Additional sex combs interacts with enhancer of zeste and trithorax and modulates levels of trimethylation on histone H3K4 and H3K27 during transcription of hsp70.

BACKGROUND: Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear. RESULTS: We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with E(z) and Trx in vivo. Drosophila Asx is required for repression of heat-shock gene hsp70 and is recruited downstream of the hsp70 promoter. Changes in the levels of H3K4me3 and H3K27me3 downstream of the hsp70 promoter in Asx mutants relative to wild type show that Asx regulates H3K4 and H3K27 trimethylation. CONCLUSIONS: We propose that during transcription Asx modulates the ratio of H3K4me3 to H3K27me3 by selectively recruiting the antagonistic HMTs, E(z) and Trx or other nucleosome-modifying enzymes to hsp70.

Chromatin proteins and RNA are associated with DNA during all phases of mitosis.

Mitosis brings about major changes to chromosome and nuclear structure. We used recently developed proximity ligation assay-based techniques to investigate the association with DNA of chromatin-associated proteins and RNAs in Drosophila embryos during mitosis. All groups of tested proteins, histone-modifying and chromatin-remodeling proteins and methylated histones remained in close proximity to DNA during all phases of mitosis. We also found that RNA transcripts are associated with DNA during all stages of mitosis. Reduction of H3K27me3 levels or elimination of RNAs had no effect on the association of the components of PcG and TrxG complexes to DNA. Using a combination of proximity ligation assay-based techniques and super-resolution microscopy, we found that the number of protein-DNA and RNA-DNA foci undergoes significant reduction during mitosis, suggesting that mitosis may be accompanied by structural re-arrangement or compaction of specific chromatin domains.

TrxG and PcG proteins but not methylated histones remain associated with DNA through replication.

Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.

Are polycomb group bodies gene silencing factories?

Polycomb group (PcG) proteins mediate long-range associations between Hox genes, which correlate with gene repression in vivo. Bantignies et al. (2011) identify a physiological role for the nuclear localization of Hox genes in PcG-mediated gene silencing, strengthening the evidence that nuclear positioning regulates gene expression.

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint.

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph(p) mutants reveals an acceleration of S phase, showing that ph(p) regulates S phase length. Like ph(p) mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph(p) are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.

Regulatory noncoding RNAs at Hox loci.

There is growing awareness of the importance of noncoding (nc)RNAs in the regulation of gene expression during pattern formation in development. Spatial regulation of Hox gene expression in development controls positional identity along the antero-posterior axis. In this review, we will focus on the role of short ncRNAs that repress Hox genes in Drosophila and mammals by RNA interference (RNAi), on long ncRNAs that may repress a Hox in cis in Drosophila by transcriptional interference, and on a novel long ncRNA that functions in trans to regulate Hox genes mammals.

Transcriptional interference: an unexpected layer of complexity in gene regulation.

Much of the genome is transcribed into long untranslated RNAs, mostly of unknown function. Growing evidence suggests that transcription of sense and antisense untranslated RNAs in eukaryotes can repress a neighboring gene by a phenomenon termed transcriptional interference. Transcriptional interference by the untranslated RNA may prevent recruitment of the initiation complex or prevent transcriptional elongation. Recent work in yeast, mammals, and Drosophila highlights the diverse roles that untranslated RNAs play in development. Previously, untranslated RNAs of the bithorax complex of Drosophila were proposed to be required for its activation. Recent studies show that these untranslated RNAs in fact silence Ultrabithorax in early embryos, probably by transcriptional interference.