149 Oral - Chromatin
Saturday April 09, 9:15 AM - 9:30 AM

Authors:
Harmony Salzler ¹; Vasudha Vandadi ¹; Sally Boerma ²; John Brown ¹; A Gregory Matera ¹

Affiliations:
1) UNC Chapel Hill; 2) Carleton College

Keywords:
g. Polycomb/trithorax complexes; j. epigenetics

Polycomb silencing is an epigenetic mechanism for determining cell-type specific gene expression programs, ensuring that genes specifying alternative lineages remain repressed. The best-studied examples of this phenomenon are the Drosophila Hox genes, which specify the body plan. To ensure proper development of body segments, Hox genes must be expressed at certain points along the body axis and repressed at others.

One critical step in this process is trimethylation of histone H3 lysine 27 (H3K27me3) by the Polycomb repressive complex 2, PRC2. Importantly, PRC2 can also “read” the H3K27me3 mark, enabling cis-linked spreading of H3K27me3, and formation of repressive chromatin domains essential for proper Hox gene expression. To antagonize the spreading of H3K27me3 into other active genes, evidence suggests that cells utilize di- and tri-methylation of H3 lysine 36 (H3K36me2/3) to abut these repressive domains. A mechanism by which H3K36me2/3 halts spreading of facultative heterochromatin was unknown until recent structural studies revealed that unmethylated H3K36 occupies a crucial role in positioning H3K27 into the active site of its cognate methyltransferase, EZH2 (the mammalian ortholog of Drosophila enhancer of zeste E(z)). This finding suggests that H3K36me2/3 sterically hinders E(z) activity and that mutation of H3K36 would elicit strong Polycomb phenotypes. One puzzle that emerged from this work is that when Hox gene expression was examined in vivo, mutants of the replication-dependent histone H3 genes (H3.2^K36R and H3.2^K36A) displayed relatively mild Polycomb phenotypes, despite inhibitory effects of these mutations on H3K27 trimethylation in vitro.

In addition to H3.2, metazoan genomes also contain the replication-independent histone variant, H3.3, which differs from H3.2 by only 4 amino acids. We hypothesized that H3.3 might function redundantly to enable Polycomb mediated gene repression in the H3.2 K36R mutants. Therefore, we generated an H3.3^K36R mutant to directly compare Polycomb mediated gene silencing between H3.3^K36R, H3.2^K36R, and H3.3^K36RH3.2^K36R combined mutant animals. Our studies in embryos, first instar larvae, and adults demonstrate that H3.2K36 and H3.3K36 cooperate to maintain H3K27 trimethylation and Hox gene silencing by different, but synergistic mechanisms.