Abstract A04: Modulation of SAGA mediated nucleosome acetylation by linker DNA and activator

Abstract

Abstract The SAGA family of co-activators are highly conserved eukaryotic protein complexes that play a major role in regulating gene expression. These complexes facilitate transcription of inducible genes, in part, by acetylating the histone H3 tails of nucleosomes. While the factors that influence nucleosome acetylation are qualitatively understood, quantitative approaches offer the potential to better understand how the interaction between complex nucleosomal substrates, a multi-subunit SAGA complex, and regulatory proteins, combine to remodel the chromatin landscape of gene promoters. Toward this end, we have developed an initial rate, steady-state, acetylation assay using immobilized nucleosomes. For a minimal 147 bp mononucleosome, we find that acetylation activity saturates at a low nucleosome concentration (50 nM KM), and that turnover is quite slow (0.25 min-1 kcat). Addition of linker DNA to the nucleosome enhances acetylation activity up to 8-fold, and does so both by changing the apparent binding constant (2x decrease in KM) and the turnover step (4x increase in kcat). This dual mechanism of stimulation is recapitulated by addition of free DNA in trans, and we are investing the minimum lengths of DNA required for stimulation, as well as the mechanism of stimulation. When the transcriptional activator Gal4-VP16 becomes bound specifically to a recognition site within the linker DNA, additional stimulation of acetylation activity is observed. We are currently working towards gaining more insights into the mechanistic basis of this effect. The results give a more in depth perspective of how different factors work in conjunction to ultimately tune inducible transcription. Citation Format: Chitvan Mittal. Modulation of SAGA mediated nucleosome acetylation by linker DNA and activator. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A04.