Research
Function of Diverse Transcription (co)Factors and Histone Modifications in Eukaryotic Transcription
The transcriptional regulatory events that are central to cell growth, differentiation and (malignant) transformation are governed by complex epigenetic mechanisms. Nuclear DNA in eukaryotic cells is organized within a hierarchical chromatin structure that restricts access to regulatory proteins that activate gene expression. Eukaryotic transcription is influenced by many transcription factors that involve general transcription factors (GTFs), positive cofactors, negative cofactors, ATP-dependent chromatin remodelers, histone modifying enzymes and so on.
1. In Vitro Chromatin Transcription
Biochemical studies with well-defined cellular machineries provide a necessary approach for an ultimate understanding of biological function at a mechanistic level. To better understand complex mechanism of transcription in eukaryotic cells, defined cell free systems for robust transcription from recombinant chromatin templates that are dependent upon (i) a DNA-bound activator, (ii) a histone acetyltransferase (p300), (iii) an ATP-dependent chromatin remodeler (ACF), (iv) Pol II and cognate general initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH) and cofactors (PC4 and Mediator) and (v) Pol II-interacting elongation factors (SII and the PAF1 complex) has been established (Kim et al., Cell 2010). Various uncharacterized transcription factors will be applied to this cell-free system to directly assess their mechanism of action in eukaryotic transcription.
2. In Vitro Chromatin Modification
The histone tails are susceptible to posttranslational modifications that include acetylation, methylation, phosphorylation, and ubiquitylation and recent years have seen an explosion of information concerning effects of covalent modifications on transcription. The biological consequences of histone modifications are mediated by evolutionarily conserved reader/effector modules that bind to histone marks in a modification- and context-specific fashion and subsequently enact chromatin changes or recruit other proteins to do so. Determining the regulatory roles of histone covalent modifications in the context of human disease will allow for a more thorough understanding of normal and pathological development, and may provide innovative therapeutic strategies wherein chromatin readers stand as potential drug targets. Of our special interest, robust in vitro chromatin modification assays have established E1, E2 (RAD6) and E3 (BRE1) enzymes and the PAF1 complex for histone H2B ubiquitylation (Kim et al., Cell 2009) that is prerequisite for active transcription-associated histone H3 lysine 4 methylation. These analyses will be immediately expanded to understand the molecular mechanism and function of histone H3 lysine 4 methylation mediated by the Set1 and/or MLL complexes.
