Eng-Seng Gan

Timing Mechanism Dependent on Cell Division Is Invoked by Polycomb Eviction in Plant Stem Cells

Introduction In plants, leaves and flowers originate from the shoot apical meristem. In an indeterminate shoot apical meristem, stem cells persist for the life of the plant. In a determinate meristem, a certain number of organs are produced before the meristem is terminated; this characterizes the floral meristem derived from the shoot apical meristem. In Arabidopsis, stem cell identity is sustained by expression of the gene WUSCHEL. Expression of WUSCHEL can be terminated by the zinc finger protein KNUCKLES (KNU), with the result that stem cell identity is inactivated. KNU expression is induced by the floral homeotic protein AGAMOUS (AG), but that induction process requires ~2 days and invokes modification of histones resident at the KNU locus. Here, we show that the 2-day time lag is a consequence of a regulated molecular mechanism and that this mechanism can be embedded in a synthetic regulatory system to invoke a similar time lag. Induction of KNU in Arabidopsis floral meristems. Synchronized inflorescences imaged by confocal microscopy and reconstructed into three-dimensional projections (red stains by a fluorescence dye show the shapes of developing flowers). In an Arabidopsis line that has been engineered so that its floral development is both inducible and synchronized, KNU expression (green) begins 1 to 3 days after the activation of flower development. The delay is mediated by repressive histone methylation at the KNU locus. Upon activation, the transcription factor AG displaces Polycomb proteins, and the repressive histone marks are lost with cell cycle progression. Scale bar, 100 μm. Methods For transgenic Arabidopsis plants, we accelerated or inhibited cell cycles with pharmacological agents and studied the resulting KNU expression in response to AG induction. We used chromatin immunoprecipitation to study the presence of Polycomb proteins on the KNU locus at specific times during flower development. We used insertional mutagenesis to alter the function of the Polycomb response element (PRE) and analyzed the response from a heterologous promoter in Arabidopsis cell cultures. We constructed a synthetic mimic in Arabidopsis floral buds of the AG function by using the DNA binding domain of the lactose operon repressor (lacI) with its cognate binding sites. To test the logic that the delay in downstream gene induction was caused by the need to evict Polycomb group (PcG) proteins from their residence, we simulated the competition between PcG proteins and DNA binding proteins by using lacI, designed transcription activator–like effector DNA binding proteins, and synthesized promoters in Arabidopsis cell lines. Results AG induces KNU with a time delay regulated by epigenetic modification. In wild-type plants, KNU expression begins in the center of the floral meristem and follows cell cycle progression. The binding sites for AG in the KNU upstream region are located within the PRE sequences required for the repressive histone modification. Binding of AG displaces PcG proteins, leading to the failure to maintain the repressive histone methylation. The combination of lacI operator sequences with a chimeric protein that contained the lacI DNA binding domain but lacked the activation domain was able to mimic the AG activity in Arabidopsis floral buds. We also reconstituted the cell division–dependent delayed-induction circuit in cell lines. Discussion Our results indicate that flower development in Arabidopsis employs cell division to provide stem cells with a window of opportunity to change fate. The competition we observed between repressive PcG proteins and an activating transcription factor may reflect a general mechanism. The logic of the molecular circuit we have uncovered here may impose timing control on diverse growth and differentiation pathways in plants and animals. A Matter of Timing Plants flower only when their developmental programs give the go-ahead; otherwise floral genes remain repressed. Sun et al. (10.1126/science.1248559; see the Perspective by Zhang) analyzed the regulatory program that controls expression of the transcription factor KNUCKLES (KNU), which is required in the control of floral genes. KNU expression was silenced by the presence of Polycomb group (PcG) proteins. The floral homeotic protein AGAMOUS competed for control of KNU and activated its expression, but with a 2-day lag time. Thus, eviction of PcG by activating DNA binding proteins can insert a lag time before a switch in gene expression takes place. A regulatory circuit controlling plant flowering genes leads the way to reconstruction of a time-delay control system. [Also see Perspective by Zhang] Plant floral stem cells divide a limited number of times before they stop and terminally differentiate, but the mechanisms that control this timing remain unclear. The precise temporal induction of the Arabidopsis zinc finger repressor KNUCKLES (KNU) is essential for the coordinated growth and differentiation of floral stem cells. We identify an epigenetic mechanism in which the floral homeotic protein AGAMOUS (AG) induces KNU at ~2 days of delay. AG binding sites colocalize with a Polycomb response element in the KNU upstream region. AG binding to the KNU promoter causes the eviction of the Polycomb group proteins from the locus, leading to cell division–dependent induction. These analyses demonstrate that floral stem cells measure developmental timing by a division-dependent epigenetic timer triggered by Polycomb eviction.

