The genome of eukaryotic cells contains several thousand genes, whose expression has to be precisely regulated to keep cells functional and alive. Regulation of eukaryotic gene expression at the level of transcription initiation depends on an intricate interplay between transcription factors, proteins that establish transcription-competent chromatin by posttranslational modification of histones and chromatin remodeling, and the general RNA polymerase II transcription machinery (GTM). That interplay culminates in the recruitment of the GTM to the promoter of target genes and transcription initiation. The GTM consists of RNA polymerase II, general transcription factors (GTF) TFIIA, -B, -D, -E, -F, -H and -I, and associated cofactors. The GTF TFIID is essential in transcription regulation by nucleating the assembly of the GTM at promoters. TFIID is a multi-protein complex that consists of the TATA-box binding protein (TBP) and at least 12 TBP-associated factors (TAFs). TAF1 is the largest TFIID-subunit and contributes to the nucleating function of TFIID by 1) interacting with TBP, most of the other TAFs, transcription regulators, and GTFs, 2) contributing to the interaction of TFIID with promoters, and 3) posttranslational modification of GTFs and histones. TAF1 acetylates histones H3 and H4, phosphorylates histone H2B, and ubiquitinates histone H1. TAF1-mediated histone modifications have been correlated with transcription activation. However, the functional importance of TAF1-mediated histone modifications in transcription initiation in the context of metazoan genomes remains unclear. The central hypothesis of this research project is that the histone modifying activities of TAF1 play a fundamental, potentially general role in metazoan transcription. The specific aims are to elucidate the functional importance of the various histone-modifying activities of TAF1 in (1) cell cycle regulation and (2) body pattern formation in the fly Drosophila, and (3) elucidate the general functional importance of TAF1-mediated histone modifications in transcription activation in the context of the Drosophila genome. In the first aim the results of RT-PCR and chromatin immunoprecipitation (XChIP) experiments will be correlated to dissect the functional importance of TAF1-mediated histone modifications in the regulation of essential cell cycle genes. In the second aim genetic analysis will be combined with biochemical assays to assess the functional importance of TAF1-mediated histone modifications in the transcription regulation of segmentation genes that establish the body pattern of Drosophila. The third aim will use genome-wide, DNA microarray-based gene expression profiling and XChIP-on-chip analyses using probes isolated from TAF1-deficient Drosophila cells and flies expressing mutant TAF1-derivatives lacking one or multiple histone-specific catalytic activities to uncover the functional importance of TAF1-mediated histone modifications in transcription in the context of a metazoan genome. Transcription initiation is one of the most fundamental cellular processes. DNA in eukaryotic cells is associated with proteins, mainly histones to form chromatin. The association of DNA with proteins in chromatin can prevent transcription initiation. The goal of this project is to gain novel insights into the molecular mechanisms that facilitate transcription initiation in the context of chromatin. The project will involve undergraduate, graduate, and postdoctoral researchers and provide novel research tools that will be made available to the scientific community.
