David Latrasse

Noncoding Transcription by Alternative RNA Polymerases Dynamically Regulates an Auxin-Driven Chromatin Loop

The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes.

The BAF60 Subunit of the SWI/SNF Chromatin-Remodeling Complex Directly Controls the Formation of a Gene Loop at FLOWERING LOCUS C in Arabidopsis

It is shown that BAF60, a subunit of the chromatin-remodeling complex SWI/SNF, induces a change in the floral repressor FLOWERING LOCUS C at the high-order chromatin level, thereby repressing the photoperiod flowering pathway in Arabidopsis. Specifically, BAF60 modulates histone density, composition, and posttranslational modification, thereby controlling gene loop formation at FLOWERING LOCUS C. SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus.

Histone acetyltransferase GCN5 interferes with the miRNA pathway in Arabidopsis

MicroRNAs (miRNA) that guide sequence-specific posttranscriptional gene silencing play an important role in gene expression required for both developmental processes and responses to environmental conditions in plants. However, little is known about the transcriptional and posttranscriptional regulation of miRNA expression. Histone acetylation plays an important role in chromatin remodeling and is required for gene activation. By analyzing the accumulation of subset of miRNAs and the corresponding primary miRNAs in mutants of Arabidopsis, we show that histone acetyltransferase GCN5 (General control non-repressed protein5) has a general repressive effect on miRNA production, while it is required for the expression of a subset of (e.g. stress-inducible) MIRNA genes. The general negative function of GCN5 in miRNA production is likely achieved through an indirect repression of the miRNA machinery genes such as DICER LIKE1 (DCL1), SERRATE (SE), HYPONASTIC LEAVES1 (HYL1) and ARGONAUTE1 (AGO1). Chromatin immunoprecipitation assays revealed that GCN5 targets to a subset of MIRNA genes and is required for acetylation of histone H3 lysine 14 at these loci. Moreover, inhibition of histone deacetylation by trichostatin A treatment or in histone deacetylase gene mutants impaired the accumulation of certain miRNAs. These data together suggest that Arabidopsis GCN5 interferes with the miRNA pathway at both the transcriptional and posttranscriptional levels and histone acetylation/deacetylation is an epigenetic mechanism involved in the regulation of miRNA production.

Arabidopsis histone deacetylase HDA9 regulates flowering time through repression of AGL19

Flowering time is tightly controlled by several regulatory pathways including photoperiod, vernalization in which epigenetic processes are involved. In this work, we have found that the Arabidopsis histone deacetylase gene HDA9 is involved in flowering time control. Mutation of the gene led to an early flowering phenotype in short day grown plants while without effect in long days. Analysis of flowering time regulatory gene expression revealed that hda9 mutations highly induced the expression of AGL19, but had no effect on CO, SOC1 or FLC. Chromatin immunoprecipitation assays indicated that the mutations led to a clear increase of histone H3K9 and H3K27 acetylation on the AGL19 gene in short days. AGL19 promotes flowering in a way independent of the CO and FLC pathways and has been shown to be repressed by polycomb group repressive complex2 (PRC2) EMF2 but activated by vernalization. The induced levels of AGL19 expression and histone acetylation by the hda9 mutations were comparable to that of the gene under long day conditions, indicating that AGL19 is regulated also by day length and that HDA9 is involved in short day repression of AGL19 by promoting histone H3 deacetylation, which may be related to the PRC2 EMF2 complex.

Characterization of a phosphatase 2C protein as an interacting partner of the histone acetyltransferase GCN5 in Arabidopsis.

Arabidopsis GCN5 is a major histone acetyltransferase. The mutation of the gene induces pleiotropic effects on plant development, and affects the expression of a large number of genes. The mechanism of action of this protein in controlling plant chromatin structure and genome expression is not understood. In this work, we report the identification of a number of potential protein interacting partners of GCN5 in Arabidopsis. In particular, GCN5 was shown to interact specifically with a phosphatase 2C protein (AtPP2C-6-6). GCN5 phosphorylated by activities in cellular extracts could be dephosphorylated by AtPP2C-6-6 in vitro. Analysis of T-DNA insertion mutants revealed a positive role of AtPP2C-6-6 in salt induction of stress-inducible genes, while the gcn5 mutation seemed to have no effect on the induction but showed up-regulation of a subset of the stress-inducible genes under non-induced conditions. In addition, the gcn5 mutation seriously reduced acetylation of histone H3K14 and H3K27, whereas the T-DNA insertions of the AtPP2C6-6 gene enhanced the acetylation of these lysine residues. Taken together, the present data suggest that AtPP2C-6-6 may function as a negative regulator of GCN5 activity in Arabidopsis.

Multiple functions of Kip-related protein5 connect endoreduplication and cell elongation.

