554A Poster - 07. Chromatin, epigenetics and genomics
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Dongsheng Han ¹; Tara O'shea ¹; Jonathan P Davies ²; Logan Richards ¹; Lars Plate ²; Jared T Nordman ¹

Affiliations:
1) Department of Biological Sciences, Vanderbilt University, Nashville, TN; 2) Department of Chemstry, Vanderbilt University, Nashville, TN

Keywords:
l. DNA replication; a. chromatin structure

Posttranslational modifications of histones impact chromatin structure and function, which underlie everything from genome stability to cell differentiation and disease. Understanding how histone modifications are established and maintained is critical to understanding these processes. BRWD3 (Bromodomain and WD repeat-containing protein 3) is a specificity factor for a CUL4 ubiquitin ligase complex. BRWD3 has been implicated in DNA replication, transcription, and regulation of histone H3 methylation and acetylation. The underlying mechanisms of BRWD3 action, however, are largely unknown. To uncover how BRWD3 functions, we performed both dBRWD3 IP-MS and BRWD3-dependent ubiquitination IP-MS. As expected, we identified the BRWD3-Cul4-DDB1 E3 ubiquitination complex and multiple histones as significantly enriched in our dBRWD3-IP. Interestingly, we identified the Origin Recognition Complex (ORC) as both associated with BRWD3 and ubiquitinated in a BRWD3-dependent manner. Bioinformatic analysis revealed that ~35% of ORC2 binding sites overlap with dBRWD3 binding sites throughout the genome. Furthermore, depletion of dBRWD3 significantly reduces DNA proliferation in the S phase. In addition to ORC, we have also identified several key chromatin-related proteins that could provide mechanistic insight into BRWD3 function. In conclusion, our approach has successfully identified likely direct targets of the BRWD3-Cul4-DDB1 E3 ubiquitin ligase complex, which could help explain how BRWD3 functions in diverse chromatin-related processes.