Transcription-replication conflicts drive R-loop-dependent nucleosome eviction and require DOT1L activity for...

Area of Science:

• Molecular Biology
• Epigenetics
• Genomics

Background:

• Transcription and replication are fundamental cellular processes that can interfere with each other, leading to transcription-replication conflicts (TRCs).
• TRCs can destabilize the genome by altering chromatin structure and epigenomic landscapes.
• Understanding the dynamic molecular events at TRC sites is crucial for comprehending genome instability and its role in diseases.

Purpose of the Study:

• To engineer a novel inducible reporter system for studying transcription-replication conflicts (TRCs).
• To characterize the dynamic changes in local chromatin structure and epigenome at TRC sites.
• To investigate the role of H3K79 methylation in the resolution of TRCs and transcription recovery.

Main Methods:

• Development of an inducible reporter system using a genome-integrated R-loop-prone sequence.
• Characterization of dynamic chromatin structure changes, including nucleosome occupancy and replication fork progression.
• Analysis of global replication stress response and specific epigenetic modifications, such as H3K79 methylation.
• Inhibition of the H3K79 methyltransferase DOT1L to assess its impact on transcription and DNA damage.

Main Results:

• Inducible TRCs lead to reduced nucleosome occupancy and replication fork blockage at the conflict sites.
• A small number of induced TRCs trigger a measurable global replication stress response.
• TRC formation is associated with a specific increase in H3K79 methylation at R-loop forming sites.
• DOT1L inhibition exacerbates DNA damage and reduces transcriptional output, indicating H3K79 methylation's role in TRC resolution.

Conclusions:

• TRCs induce significant dynamic changes in local chromatin structure and epigenome.
• H3K79 methylation is a TRC-dependent epigenetic mark that facilitates transcription recovery and resolution.
• This epigenetic bookmarking mechanism at TRC sites is relevant to diseases like cancer.