The cell cycle inhibitor KRP5 binds chromatin to coordinately control endoreduplication and chromatin structure and to allow the expression of genes required for cell elongation. Despite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclin-dependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis (Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication, indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization. Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithorax-related protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required for cell elongation.

LHP1 Regulates H3K27me3 Spreading and Shapes the Three-Dimensional Conformation of the Arabidopsis Genome

Precise expression patterns of genes in time and space are essential for proper development of multicellular organisms. Dynamic chromatin conformation and spatial organization of the genome constitute a major step in this regulation to modulate developmental outputs. Polycomb repressive complexes (PRCs) mediate stable or flexible gene repression in response to internal and environmental cues. In Arabidopsis thaliana, LHP1 co-localizes with H3K27me3 epigenetic marks throughout the genome and interacts with PRC1 and PRC2 members as well as with a long noncoding RNA. Here, we show that LHP1 is responsible for the spreading of H3K27me3 towards the 3’ end of the gene body. We also identified a subset of LHP1-activated genes and demonstrated that LHP1 shapes local chromatin topology in order to control transcriptional co-regulation. Our work reveals a general role of LHP1 from local to higher conformation levels of chromatin configuration to determine its accessibility to define gene expression patterns.

Put your 3D glasses on: plant chromatin is on show

The three-dimensional organization of the eukaryotic nucleus and its chromosomal conformation have emerged as important features in the complex network of mechanisms behind gene activity and genome connectivity dynamics, which can be evidenced in the regionalized chromosomal spatial distribution and the clustering of diverse genomic regions with similar expression patterns. The development of chromatin conformation capture (3C) techniques has permitted the elucidation of commonalities between the eukaryotic phyla, as well as important differences among them. The growing number of studies in the field performed in plants has shed light on the structural and regulatory features of these organisms. For instance, it has been proposed that plant chromatin can be arranged into different conformations such as Rabl, Rosette-like, and Bouquet, and that both short- and long-range chromatin interactions occur in Arabidopsis. In this review, we compile the current knowledge about chromosome architecture characteristics in plants, as well as the molecular events and elements (including long non-coding RNAs, histone and DNA modifications, chromatin remodeling complexes, and transcription factors) shaping the genome three-dimensional conformation. Furthermore, we discuss the developmental outputs of genome topology-mediated gene expression regulation. It is becoming increasingly clear that new tools and techniques with higher resolution need to be developed and implemented in Arabidopsis and other model plants in order to better understand chromosome architecture dynamics, from an integrative perspective with other fields of plant biology such as development, stress biology, and finally agriculture.

Dual function of MIPS1 as a metabolic enzyme and transcriptional regulator.

Because regulation of its activity is instrumental either to support cell proliferation and growth or to promote cell death, the universal myo-inositol phosphate synthase (MIPS), responsible for myo-inositol biosynthesis, is a critical enzyme of primary metabolism. Surprisingly, we found this enzyme to be imported in the nucleus and to interact with the histone methyltransferases ATXR5 and ATXR6, raising the question of whether MIPS1 has a function in transcriptional regulation. Here, we demonstrate that MIPS1 binds directly to its promoter to stimulate its own expression by locally inhibiting the spreading of ATXR5/6-dependent heterochromatin marks coming from a transposable element. Furthermore, on activation of pathogen response, MIPS1 expression is reduced epigenetically, providing evidence for a complex regulatory mechanism acting at the transcriptional level. Thus, in plants, MIPS1 appears to have evolved as a protein that connects cellular metabolism, pathogen response and chromatin remodeling.

Chromatin meets the cell cycle.

The cell cycle is one of the most comprehensively studied biological processes, due primarily to its significance in growth and development, and its deregulation in many human disorders. Studies using a diverse set of model organisms, including yeast, worms, flies, frogs, mammals, and plants, have greatly expanded our knowledge of the cell cycle and have contributed to the universally accepted view of how the basic cell cycle machinery is regulated. In addition to the oscillating activity of various cyclin-dependent kinase (CDK)-cyclin complexes, a plethora of proteins affecting various aspects of chromatin dynamics has been shown to be essential for cell proliferation during plant development. Furthermore, it was reported recently that core cell cycle regulators control gene expression by modifying histone patterns. This review focuses on the intimate relationship between the cell cycle and chromatin. It describes the dynamics and functions of chromatin structures throughout cell cycle progression and discusses the role of heterochromatin as a barrier against re-replication and endoreduplication. It also proposes that core plant cell cycle regulators control gene expression in a manner similar to that described in mammals. At present, our challenge in plants is to define the complete set of effectors and actors that coordinate cell cycle progression and chromatin structure and to understand better the functional interplay between these two processes.