Genetic interactions between variant histone H2.A.Z and putative targets

Abstract

In eukaryotic cells, the DNA is compacted into a complex structure known as chromatin. The individual unit of chromatin is the nucleosome, which is composed of 146 bp of DNA wrapped 1,75 times around a protein complex called the core histone octamer. The octamer is formed of two copies each of the histone proteins H2A, H2B, H3 and H4. Nucleosomes impose a barrier to transcription that can be overcome in vivo by remodeling complexes such as Swi/Snf and historic modification complexes such as SAGA. Mutations in the major core histories relieve transcriptional repression and bypass the requirement for Swi/Snf and SAGA. It was found that the variant histone H2A.Z regulates gene transcription. H2A.Z is highly conserved with ~90% sequence identity among different organisms. However, H2A.Z shares only ~60% sequence identity with major H2A, suggesting a unique function. H2A.Z histories are essential for the viability of Tetrahymena, Drosophila , and mice. Research in Drosophila and in yeast shows that the unique feature of H2A.Z does not reside in its histone fold but in its carboxy-terminal domain, termed M6, which is essential for the survival of that organism. Some experiments carried out with Saccharomyces cerevisiae have shown that H2A.Z could regulate transcription and that its function was partially redundant with certain nucleosome-remodeling complexes. My project was aimed at identifying a H2A.Z M6 mutant that could confer H2A.Z loss-of-function. Amino acids 98-108 in yeast H2A.Z M6 region correspond to the conserved amino acids in Drosophila H2A.Z residues that are essential for its viability. In this work, after substitution of these amino acids with that of H2A using site-directed mutagenesis, I identified one mutant M6-G98N that expressed loss-of-function of H2A.Z. We concluded that the M6 region, in particular amino acid 98, is an important determinant for the special function of H2A.Z in transcription. I next attempted to identify targets of the H2A.Z C-terminal region that are important for its function in gene transcription. The H2A.Z loss of function mutant, M6-G98N, was used in a complementation assay using a yeast genomic expression library to identify suppressors of the mutant. Since htz1 _ cells were highly sensitive to hydroxyurea, we used this reagent for screening functional complementation. I isolated two suppressors that could rescue the deficiency. After sequencing, the SWI1 and SRF6 genes were identified, respectively. This result suggests that SRF6 was possibly a target of the H2A.Z C-terminal region in DNA repair and gene transcription. However, how SRF6 affects the function of H2A.Z needs further investigation. SWI1 is a component of the Swi/Snf complex, which utilizes the energy of ATP hydrolysis to remodel chromatin structures. I identified SWI1 as a potential functional target of H2A.Z C-terminal region. This finding supports the special function of H2A.Z in gene transcription. However, the mechanism on the interaction between SWI1 and H2A.Z needs to be investigated in the future.