Roles of Polycomb complexes in regulating gene expression and chromatin structure in plants.

The evolutionary conserved Polycomb Group (PcG) repressive system comprises two central protein complexes, PcG repressive complex 1 (PRC1) and PRC2. These complexes, through the incorporation of histone modifications on chromatin, have an essential role in the normal development of eukaryotes. In recent years, a significant effort has been made to characterize these complexes in the different kingdoms, and despite there being remarkable functional and mechanistic conservation, some key molecular principles have diverged. In this review, we discuss current views on the function of plant PcG complexes. We compare the composition of PcG complexes between animals and plants, highlight the role of recently identified plant PcG accessory proteins, and discuss newly revealed roles of known PcG partners. We also examine the mechanisms by which the repression is achieved and how these complexes are recruited to target genes. Finally, we consider the possible role of some plant PcG proteins in mediating local and long-range chromatin interactions and, thus, shaping chromatin 3D architecture.

Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein-protein interactions.

Fibrillins (FBNs) are plastidial proteins found in photosynthetic organisms from cyanobacteria to higher plants. The function of most FBNs remains unknown. Here, we focused on members of the FBN subgroup comprising FBN1a, FBN1b, and FBN2. We show that these three polypeptides interact between each other, potentially forming a network around the plastoglobule surface. Both FBN2 and FBN1s interact with allene oxide synthase, and the elimination of any of these FBNs results in a delay in jasmonate-mediated anthocyanin accumulation in response to a combination of moderate high light and low temperature. Mutations in the genes encoding FBN1s or FBN2 also affect the protection of PSII under the combination of these stresses. Fully developed leaves of these mutants have lower maximum quantum efficiency of PSII (Fv/Fm) and higher oxidative stress than wild-type plants. These effects are additive, and the fbn1a-1b-2 triple mutant shows a stronger decrease in Fv/Fm and a greater increase in oxidative stress than fbn1a-1b or fbn2 mutants. Co-immunoprecipitation analysis indicated that FBN2 also interacts with other proteins involved in different metabolic processes. We propose that these fibrillins facilitate accurate positioning of different proteins involved in distinct metabolic processes, and that their elimination leads to dysfunction of those proteins.

DNA polymerase epsilon is required for heterochromatin maintenance in Arabidopsis.

BACKGROUND: Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. RESULTS: We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin. CONCLUSION: These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

The repressive role of Arabidopsis H2A.Z in transcriptional regulation depends on AtBMI1 activity.

H2A.Z variant has emerged as a critical player in regulating plant responses to environment; however, the mechanism by which H2A.Z mediates this regulation remains unclear. In Arabidopsis, H2A.Z has been proposed to have opposite effects on transcription depending on its localization within the gene. These opposite roles have been assigned by correlating gene expression and H2A.Z enrichment analyses but without considering the impact of possible H2A.Z post-translational modifications. Here, we show that H2A.Z can be monoubiquitinated by the PRC1 components AtBMI1A/B/C. The incorporation of this modification is required for H2A.Z-mediated transcriptional repression through a mechanism that does not require PRC2 activity. Our data suggest that the dual role of H2A.Z in regulating gene expression depends on the modification that it carries, while the levels of H2A.Z within genes depend on the transcriptional activity.

Arabidopsis YAF9 histone readers modulate flowering time through NuA4-complex-dependent H4 and H2A.Z histone acetylation at FLC chromatin.

Posttranslational histone modifications and the dynamics of histone variant H2A.Z are key mechanisms underlying the floral transition. In yeast, SWR1-C and NuA4-C mediate the deposition of H2A.Z and the acetylation of histone H4, H2A and H2A.Z, respectively. Yaf9 is a subunit shared by both chromatin-remodeling complexes. The significance of the two Arabidopsis YAF9 homologues, YAF9A and YAF9B, is unknown. To get an insight into the role of Arabidopsis YAF9 proteins in plant developmental responses, we followed physiological, genetic, genomic, epigenetic, proteomics and cell biology approaches. Our data revealed that YAF9A and YAF9B are histone H3 readers with unequally redundant functions. Double mutant yaf9a yaf9b plants display pleiotropic developmental phenotypic alterations as well as misregulation of a wide variety of genes. We demonstrated that YAF9 proteins regulate flowering time by both FLC-dependent and independent mechanisms that work in parallel with SWR1-C. Interestingly, we show that YAF9A binds FLC chromatin and that YAF9 proteins regulate FLC expression by modulating the acetylation levels of H2A.Z and H4 but not H2A.Z deposition. Our work highlights the key role exerted by YAF9 homologues in the posttranslational modification of canonical histones and variants that regulate gene expression in plants to control development.

A role for MED14 and UVH6 in heterochromatin transcription upon destabilization of silencing.

Constitutive heterochromatin is associated with repressive epigenetic modifications of histones and DNA which silence transcription. Yet, particular mutations or environmental changes can destabilize heterochromatin-associated silencing without noticeable changes in repressive epigenetic marks. Factors allowing transcription in this nonpermissive chromatin context remain poorly known. Here, we show that the transcription factor IIH component UVH6 and the mediator subunit MED14 are both required for heat stress-induced transcriptional changes and release of heterochromatin transcriptional silencing in Arabidopsis thaliana. We find that MED14, but not UVH6, is required for transcription when heterochromatin silencing is destabilized in the absence of stress through mutating the MOM1 silencing factor. In this case, our results raise the possibility that transcription dependency over MED14 might require intact patterns of repressive epigenetic marks. We also uncover that MED14 regulates DNA methylation in non-CG contexts at a subset of RNA-directed DNA methylation target loci. These findings provide insight into the control of heterochromatin transcription upon silencing destabilization and identify MED14 as a regulator of DNA methylation.

