow arrows). Scale bar, 1 μm. video/mp4 6.58 MB MD5: ef1d4d7f6bae0868ed4d78331ad6d593 |
| [920] sciadv.adu8400_movie_s24.mp4
Dual-color single-nucleosome imaging with the heterochromatin-specific (class II) Repli-Histo labeling in a live HeLa cell. The two channels were recorded simultaneously. Scale bar: 5 μm. video/mp4 7.21 MB MD5: b958112930c993cb761ae7fad382992b |
| [921] sciadv.adu8400_movie_s25.mp4
Dual-color single-nucleosome imaging with the euchromatin-specific (class IA) Repli-Histo labeling in a formaldehyde-fixed HeLa cell. The two channels were recorded simultaneously. Scale bar: 5 μm. video/mp4 6.96 MB MD5: cbf9d4e5309bcacb65732e3e504cebf0 |
| [922] sciadv.adu8400_movie_s26.mp4
Dual-color single-nucleosome imaging with the heterochromatin-specific (class II) Repli-Histo labeling in a formaldehyde-fixed HeLa cell. The two channels were recorded simultaneously. Scale bar: 5 μm. video/mp4 7.10 MB MD5: 74327623618d3d446f574efcd43e6012 |
