DROSHA、DICER以及由损伤诱导的长链非编码RNA（long ncRNA）在DNA双链断裂处调控依赖于BMI1的转录抑制机制

《Cell Reports》：DROSHA, DICER, and damage-induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break

DROSHA和DICER通过招募BMI1到DNA双链断裂（DSB）位点，诱导染色质修饰和转录沉默（DISC），调控DNA损伤响应。

　　
The study investigates the regulatory role of DROSHA and DICER in DNA damage response (DDR) and transcriptional silencing at double-strand break (DSB) sites. Here’s a structured interpretation of the findings:

### Key Mechanisms of DROSHA and DICER in DISC
1. **Recruitment to DSBs via MRN Complex**:
- The MRN complex (MRE11-RAD50-NBS1) is essential for recruiting DROSHA and DICER to DNA damage sites. This recruitment is ATM-dependent, as inhibiting ATM disrupts the proximity of MRN with these RNAi factors.
- DROSHA and DICER interact physically, and their recruitment to DSBs is spatially and temporally coordinated, requiring MRN activity.

2. **Role in Chromatin Modification**:
- DROSHA and DICER facilitate the recruitment of BMI1, a component of the polycomb repressive complex 1 (PRC1), to DSBs. BMI1 mediates histone H2A-K119 ubiquitination, a mark of chromatin compaction and transcriptional repression.
- Inhibition of DROSHA or DICER reduces ubH2A-K119 deposition, thereby weakening silencing of adjacent genes. This effect is independent of miRNA-mediated repression, as miRNA pathway components (e.g., GW182 proteins) do not influence DISC when knocked down.

3. **dilncRNAs and DDRNAs as Mediators**:
- DSBs induce the transcription of damage-specific long non-coding RNAs (dilncRNAs) and small RNAs (DDRNAs). These RNAs interact with DROSHA and DICER to stabilize the BMI1-ubH2A-K119 complex at DSBs.
-实验证明，敲除DROSHA或DICER会减少dilncRNAs在BMI1结合位点上的积累，从而削弱DISC。相反，增强DICER活性（如使用激活剂enoxacin）可增强DDR信号并促进DISC。

4. **ATM and MRN in Cross-Regulation**:
- ATM activation by MRN is required for DROSHA recruitment. However, enoxacin can bypass ATM inhibition by directly stimulating DICER nuclease activity, highlighting a dual pathway: one dependent on ATM and MRN, and another driven by DICER’s endonuclease function.
- MRN inhibition (e.g., with mirin) blocks both DROSHA recruitment and ATM activation, showing MRN’s central role in linking RNAi factors to DDR signaling.

### Experimental Validation
- ** Reporter Systems**:
- U2OS 2-6-3 cells with clustered DSBs and LacO repeats showed DISC dependent on DICER but less affected by DROSHA alone. Combined RNAi of both factors mimicked ATM inhibition.
- DIvA cells (with endogenous DSBs induced by AsiSI enzyme) confirmed that individual DSBs require DROSHA/DICER for silencing genes within 2 kb.

- **Chromatin Immunoprecipitation (ChIP) and Proximity Ligation Assays (PLA)**:
- ChIP-qPCR demonstrated that BMI1 recruitment to DSBs is contingent on DROSHA and DICER. Knocking down either factor reduced ubH2A-K119 at DSBs.
- PLA revealed direct interaction between BMI1 and DROSHA at DSBs, mediated by dilncRNAs. This interaction persists even when RNase III is used, indicating it is RNA-independent.

- **RNA Sequencing and Functional Assays**:
- RNA-seq of DIvA cells showed downregulation of 98 genes near DSBs, with DROSHA/DICER co-KD restoring expression. This confirms their role in silencing.
- Cas13d-based RNA cleavage of dilncRNAs in HeLa-GFP cells and DIvA cells abolished DISC, proving dilncRNAs are necessary for silencing.

### Model of DISC
The study proposes a model where:
1. **MRN Complex Activation**: DSBs recruit MRN, which activates ATM and recruits DROSHA/DICER.
2. **RNA Processing and Chromatin Modification**:
- DROSHA processes long RNAs into siRNAs and DDRNAs.
- DICER’s endonuclease activity generates DDRNAs, which anneal with dilncRNAs to form RNA-DNA hybrids.
3. **BMI1 Recruitment and Ubiquitination**:
- DROSHA/DICER-processed RNAs interact with BMI1, recruiting it to DSBs.
- BMI1 catalyzes ubH2A-K119 deposition, compacting chromatin and silencing adjacent genes.

### Implications for Disease and Therapy
- **DNA Repair Pathways**: The findings highlight a previously uncharacterized RNAi-DDB pathway where non-coding RNAs and RNAi factors directly influence chromatin silencing.
- **Therapeutic Targets**: Inhibitors of DROSHA/DICER or dilncRNAs could disrupt DISC, potentially treating conditions where excessive DNA repair silencing causes pathologies (e.g., cancer, fibrosis). Conversely, enoxacin may enhance DDR in damaged tissues.

### Limitations and Future Directions
- **Mechanistic Gaps**: The exact structural basis for BMI1-DROSHA interaction and the temporal dynamics of DSB-induced silencing remain unclear.
- **System Specificity**: The study focuses on human cell systems; validation in other organisms (e.g., mice) is needed.
- **Alternative Pathways**: While ATM is critical, the independence of DICER’s nuclease activity suggests parallel DDR pathways.

### Conclusion
DROSHA and DICER orchestrate transcriptional silencing at DSBs by recruiting BMI1 and modulating chromatin states. Their activity is MRN-dependent but can partially compensate for ATM pathways. This mechanism integrates RNA processing with chromatin modification, offering a new frontier in understanding DNA repair and transcriptional regulation.