CHARACTERIZATION OF TWO DISTINCT dGCN5-CONTAINING HAT COMPLEXES IN DROSOPHILA

Open Access
Author:
Guelman, Sebastian
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 17, 2005
Committee Members:
  • Jerry L Workman, Committee Chair
  • Susan M. Abmayr, Committee Member
  • Song Tan, Committee Member
  • Richard W Ordway, Committee Member
  • David Scott Gilmour, Committee Chair
Keywords:
  • histone
  • HAT
  • ATAC
  • dSAGA
  • Gcn5
  • Chromatin
  • Drosophila
Abstract:
Eukaryotes organize their genomes in the form of chromatin, a filament composed of DNA, histones and non-histone proteins. Chromatin structure plays crucial roles in the regulation of nuclear processes that employ DNA as template, including transcription, DNA replication, repair and recombination. Chromatin structure can be altered to allow such processes to occur in the cell. One way to do so is by covalently modifying different amino acid residues present at the histone N-terminal tails, which protrude away from the nucleosomes. These modifications can affect inter-nucleosomal interactions and result in changes in the compaction of chromatin. In addition, covalently modified histone tails can recruit additional factors which can trigger downstream events. Acetylation is one of the best characterized histone modifications. This modification is performed by histone acetyltransferases (HATs), which transfer an acetyl group from acetyl CoA to lysine residues of the histone tails. This modification is reversible and can be removed by histone deacetylases. The yeast coactivator SAGA provides a good example of a HAT and has been studied in detail. Different experiments demonstrated that histone acetylation by the SAGA complex is required for transcription of a subset of genes. Gcn5 is the catalytic subunit of this complex and is highly conserved throughout eukaryotes. At the time we began this project, the dGcn5 (Drosophila Gcn5) gene had been cloned but no additional information was available about dGcn5. Therefore, we sought to determine if dGcn5 forms part of multi-subunit protein complexes in flies, which would enable us to understand the role of dGcn5 in fly development. This thesis describes the purification of dGcn5 as part of two distinct HAT complexes. One of these complexes is dSAGA (Drosophila SAGA), which shares homologous subunits with yeast SAGA and the mammalian complexes STAGA, PCAF and TFTC. We identified a novel WD-repeat containing protein in dSAGA and demonstrated that this protein is essential for viability and required for histone H3 acetylation in the embryo. The other dGcn5-containing complex we purified, ATAC (Ada Two A Containing), harbors an orthologue of the human VP16-interacting protein HCF-1, as well as other uncharacterized proteins with conserved domains. ATAC is very different from SAGA, both in size and biochemical composition, which suggests that ATAC and dSAGA carry out distinct functions during Drosophila development