Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene

Abstract

Patterned expression of many developmental genes is specified by transcription factor gene expression, but is thought to be refined by chromatin-mediated repression. Regulatory DNA sequences called Polycomb Response Elements (PREs) are required to repress some developmental target genes, and are widespread in genomes, suggesting that they broadly affect developmental programs. While PREs in transgenes can nucleate trimethylation on lysine 27 of the histone H3 tail (H3K27me3), none have been demonstrated to be necessary at endogenous chromatin domains. This failure is thought to be due to the fact that most endogenous H3K27me3 domains contain many PREs, and individual PREs may be redundant. In contrast to these ideas, we show here that PREs near the wing selector gene vestigial have distinctive roles at their endogenous locus, even though both PREs are repressors in transgenes. First, a PRE near the promoter is required for vestigial activation and not for repression. Second, only the distal PRE contributes to H3K27me3, but even removal of both PREs does not eliminate H3K27me3 across the vestigial domain. Thus, endogenous chromatin domains appear to be intrinsically marked by H3K27me3, and PREs appear required to enhance this chromatin modification to high levels at inactive genes.\n\nAuthor summaryEukaryotic genes are packaged in chromatin, and their transcription relies on activators that recruit RNA polymerases and on repressive factors. Some of these factors factors that modulate chromatin structure to promote or inhibit gene transcription. In multicellular organisms, cell types have distinct patterns of gene expression, and these patterns are controlled by by the expression of cell-type-specific transcription factors and by modulating chromatin structure. The Polycomb system is one major system for the chromatin-mediated silencing of developmental gene expression, where a histone methyltransferase marks extended chromatin domains with trimethylation of lysine-27 of the histone H3 tail (H3K27me3) and forms repressed chromatin. In Drosophila, repressive regulatory elements called Polycomb Response Elements (PREs) are thought to nucleate histone methyltransferase binding which then spreads across these domains. In this study, we demonstrate that two PREs near the developmental vestigial gene have distinct and separable effects on gene activation and chromatin structure. Both PREs are functional repressors in transgenes, but the PRE located near the vestigial promoter is required for gene transcription. This PRE has no effect on histone methylation of the domain. The second PRE, located in the middle of the chromatin domain is required for high-level H3K27me3 of the domain, and this methylation is not required to refine vestigial gene expression. Strikingly, a significant chromatin methylation remains when both PREs are deleted. Our findings imply that PREs near promoters may often play activating roles in gene expression in the Drosophila genome. We suggest that some domains of H3K27me3 occur in regions that lack histone acetylation, and has little consequence for correctly patterning gene expression.