Poster abstracts
Poster number 51 submitted by Liudmila Popova
Using Proximity-Dependent Biotinylation to Identify Proteins Proximal to Histone Acetyltransferase 1 in vivo
Liudmila Popova (MCDB), Mark Parthun (Biological Chemistry and Pharmaclogy), Miranda Gardner (OSBP), Michael Freitas (Cancer Biology and Genetics)
Abstract:
Histone Acetyltransferase 1 (Hat1) is an evolutionarily conserved enzyme known to acetylate lysines 5 and 12 in the tail of the newly synthesized histone H4. Besides playing this role in replication-coupled chromatin assembly, Hat1 has been shown to contribute to DNA damage repair in a variety of organisms. However, much remains unknown about Hat1 functions in vivo. In order to gain detailed insight into cellular roles of Hat1 in mammalian cells we utilized a proximity-dependent biotinylation approach (BioID), which relies on fusing a mutant biotin ligase BirA (R118G) to the protein of interest (Hat1) in order to allow for biotinylation of proteins vicinal to the protein of interest in vivo. For this experiment, a triplicate of HEK 293 cells was transfected i with Hat1-BirA (R118G) construct and treated with exogenous biotin for 24 hours. Biotinylated proteins were isolated, and the pull-downs were analyzed by mass spectrometry. Statistical analysis yielded a list of ~60 proteins enriched in the Hat1-BirA (R118G) transfected-exogenous biotin treated sample compared to the control. The list of enriched proteins included actin-related proteins, transcriptional regulator KAISO, RNA-splicing-related protein GEMIN5 and other proteins. Further experiments are necessary to uncover the nature of Hat1’s relationship to the aforementioned proteins.
References:
Lambert et al. Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes.J Proteomics. 2015 Apr 6;118:81-94.
Keywords: Hat1, BioID