Arabidopsis SWC4 Binds DNA and Recruits the SWR1 Complex to Modulate Histone H2A.Z Deposition at Key Regulatory Genes.

Deposition of the H2A.Z histone variant by the SWR1 complex (SWR1-C) in regulatory regions of specific loci modulates transcription. Characterization of mutations in Arabidopsis thaliana homologs of yeast SWR1-C has revealed a role for H2A.Z exchange in a variety of developmental processes. Nevertheless, the exact composition of plant SWR1-C and how it is recruited to target genes remains to be established. Here we show that SWC4, the Arabidopsis homolog of yeast SANT domain protein Swc4/Eaf2, is a DNA-binding protein that interacts with SWR1-C subunits. We demonstrate that the swc4-1 knockout mutant is embryo-lethal, while SWC4 RNAi knockdown lines display pleiotropic phenotypic alterations in vegetative and reproductive traits, including acceleration of flowering time, indicating that SWC4 controls post-embryonic processes. Transcriptomic analyses and genome-wide profiling of H2A.Z indicate that SWC4 represses transcription of a number of genes, including the floral integrator FT and key transcription factors, mainly by modulating H2A.Z deposition. Interestingly, SWC4 silencing does not affect H2A.Z deposition at the FLC locus nor expression of this gene, a master regulator of flowering previously shown to be controlled by SWR1-C. Importantly, we find that SWC4 recognizes specific AT-rich DNA elements in the chromatin regions of target genes and that SWC4 silencing impairs SWR1-C binding at FT. Collectively, our data suggest that SWC4 regulates plant growth and development by aiding SWR1-C recruitment and modulating H2A.Z deposition.

H2A monoubiquitination in Arabidopsis thaliana is generally independent of LHP1 and PRC2 activity.

BACKGROUND: Polycomb group complexes PRC1 and PRC2 repress gene expression at the chromatin level in eukaryotes. The classic recruitment model of Polycomb group complexes in which PRC2-mediated H3K27 trimethylation recruits PRC1 for H2A monoubiquitination was recently challenged by data showing that PRC1 activity can also recruit PRC2. However, the prevalence of these two mechanisms is unknown, especially in plants as H2AK121ub marks were examined at only a handful of Polycomb group targets. RESULTS: By using genome-wide analyses, we show that H2AK121ub marks are surprisingly widespread in Arabidopsis thaliana, often co-localizing with H3K27me3 but also occupying a set of transcriptionally active genes devoid of H3K27me3. Furthermore, by profiling H2AK121ub and H3K27me3 marks in atbmi1a/b/c, clf/swn, and lhp1 mutants we found that PRC2 activity is not required for H2AK121ub marking at most genes. In contrast, loss of AtBMI1 function impacts the incorporation of H3K27me3 marks at most Polycomb group targets. CONCLUSIONS: Our findings show the relationship between H2AK121ub and H3K27me3 marks across the A. thaliana genome and unveil that ubiquitination by PRC1 is largely independent of PRC2 activity in plants, while the inverse is true for H3K27 trimethylation.

The Arabidopsis Polycomb Repressive Complex 1 (PRC1) Components AtBMI1A, B, and C Impact Gene Networks throughout All Stages of Plant Development.

Polycomb Group regulation in Arabidopsis (Arabidopsis thaliana) is required to maintain cell differentiation and allow developmental phase transitions. This is achieved by the activity of three PcG repressive complex 2s (PRC2s) and the participation of a yet poorly defined PRC1. Previous results showed that apparent PRC1 components perform discrete roles during plant development, suggesting the existence of PRC1 variants; however, it is not clear in how many processes these components participate. We show that AtBMI1 proteins are required to promote all developmental phase transitions and to control cell proliferation during organ growth and development, expanding their proposed range of action. While AtBMI1 function during germination is closely linked to B3 domain transcription factors VAL1/2 possibly in combination with GT-box binding factors, other AtBMI1 regulatory networks require participation of different factor combinations. Conversely, EMF1 and LHP1 bind many H3K27me3 positive genes up-regulated in atbmi1a/b/c mutants; however, loss of their function affects expression of a different subset, suggesting that even if EMF1, LHP1, and AtBMI1 exist in a common PRC1 variant, their role in repression depends on the functional context.

Mutations in the Arabidopsis SWC6 gene, encoding a component of the SWR1 chromatin remodelling complex, accelerate flowering time and alter leaf and flower development.

Mutations affecting the Arabidopsis SWC6 gene encoding a putative orthologue of a component of the SWR1 chromatin remodelling complex in plants have been characterized. swc6 mutations cause early flowering, shortened inflorescence internodes, and altered leaf and flower development. These phenotypic defects resemble those of the photoperiod independent early flowering 1 (pie1) and early in short days 1 (esd1) mutants, also affected in homologues of the SWR1 complex subunits. SWC6 is a ubiquitously expressed nuclear HIT-Zn finger-containing protein, with the highest levels found in pollen. Double mutant analyses suggest that swc6 abolishes the FLC-mediated late-flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. It was found that SWC6 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of swc6 in an flc null background and the down-regulation of other FLC-like/MAF genes in swc6 mutants suggest that flowering inhibition mediated by SWC6 occurs through both FLC- and FLC-like gene-dependent pathways. Both genetic and physical interactions between SWC6 and ESD1 have been demonstrated, suggesting that both proteins act in the same complex. Using chromatin immunoprecipitation, it has been determined that SWC6, as previously shown for ESD1, is required for both histone H3 acetylation and H3K4 trimethylation of the FLC chromatin. Altogether, these results suggest that SWC6 and ESD1 are part of an Arabidopsis SWR1 chromatin remodelling complex involved in the regulation of diverse aspects of plant development, including floral repression through the activation of FLC and FLC-like genes.