Frédéric Pontvianne

Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation

The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.

PHYTOCHROME B and HISTONE DEACETYLASE 6 Control Light-Induced Chromatin Compaction in Arabidopsis thaliana

Natural genetic variation in Arabidopsis thaliana exists for many traits and often reflects acclimation to local environments. Studying natural variation has proven valuable in the characterization of phenotypic traits and, in particular, in identifying genetic factors controlling these traits. It has been previously shown that chromatin compaction changes during development and biotic stress. To gain more insight into the genetic control of chromatin compaction, we investigated the nuclear phenotype of 21 selected Arabidopsis accessions from different geographic origins and habitats. We show natural variation in chromatin compaction and demonstrate a positive correlation with latitude of geographic origin. The level of compaction appeared to be dependent on light intensity. A novel approach, combining Quantitative Trait Locus (QTL) mapping and microscopic examination, pointed at PHYTOCHROME-B (PHYB) and HISTONE DEACETYLASE-6 (HDA6) as positive regulators of light-controlled chromatin compaction. Indeed, mutant analyses demonstrate that both factors affect global chromatin organization. HDA6, in addition, strongly promotes the light-mediated compaction of the Nucleolar Organizing Regions (NORs). The accession Cape Verde Islands-0 (Cvi-0), which shows sequence polymorphism in the PHYB gene and in the HDA6 promotor, resembles the hda6 mutant in having reduced chromatin compaction and decreased methylation levels of DNA and histone H3K9 at the NORs. We provide evidence that chromatin organization is controlled by light intensity. We propose that chromatin plasticity is associated with acclimation of Arabidopsis to its environment. The polymorphic alleles such as PHYB and HDA6 control this process.

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

Eukaryotes can have thousands of 45S ribosomal RNA (rRNA) genes, many of which are silenced during development. Using fluorescence-activated sorting techniques, we show that active rRNA genes in Arabidopsis thaliana are present within sorted nucleoli, whereas silenced rRNA genes are excluded. DNA methyltransferase (met1), histone deacetylase (hda6), or chromatin assembly (caf1) mutants that disrupt silencing abrogate this nucleoplasmic-nucleolar partitioning. Bisulfite sequencing data indicate that active nucleolar rRNA genes are nearly completely demethylated at promoter CGs, whereas silenced genes are nearly fully methylated. Collectively, the data reveal that rRNA genes occupy distinct but changeable nuclear territories according to their epigenetic state.

Arabidopsis Histone Lysine Methyltransferases

In eukaryotes, changes in chromatin structure regulate the access of gene regulatory sequences to the transcriptional machinery and play important roles in the repression of transposable elements, thereby protecting genome integrity. Chromatin dynamics and gene expression states are highly correlated, with DNA methylation and histone post-translational modifications playing important roles in the establishment or maintenance of chromatin states in plants. Histones can be covalently modified in a variety of ways, thereby affecting nucleosome spacing and/or higher-order nucleosome interactions directly or via the recruitment of histone-binding proteins. An extremely important group of chromatin modifying enzymes are the histone lysine methyltransferases (HKMTs). These enzymes are involved in the establishment and/or maintenance of euchromatic or heterochromatic states of active or transcriptionally repressed sequences, respectively. The vast majority of HKMTs possess a SET domain named for the three Drosophila proteins that are the founding members of the family: Suppressor of variegation, Enhancer of zeste and Trithorax. It is the SET domain that is responsible for HKMT enzymatic activity. Mutation of Arabidopsis HKMT genes can result in phenotypic abnormalities due to the improper regulation of important developmental genes. Here, we review the different classes of HKMTs present in the model plant Arabidopsis thaliana and discuss what is known about their biochemical and biological functions.

Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

A Two-Step Process for Epigenetic Inheritance in Arabidopsis

In Arabidopsis, multisubunit RNA polymerases IV and V orchestrate RNA-directed DNA methylation (RdDM) and transcriptional silencing, but what identifies the loci to be silenced is unclear. We show that heritable silent locus identity at a specific subset of RdDM targets requires HISTONE DEACETYLASE 6 (HDA6) acting upstream of Pol IV recruitment and siRNA biogenesis. At these loci, epigenetic memory conferring silent locus identity is erased in hda6 mutants such that restoration of HDA6 activity cannot restore siRNA biogenesis or silencing. Silent locus identity is similarly lost in mutants for the cytosine maintenance methyltransferase, MET1. By contrast, pol IV or pol V mutants disrupt silencing without erasing silent locus identity, allowing restoration of Pol IV or Pol V function to restore silencing. Collectively, these observations indicate that silent locus specification and silencing are separable steps that together account for epigenetic inheritance of the silenced state.

Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis

In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2.

A Duplicated NUCLEOLIN Gene with Antagonistic Activity Is Required for Chromatin Organization of Silent 45S rDNA in Arabidopsis

The nucleolus, the most prominent nuclear structure, is involved in ribosome biogenesis and nuclear and nucleolar chromatin organization. Nucleolins are among the most abundant nucleolar proteins. In contrast with mammals, plants encode two nucleolins. Characterization of Arabidopsis thaliana nuc2 mutants shows the importance of these proteins in chromatin silencing and ribosomal DNA organization. In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.

Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome

The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

Hybrid incompatibility caused by an epiallele

Significance Deleterious mutations in different copies of a duplicated gene pair have the potential to cause hybrid incompatibility between diverging subpopulations, contributing to reproductive isolation and speciation. This study demonstrates a case of epigenetic gene silencing rather than pseudogene creation by mutation, contributing to a lethal gene combination on hybridization of two ecotypes of Arabidopsis thaliana. Our findings provide direct evidence that naturally occurring epigenetic variation can contribute to incompatible hybrid genotypes, reducing gene flow between subtypes of the same species. Hybrid incompatibility resulting from deleterious gene combinations is thought to be an important step toward reproductive isolation and speciation. Here, we demonstrate involvement of a silent epiallele in hybrid incompatibility. In Arabidopsis thaliana accession Cvi-0, one of the two copies of a duplicated histidine biosynthesis gene, HISN6A, is mutated, making HISN6B essential. In contrast, in accession Col-0, HISN6A is essential because HISN6B is not expressed. Owing to these differences, Cvi-0 × Col-0 hybrid progeny that are homozygous for both Cvi-0 HISN6A and Col-0 HISN6B do not survive. We show that HISN6B of Col-0 is not a defective pseudogene, but a stably silenced epiallele. Mutating HISTONE DEACETYLASE 6 (HDA6), or the cytosine methyltransferase genes MET1 or CMT3, erases HISN6Bs silent locus identity, reanimating the gene to circumvent hisn6a lethality and hybrid incompatibility. These results show that HISN6-dependent hybrid lethality is a revertible epigenetic phenomenon and provide additional evidence that epigenetic variation has the potential to limit gene flow between diverging populations of a species.