Poster abstracts
Poster number 45 submitted by Liudmila Popova
Using Proximity-Dependent Biotinylation to Identify Proteins Proximal to Histone Acetyltransferase 1 in vivo
Liudmila Popova (MCDB), Miranda Gardner (OSBP), Michael Freitas (Cancer Biology and Genetics), Mark Parthun (Biological Chemistry and Pharmacology)
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 several actin-related proteins, transcriptional regulator KAISO, and other proteins.
References:
Lambert J.P. et al. Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes. (2015) J Proteomics. 118, 81-94.
Parthun, M.R. (2012) Histone acetyltransferase 1: More than just an enzyme? Biochim Biophys Acta, 1819, 256-63.
Keywords: Hat1 , BioID