UC San Diego

The eukaryotic DNA is packaged into chromatin that is compartmentalized in the nucleus. Enzymatic activities directed towards chromatin and chromatin-associated proteins thus directly determine the accessibility of the DNA to various cellular machineries. A growing field in the study of chromatin is the interplay between metabolic proteins and chromatin regulation. Previous work on the yeast homocitrate synthase Lys20 demonstrated that this lysine biosynthetic enzyme has a moonlighting function in DNA damage repair. This raised the question of whether other amino acid metabolic proteins participate in chromatin regulation. In this work, an in silico screen was conducted to search for candidate proteins that potentially function in chromatin regulation. Silencing reporter assays revealed that four new candidate proteins, Gdh1, Arg82, Hom2 and Hom6 are involved in chromatin silencing. Work focused on Gdh1, the broadly conserved glutamate dehydrogenase enzyme, demonstrated a positive regulatory role of this protein in telomeric silencing. Its silencing function is dependent on a catalytic residue required for its metabolic function, suggesting that Gdh1 regulates silencing in a metabolism- dependent manner. Further analyses showed that high [alpha]-ketoglutarate levels, such as that associated with the deletion of GDH1 are in general detrimental to telomeric silencing. Moreover, deletion of GDH1 results in decreased binding of the silent information regulator (SIR complex) and increased H3 N-terminal clipping at the telomeres. A histone mutant with reduced clipping improved silencing at some telomeres in the gdh1[delta] mutant, indicating that Gdh1 at least partially regulates telomeric silencing by controlling H3 clipping. Further analysis was also undertaken for Hom2 and Hom6, which suggested a role of threonine metabolism in the regulation of rDNA silencing. Also, works on Hom6 and Arg82 revealed possible moonlighting functions of these proteins in chromatin silencing. Another work related to the general theme of chromatin regulation is the characterization of a new tyrosine phosphorylation site on histone H2A and its role in transcriptional elongation. The results presented in this thesis add to the growing recognition that chromatin function is tightly regulated by metabolism and metabolic proteins, and that histone tyrosine phosphorylation has important roles in chromatin regulation