The Arabidopsis SWI2/SNF2 Chromatin Remodeler BRAHMA Regulates Polycomb Function during Vegetative Development and Directly Activates the Flowering Repressor Gene SVP

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.     

Seed dormancy cycling in Arabidopsis: chromatin remodelling and regulation of DOG1 in response to seasonal environmental signals

The involvement of chromatin remodelling in dormancy cycling in the soil seed bank (SSB) is poorly understood. Natural variation between the winter and summer annual Arabidopsis ecotypes Cvi and Bur was exploited to investigate the expression of genes involved in chromatin remodelling via histone 2B (H2B) ubiquitination/de‐ubiquitination and histone acetylation/deacetylation, the repressive histone methyl transferases CURLY LEAF (CLF) and SWINGER (SWN), and the gene silencing repressor ROS1 (REPRESSOR OF SILENCING1) and promoter of silencing KYP/SUVH4 (KRYPTONITE), during dormancy cycling in the SSB. ROS1 expression was positively correlated with dormancy while the reverse was observed for CLF and KYP/SUVH4. We propose ROS1 dependent repression of silencing and a sequential requirement of CLF and KYP/SUVH4 dependent gene repression and silencing for the maintenance and suppression of dormancy during dormancy cycling. Seasonal expression of H2B modifying genes was correlated negatively with temperature and positively with DOG1 expression, as were histone acetyltransferase genes, with histone deacetylases positively correlated with temperature. Changes in the histone marks H3K4me3 and H3K27me3 were seen on DOG1 (DELAY OF GERMINATION1) in Cvi during dormancy cycling. H3K4me3 activating marks remained stable along DOG1. During relief of dormancy, H3K27me3 repressive marks slowly accumulated and accelerated on exposure to light completing dormancy loss. We propose that these marks on DOG1 serve as a thermal sensing mechanism during dormancy cycling in preparation for light repression of dormancy. Overall, chromatin remodelling plays a vital role in temporal sensing through regulation of gene expression.     

Polycomb Domain Formation Depends on Short and Long Distance Regulatory Cues

Background

Polycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental genes. In Drosophila, cis-regulatory regions termed PcG response elements (PREs) act as nucleation sites for PcG proteins to create large repressive PcG domains that are marked by trimethylation of lysine 27 on histone H3 (H3K27me3). In addition to an action in cis, PREs can interact over long distances, thereby enhancing PcG dependent silencing. How PcG domains are established, which factors limit their propagation in cis, and how long range interactions of PREs in trans affect the chromatin structure is largely unknown.

Principal Findings

We demonstrate that the insertion of a PRE-containing transgene in the Drosophila genome generates an artificial PcG domain and we analyze its organization by quantitative ChIP and ChIP-on-chip experiments. Intriguingly, a boundary element and known insulator proteins do not necessarily interfere with spreading of H3K27me3. Instead, domain borders correlate with the presence of promoter regions bound by RNA Polymerase II and active chromatin marks. In contrast, genes that are silent during early fly development get included within the PcG domain and this incorporation interferes with gene activation at later developmental stages. Moreover, trans-interaction of the transgenic PRE with its homologous endogenous PRE results in increased PcG binding, correlating with reinforced silencing of genes within the domain borders.

Conclusions

Our results suggest that higher-order organization of PcG-bound chromatin can stabilize gene silencing within PcG domains. Further we propose that multi-protein complexes associated with active promoters are able to define the limits of PcG domains. Future work aimed to pinpoint the factors providing this barrier function will be required to understand the precise molecular mechanism by which active promoter regions can act as boundaries to stop spreading of H3K27me3.     

Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb repressive complex 2 components

Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.     

Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements

Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.     

Polycomb group proteins: remembering how to catch chromatin during replication

Polycomb group (PcG) proteins maintain the expression state of PcG‐responsive genes during development of multicellular organisms. Recent observations suggest that “the H3K27me3 modification” acts to maintain Polycomb repressive complex (PRC) 2, the enzyme that creates this modification, on replicating chromatin. This could in turn promote propagation of H3K27me3 on newly replicated daughter chromatin, and promote recruitment of PRC1. Other work suggests that PRC1‐class complexes can be maintained on replicating chromatin, at least in vitro, independently of H3K27me3. Thus, histone modifications and PcG proteins themselves may both be maintained through replication.