哺乳類のDNA複製タイミング制御におけるゲノムとエピゲノムの機能的リンク

2021 Fiscal Year Research-status Report

• Project/Area Number: 21K06129
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: SHARIF JAFAR 国立研究開発法人理化学研究所, 生命医科学研究センター, 専任研究員 (00577968)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: Replication timing / Transcription / Epigenome

Outline of Annual Research Achievements

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.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

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.

Strategy for Future Research Activity

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.

Causes of Carryover

The current research has progressed well during the first two years. Therefore, the necessity of buying reagents were reduced than previously planned. This is the reason for the incurring amount to be used next fiscal year. The incurring amount will be used for buying reagents and performing next generation sequencing next year.