Animal development depends on the capacity of cells to respond to transient spatial and temporal cues by establishing heritable states of gene expression that specify cell fate. A long-standing question is whether such epigenetic states can be maintained by the inheritance of cis-acting chromatin modifications. To answer this question, it is necessary to identify those modifications that play a causal role in epigenetic memory and investigate how they are maintained and propagated through multiple rounds of replication.
Many animals are composed of diverse body segments governed by evolutionarily conserved HOX gene complexes. Early in embryogenesis, cells in each segment heritably activate some HOX genes while silencing others. This provides a paradigm of epigenetic memory in which transient cues establish permanent ON and OFF states of gene expression. In the OFF state, HOX gene chromatin is marked with trimethylation of lysine 27 of histone H3 (H3K27me3) that is catalyzed by Polycomb repressive complex 2 (PRC2). Here, we investigated whether H3K27me3 nucleosomes are the carriers of epigenetic memory of the OFF state. To this end, we generated a transgenic lacZ reporter of the Drosophila HOX gene Ultrabithorax (Ubx), which recapitulates Ubx regulation throughout development. Deposition of H3K27me3 at this reporter depends on the presence of a Polycomb response element (PRE), the cis-acting DNA to which PRC2 is targeted. We monitored both the OFF state and H3K27me3 after inducing excision of the PRE at different time points during development. Using this strategy, in combination with manipulation of PRC2 activity and H3K27 substrate availability, we assessed the capacity of PRC2 that is not recruited to the PRE to propagate H3K27me3 and investigated whether there is a causal relationship between H3K27me3 inheritance and maintenance of the OFF state.
Focusing on the developing wing imaginal disc where the transgene is normally OFF, we find that PRE excision results in a release from silencing that depends on the cell-division–coupled dilution of H3K27me3 and occurs after a stereotyped number of cell divisions. Further, we show that the rate of dilution depends on the residual capacity of PRC2 that is not recruited by the PRE to propagate the mark. Reducing this capacity increases the rate of dilution and leads to a corresponding decrease in the number of times a cell can divide while maintaining the OFF state. These findings establish a causal relationship between inheritance of the H3K27me3 mark and memory of the OFF state.
Our results demonstrate (i) that H3K27me3, once established at a repressed Drosophila HOX gene, remains heritably associated with that gene; (ii) that local inheritance of the mark transmits the memory of the OFF state from one cell generation to the next; and (iii) that long-term memory depends on efficient copying of the mark after each replication cycle, a process that requires recruitment of PRC2 by the PRE. Taken together, these findings establish H3K27me3 as a determinant of epigenetic memory and identify efficient propagation of the mark by PRE-associated PRC2 as a requirement for perpetuating the memory.