| Chromatin is intimately connected to the regulation of gene expression in eukaryotes. Modification of histone tails by methylation has been particularly important in determining the dynamic transitions between transcriptionally-active and transcriptionally-silent chromatin states. The crucial question is how the different histone modifications cooperate in determining the chromatin state. This aspect of the ?histone code? is crucial for transcription regulation. We have recently discovered that tri-methylation of lysine-4 of histone H3 (H3K4me3) can act as an anchor for the basal transcription factor TFIID. As an extension of these studies we developed a highly-selective affinity-based protocol to purify nucleosomes marked by H3K4me3. This enables a detailed proteomics study to determine the histone modifications co-occuring or mutually-exclusive with H3K4me3. We will compare differentiated cells with embryonic stem cells to examine differences in the epigenetic marks. This detailed information will be used to determine the molecular consequences of this histone ?crosstalk?. We will employ genomics and bioinformatics tools to investigate the contributions to promoter activity of genetic elements like core promoter sequences and of epigenetic elements like H3K4 methylation. TFIID and its TAF3 subunit, in particular, represent an important focus of our study to understand how the combinatorial marks influence transcription. We will use protein engineering to alter the substrate specificity of the PHD of TAF3 and examine its effect on gene expression. Together, the results of our study will provide a detailed molecular understanding of the epigenetic and genetic principles, which govern gene activity in differentiated and undifferentiated mammalian cells. |
