Eukaryotic DNA is packaged into chromatin, which consists of DNA wound around histone proteins to form nucleosomes. Dynamic changes to chromatin structure, essential for regulating gene expression, are mediated by chromatin-associated factors, including histone modifiers, chromatin remodelers, and histone chaperones. Mutation in these complexes, or their altered expression, is among the major causes for inception as well progression of many diseases, including cancer. Changes in chromatin involve reorganization of nucleosomal structure, including complete disassembly of nucleosomes during transcription by RNA polymerase II. FACT, a heterodimer of Spt16 and Pob3, is an essential and highly conserved histone chaperone, which can bind to all four histones, and harbor specific activity to incorporate H2A/H2B in nucleosomes. Loss of its activity impairs transcription, reduces histone density, and results in widespread aberrant transcription. The goal of this project is to define the mechanisms by which FACT associates with chromatin to promote productive transcription elongation in coordination with other histone chaperones and chromatin remodelers. We will use high-resolution genomic approaches in the budding yeast Saccharomyces cerevisiae to define the role of FACT in transcription elongation. In the first specific aim, we will examine defects in Pol II elongation caused by FACT deficiency. Further, we will determine whether FACT stimulates transcription by reorganizing nucleosomal structure or by preferentially evicting H2A/H2B dimer. Cooperation between FACT and Spt6, a H3/H4 chaperone, in regulating transcription elongation and chromatin structure will also be determined. In the second aim, we will examine the mechanisms that enhance FACT recruitment to transcribed genes. Our preliminary data implicate histone acetylation in FACT recruitment. Histone acetylation could enhance recruitment directly by interacting with FACT or indirectly by promoting open chromatin structure. Using histone tail deletion and site-specific mutants, we will examine the impact of histone acetylation in enhancing FACT occupancy, genome-wide. FACT occupancy will be also be determined in cells defective for chromatin remodeling to assess the extent to which open chromatin assists in recruiting FACT. Specific hydrophobic and acidic residues are utilized by FACT to interact with histones. The importance of these specific residues in regulating FACT function will also be examined, genome- wide. The experiments outlined in this proposal are expected to provide valuable insight into the mechanisms by which histones and elongation factors cooperate to recruit FACT to chromatin under in vivo conditions, but also provide important information by which FACT control chromatin structure to enhance transcription.
Changes in gene expression due to altered chromatin structure are one of the major causes for many human diseases, including cancer. This proposal will provide important insight into the mechanisms by which histone chaperones cooperate with other chaperones and remodelers to regulate chromatin structure and promote productive transcription while suppressing aberrant transcription. This study will help in understanding the underlying causes for diseases associated with impaired function of these chaperones and in developing strategies to prevent and cure human diseases.
