Chromatin dynamics of mutated H3.3 in ESC and differentiating cells

author.DisplayName ¹ author.DisplayName ² author.DisplayName ^1,5 author.DisplayName ³ author.DisplayName ⁴ author.DisplayName ^1,5

¹Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Israel
²Department of Animal Science, the Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Israel
³Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, USA
⁴School of Computer Science and Engineering, The Hebrew University of Jerusalem, Israel
⁵The Edmond and Lily Safra Center for Brain Sciences (ELSC), The Hebrew University of Jerusalem, Israel

The histone variant H3.3 plays an important role in pluripotency and differentiation, and is critical for normal development. Its importance has further emerged lately with the finding of a significant percentage of H3.3 mutations in pediatric glioblastoma (GBM). In order to elucidate the effect of the H3.3 cancer-causing mutations on the histone turnover, we established a system to analyze H3.3 turnover dynamics in a genome-wide manner. We generated mouse ESC lines carrying a single copy of a doxycycline (Dox)-inducible HA-tagged version of WT or mutant H3.3 (H3.3K27M/L and H3.3G34R/V), and monitored the rate of H3.3 incorporation by ChIP-seq at varying time points following Dox induction (Time-seq). We found that K27 mutations, but not G34, harbor faster turnover dynamics. Gene expression in the different H3.3 mutants is barely affected 72 hours following Dox addition, despite higher turnover and global reduction of K27me3 in the K27M mutant. Nevertheless, genes that are differentially expressed in the K27M mutant lines show higher H3.3 turnover at their promoters, hinting at an early link between H3.3 turnover and gene expression. These results suggest that changes in H3.3 turnover dynamics are among the early steps in GBM formation.