Histones are extensively modified in chromatin. Each modification plays an important role in chromatin organization and/or gene expression. Recently, histones were found to be modified by the attachment of biotin, a water-soluble vitamin. Biotinylation is unique among histone modifications, in that a metabolic co-factor also functions as a chromatin mark. Histone biotinylation could act as a metabolic sensor, mediating changes in transcription in response to changes in nutritional status.
The long-term goal of this project is to determine the genetic role of histone biotinylation. Histone biotinylation is mediated by holocarboxylase synthetase (HCS) and by biotinidase (BTD). Both enzymes are found in the nucleus. HCS is found at specific sites on Drosophila chromosomes, consistent with a role for targeted HCS in histone biotinylation. Knockdown of HCS in transgenic flies results in shorter lifespan, reduction in certain biotinylated histones and altered gene expression, including ribosomal RNA. Certain biotinylated histones are concentrated in the nucleolus, suggesting that histone biotinylation could regulate rDNA transcription. The working hypothesis for this project is that histone biotinylation plays multiple roles in gene expression. The project has three specific aims: Aim 1. Test the roles of HCS and BTD in gene expression. Genetic assays will be used to test whether knockdown of BTD and/or HCS affect ribosome function. Genetic assays will be used to test the effects of HCS and BTD levels on heterochromatin function. The effect of targeted HCS and BTD on reporter expression and histone biotinylation at a transgene will be tested. Aim 2. Determine the mechanism of HCS targeting to chromosomes. The domain(s) necessary for chromosome binding of HCS will be mapped. Purification of HCS-containing protein complexes that may target proteins to chromatin will be initiated. Aim 3. Determine the chromosomal distribution of biotinylated histone isoforms. Antibodies to specifically biotinylated isoforms will be used to test (1) the genomic distribution of biotinylated histones, (2) the effect of HCS and BTD knockdown on the distributions of each modification and (3) the DNA sequences associated with specific isoforms.
Broader Impact: The proposed studies will have broad implications for our understanding of gene regulation. Additionally, the proposed project will provide training for two high school students per year. The PI has trained graduate students, postdocs, undergrads and high school students. Eight high school students and one high school teacher have done research in the PI's lab. The PI has participated in the "Students and Teachers As Research Scientists" (STARS) program, a highly selective program (funded by Solutia and Pfizer) administered through U. Missouri-St. Louis. Two students who worked in the PI's lab received awards based on their research. In addition, two high school students worked in the lab on the PI's previous NSF-funded research project last spring as part of a senior project and returned to work on the project in the summer after their freshman years in college. The PI will continue to involve two high school students or teachers in the proposed project per year throughout the funding period.
