| 研究実績の概要 |
Replication of the mammalian DNA is mediated by a defined spatio-temporal program know as replication timing (RT), by which transcriptionally active regions in the genome replicate early while the transcriptionally inactive regions replicate late in the S-phase. To elucidate the molecular mechanisms that regulate RT, I have focused on a transcription coupled epigenetic mark known as H2Bub. By performing ChIP-seq and Repli-seq, I have found that H2Bub marks are enriched in the early replicating A-compartments. To manipulate H2Bub marks in mouse ES cells, I knocked out Rnf20 which is the E3 ubiquitin ligase for H2Bub. In Rnf20 knocked out cells H2Bub was depleted, as expected. Strikingly, early replication was perturbed in Rnf20 knocked out cells, revealing a link between RT and H2Bub. It is known that RT is strongly linked with 3D chromatin structure, and therefore I performed Hi-C analysis to reveal the 3D organization of the genome in WT or Rnf20 knocked out cells. I found that Rnf20 promotes homotypic interactions between A-A or B-B compartments, but represses heterotypic interactions between A-B or B-A compartment. Taken together, my study provides a molecular link mediated by Rnf20 and H2Bub to connect 3D genome folding with replication timing.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
I was able to construct conditional knockout ES cells for Rnf20, a protein that regulates H2Bub in mammals and other species. This cell line enabled me to directly test the impact of H2Bub depletion, by ablation of Rnf20, on replication timing. I was also able to carry out replication timing analysis in WT or Rnf20 knockout cells, and find that early replication is perturbed in the absence of Rnf20.
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| 今後の研究の推進方策 |
I plan to perform Hi-C analysis in WT or Rnf20 knockout cells to reveal if changes in the 3D genome structure is altered in the absence of Rnf20. 3D genome plays a direct role in replication timing, therefore if my experiments reveal perturbations in 3D genome folding in Rnf20 knockout cells, this will indicate that Rnf20 regulates replication timing via modulating the 3D genome.
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