Tagging Endogenous H2A.Z1 and H2A.Z2 Histone Variant Isoforms Using CRISPR-Cas9 and Identifying SUMOylated Proteins in Proximity to H2A.Z-Containing Nucleosomes
Ma, Myat Su Nwe
MetadataShow full item record
H2A.Z is a variant of the H2A core histone, and is well known to be important for transcriptional regulation in eukaryotic cells. It was recently discovered that higher eukaryotes have two almost identical isoforms of H2A.Z that differ only by 3 amino acids. Although current biochemical studies are unable to distinguish between the native H2A.Z isoforms, several studies have shown potential non-redundant functions between the two. For my thesis work, CRISPR-Cas9 was used to insert a small epitope tag to the C-terminus region of either H2A.Z1 and H2A.Z2 in 293T cells to allow us to study the two isoforms individually in their native states. We successfully generated 3 H2A.Z1-Strep and 3 H2A.Z2-Strep clones, and confirmed that there were minimal off-target effects by Cas9 nuclease. However, the H2A.Z1-Strep clones were found to have much lower than normal H2A.Z1 expression whereas H2A.Z2-Strep clones have normal levels of H2A.Z expression. Strep-tag ChIP data showed H2A.Z1 being enriched on the proximal region of the transcription start site (TSS) of the MTA1 gene compared to H2A.Z2. For a second project, an Avi-tag BirA system was used to test for SUMOylation of H2A.Z in mammalian cells. This method was also used to determine whether there are proteins in proximity to H2A.Z that can be SUMOylated. Our assay did not detect any H2A.Z SUMOylation; however, there were a few proteins within close proximity to H2A.Z that appeared to be SUMOylated. These putative SUMOylated proteins (which are biotinylated by the fusion H2A.Z-BirA) were purified by Streptavidin-coupled beads and awaiting identification by mass spectrometry.