Mutations in the Charged Domain of CBX2 Disrupt PRC1 Function in Vivo

Abstract

Epigenetics inheritance is a phenomenon where a cell state is inherited across cellular divisions. It is important for maintaining differentiated cells types, and thus is fundamental to the development of multicellular organisms. Failure in the system can lead to deleterious effects that range from embryonic lethality to the development of diseases such as cancer. The Polycomb group (PcG) proteins are important regulators of epigenetic inheritance: they maintain the repression of the Hox genes, which specify the body axes of an organism, throughout development and into adulthood. The loss of function of PcG genes therefore leads to homeotic transformations. A mechanistic understanding of PcG function will yield insight into the mechanisms involved in epigenetics inheritance. The Polycomb Repressive Complex 1 (PRC1) is able to inhibit chromatin remodeling and compact polynucleosomes in vitro; these activities could represent how stable gene repression is achieved in vivo. However, it has been difficult to determine the biological relevance of these activities because the nature of nucleosomal-level compaction in the nucleus is unknown and there is no direct assay for it. To circumvent this problem, we adopted an approach where we introduced into mouse embryonic stem cells (mESCs) and mice the mutations that disrupt the in vitro activities. The mutations are specific lysine-to-alanine and arginine-to-alanine substitutions in a positively charged domain of the CBX2 subunit of PRC1. These are known to disrupt PRC1 polynucleosmal compaction activities from previous studies. We compared the mutant mESCs and mice to wild type counterparts for PcG-related phenotypes. We observed that the mutations prevented the repression of CBX2 (also PRC1) target genes in mESCs. They did not prevent CBX2 from forming PRC1 or binding to chromatin. This suggests that the in vitro activities are specifically involved in inhibiting transcription. Moreover, we observed that the Cbx2 mutant mice exhibit the classic PcG phenotype, which is homeotic transformation in the anterior-posterior axis. The posterior transformations are similar to those observed in the Cbx2-/- mice, indicating that our mutations mimic loss-of-function of Cbx2. Notably, the Cbx2-/- mice had other phenotypes in addition to the PcG phenotype, which we did not observe in our mutants. This indicates that the mutations in the compaction domain of CBX2 specifically disrupted a PcG function during development. These experimental outcomes are consistent with the hypothesis that the in vitro compaction activity of PRC1 is important for how heritable gene repression can be achieved in vivo.