Inner workings and regulatory inputs that control Polycomb repressive complex 2.

Polycomb repressive complex 2 (PRC2) is a conserved multisubunit enzyme that methylates histone H3 on lysine-27. This chromatin modification is a hallmark of target genes transcriptionally silenced by the Polycomb system. At its core, PRC2 activity depends upon the SET domain active site of its catalytic subunit, EZH2, as well as critical stimulatory inputs from noncatalytic subunits, especially EED and SU(Z)12. We review recent progress on this core PRC2 machinery, including key features of the active site, control mechanisms that operate via EZH2 phosphorylation, and subunit elements and architectures that influence PRC2 function. Among these, we highlight work identifying an EED regulatory site that enables PRC2 to bind pre-existing methylated H3-K27 and stimulate enzyme output. These advances illuminate basic inner workings of PRC2 and also provide insights that could aid design of PRC2 inhibitors. The chromatin landscape that PRC2 encounters in vivo is decorated with many histone modifications that accompany active transcription, such as H3-K4 methylation. It has long been assumed that these "active" modifications oppose PRC2 at some level but, until recently, mechanisms of this antagonistic cross-talk have been elusive. We discuss new findings that illuminate how H3-K4 and H3-K36 methylation, H3-K27 acetylation, and H3-S28 phosphorylation each exert a negative impact on PRC2 function. The emerging picture presents PRC2 as a cooperative multipart machine, intricately outfitted to sense and respond to the local chromatin environment and other cues. This PRC2 design ensures flexibility and fine tuning of its fundamental gene silencing roles in diverse biological contexts.

A left/right asymmetric neuronal differentiation program is controlled by the Caenorhabditis elegans lsy-27 zinc-finger transcription factor.

Functional diversification across the left/right axis is a common feature of many nervous systems. The genetic programs that control left/right asymmetric neuron function and gene expression in the nervous system are, however, poorly understood. We describe here the molecular characterization of two phenotypically similar mutant Caenorhabditis elegans strains in which left/right asymmetric gene expression programs of two gustatory neurons, called ASEL and ASER, are disrupted such that the differentiation program of the ASER neuron is derepressed in the ASEL neuron. We show that in one mutant strain the LIM homeobox gene lim-6 is defective whereas in another strain a novel member of a nematode-specific, fast-evolving family of C2H2 zinc-finger transcription factors, lsy-27, is mutated, as revealed by whole-genome sequencing. lsy-27 is broadly and exclusively expressed in the embryo and acts during the initiation, but not during the maintenance phase of ASE asymmetry control to assist in the initiation of lim-6 expression.

Maintenance of neuronal laterality in Caenorhabditis elegans through MYST histone acetyltransferase complex components LSY-12, LSY-13 and LIN-49.

Left/right asymmetrically expressed genes permit an animal to perform distinct tasks with the right vs. left side of its brain. Once established during development, lateralized gene expression patterns need to be maintained during the life of the animal. We show here that a histone modifying complex, composed of the LSY-12 MYST-type histone acetyltransferase, the ING-family PHD domain protein LSY-13, and PHD/bromodomain protein LIN-49, is required to first initiate and then actively maintain lateralized gene expression in the gustatory system of the nematode Caenorhabditis elegans. Similar defects are observed upon postembryonic removal of two C2H2 zinc finger transcription factors, die-1 and che-1, demonstrating that a combination of transcription factors, which recognize DNA in a sequence-specific manner, and a histone modifying enzyme complex are responsible for inducing and maintaining neuronal laterality.

Cis-regulatory mutations in the Caenorhabditis elegans homeobox gene locus cog-1 affect neuronal development.

We apply here comparative genome hybridization as a novel tool to identify the molecular lesion in two Caenorhabditis elegans mutant strains that affect a neuronal cell fate decision. The phenotype of the mutant strains resembles those of the loss-of-function alleles of the cog-1 homeobox gene, an inducer of the fate of the gustatory neuron ASER. We find that both lesions map to the cis-regulatory control region of cog-1 and affect a phylogenetically conserved binding site for the C2H2 zinc-finger transcription factor CHE-1, a previously known regulator of cog-1 expression in ASER. Identification of this CHE-1-binding site as a critical regulator of cog-1 expression in the ASER in vivo represents one of the rare demonstrations of the in vivo relevance of an experimentally determined or predicted transcription-factor-binding site. Aside from the mutationally defined CHE-1-binding site, cog-1 contains a second, functional CHE-1-binding site, which in isolation is sufficient to drive reporter gene expression in the ASER but in an in vivo context is apparently insufficient for promoting appropriate ASER expression. The cis-regulatory control regions of other ASE-expressed genes also contain ASE motifs that can promote ASE neuron expression when isolated from their genomic context, but appear to depend on multiple ASE motifs in their normal genomic context. The multiplicity of cis-regulatory elements may ensure the robustness of gene expression.

Caenorhabditis elegans mutant allele identification by whole-genome sequencing.

Identification of the molecular lesion in Caenorhabditis elegans mutants isolated through forward genetic screens usually involves time-consuming genetic mapping. We used Illumina deep sequencing technology to sequence a complete, mutant C. elegans genome and thus pinpointed a single-nucleotide mutation in the genome that affects a neuronal cell fate decision. This constitutes a proof-of-principle for using whole-genome sequencing to analyze C. elegans mutants.

Genetic screens for Caenorhabditis elegans mutants defective in left/right asymmetric neuronal fate specification.

We describe here the results of genetic screens for Caenorhabditis elegans mutants in which a single neuronal fate decision is inappropriately executed. In wild-type animals, the two morphologically bilaterally symmetric gustatory neurons ASE left (ASEL) and ASE right (ASER) undergo a left/right asymmetric diversification in cell fate, manifested by the differential expression of a class of putative chemoreceptors and neuropeptides. Using single cell-specific gfp reporters and screening through a total of almost 120,000 haploid genomes, we isolated 161 mutants that define at least six different classes of mutant phenotypes in which ASEL/R fate is disrupted. Each mutant phenotypic class encompasses one to nine different complementation groups. Besides many alleles of 10 previously described genes, we have identified at least 16 novel "lsy" genes ("laterally symmetric"). Among mutations in known genes, we retrieved four alleles of the miRNA lsy-6 and a gain-of-function mutation in the 3'-UTR of a target of lsy-6, the cog-1 homeobox gene. Using newly found temperature-sensitive alleles of cog-1, we determined that a bistable feedback loop controlling ASEL vs. ASER fate, of which cog-1 is a component, is only transiently required to initiate but not to maintain ASEL and ASER fate. Taken together, our mutant screens identified a broad catalog of genes whose molecular characterization is expected to provide more insight into the complex genetic architecture of a left/right asymmetric neuronal cell fate decision.