Brwd3’s Function in Epigenome Maintenance and Replication Regulation

Abstract

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modification levels may contribute to disease development and cancer. However, little is known about how histone modification levels are maintained. The chromatin-binding protein BRWD3, a known substrate specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, is required for maintaining active histone modification levels. Loss of BRWD3 function causes an increase in H3K4me1 levels. The underline mechanism, however, is unknown. We found that BRWD3 depletion causes decreased H3K4me3 levels. We identified an interaction between BRWD3 and the lysine-specific demethylase 5 (KDM5), an enzyme that removes tri- and di- methyl marks from lysine 4 on histone H3. Moreover, ChIP-Seq data analysis revealed that BRWD3 and KDM5 are significantly co-localized throughout the genome. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels. In addition to its role in epigenetic regulation, BRWD3 also contributes to DNA replication. We found that BRWD3 physically associates and co-localizes with the Origin Recognition Complex (ORC) throughout the genome, yet regulates replication initiation independently of ORC loading. Moreover, BRWD3 is essential for replication fork progression and genome stability. To further explore its role in replication dynamics, we are developing a novel approach to profile replication genome-wide using ultra-long single-molecule Nanopore sequencing in Drosophila. Together, our findings reveal a dual role for BRWD3 in maintaining both epigenetic states and replication integrity, highlighting its importance in genome regulation and stability.