Jumonji demethylases moderate precocious flowering at elevated temperature via regulation of FLC in Arabidopsis

As sessile organisms, plants have evolved multiple mechanisms to respond to environmental changes to improve survival. Arabidopsis plants show accelerated flowering at increased temperatures. Here we show that Jumonji-C domain-containing protein JMJ30 directly binds to the flowering-repressor FLOWERING LOCUS C (FLC) locus and removes the repressive histone modification H3 lysine 27 trimethylation (H3K27me3). At elevated temperatures, the JMJ30 RNA and protein are stabilized, and FLC expression is maintained at high levels to prevent extreme precocious flowering. The double mutant of JMJ30 and its homologue JMJ32, grown at elevated temperatures, exhibits an early-flowering phenotype similar to the flc mutant, which is associated with increased H3K27me3 levels at the FLC locus and decreased FLC expression. Furthermore, ectopic expression of JMJ30 causes an FLC-dependent late-flowering phenotype. Taken together, JMJ30/JMJ32-mediated histone demethylation at the FLC locus constitutes a balancing mechanism in flowering control at warm temperatures to prevent premature early flowering.

Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes

Trimethylation of lysine 36 of histone H3 (H3K36me3) is found to be associated with various transcription events. In Arabidopsis, the H3K36me3 level peaks in the first half of coding regions, which is in contrast to the 3′-end enrichment in animals. The MRG15 family proteins function as ‘reader’ proteins by binding to H3K36me3 to control alternative splicing or prevent spurious intragenic transcription in animals. Here, we demonstrate that two closely related Arabidopsis homologues (MRG1 and MRG2) are localised to the euchromatin and redundantly ensure the increased transcriptional levels of two flowering time genes with opposing functions, FLOWERING LOCUS C and FLOWERING LOCUS T (FT). MRG2 directly binds to the FT locus and elevates the expression in an H3K36me3-dependent manner. MRG1/2 binds to H3K36me3 with their chromodomain and interact with the histone H4-specific acetyltransferases (HAM1 and HAM2) to achieve a high expression level through active histone acetylation at the promoter and 5′ regions of target loci. Together, this study presents a mechanistic link between H3K36me3 and histone H4 acetylation. Our data also indicate that the biological functions of MRG1/2 have diversified from their animal homologues during evolution, yet they still maintain their conserved H3K36me3-binding molecular function.

A matrix protein silences transposons and repeats through interaction with retinoblastoma-associated proteins.

Epigenetic regulation helps to maintain genomic integrity by suppressing transposable elements (TEs) and also controls key developmental processes, such as flowering time. To prevent TEs from causing rearrangements and mutations, TE and TE-like repetitive DNA sequences are usually methylated, whereas histones are hypoacetylated and methylated on specific residues (e.g., H3 lysine 9 dimethylation [H3K9me2]). TEs and repeats can also attenuate gene expression. However, how various histone modifiers are recruited to target loci is not well understood. Here we show that knockdown of the nuclear matrix protein with AT-hook DNA binding motifs TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) in Arabidopsis Landsberg erecta results in robust activation of various TEs, the TE-like repeat-containing floral repressor genes FLOWERING LOCUS C (FLC) and FWA. This derepression is associated with chromatin conformational changes, increased histone acetylation, reduced H3K9me2, and even TE transposition. TEK directly binds to an FLC-repressive regulatory region and the silencing repeats of FWA and associates with Arabidopsis homologs of the Retinoblastoma-associated protein 46/48, FVE and MSI5, which mediate histone deacetylation. We propose that the nuclear matrix protein TEK acts in the maintenance of genome integrity by silencing TE and repeat-containing genes.

Functional Roles of Histone Modification, Chromatin Remodeling and MicroRNAs in Arabidopsis Flower Development

Flowers are the reproductive units of angiosperms and originate from small number of stem cells maintained at the growing tips of shoots. Flower development is a multistep process starting from an environmental response, followed by the meristem identity change, termination of the stem cell activity, organ polarity control, organ identity determination, and organogenesis. It is regulated through many hard-wired genetic pathways, composed of transcription factors, signaling molecules, catalytic enzymes, and structural proteins. Epigenetic regulators play essential roles for the initiation and maintenance of the genetic pathways by controlling gene expression through chromosomes. Histone modification, ATP-dependent chromatin remodeling, and microRNAs are involved in the regulation of spatiotemporal-specific expression of huge numbers of genes that lead to patterning, specification, and morphogenesis of flowers. In contrast, DNA methylation mainly works for genome stability and integrity, silencing transposons, and repeats. This review will describe the recent progress on functional roles of epigenetic regulators and their crosstalks in Arabidopsis flower development.

Co-ordination of Flower Development Through Epigenetic Regulation in Two Model Species: Rice and Arabidopsis

Angiosperms produce flowers for reproduction. Flower development is a multistep developmental process, beginning with the initiation of the floral meristems, followed by floral meristem identity specification and maintenance, organ primordia initiation, floral organ identity specification, floral stem cell termination and finally floral organ maturation. During flower development, each of a large number of genes is expressed in a spatiotemporally regulated manner. Underlying these molecular and phenotypic events are various genetic and epigenetic pathways, consisting of diverse transcription factors, chromatin-remodeling factors and signaling molecules. Over the past 30 years, genetic, biochemical and genomic assays have revealed the underlying genetic frameworks that control flower development. Here, we will review the transcriptional regulation of flower development in two model species: Arabidopsis thaliana and rice (Oryza sativa). We focus on epigenetic regulation that functions to co-ordinate transcription pathways in flower development.

Dynamics of H3K27me3 methylation and demethylation in plant development

Epigenetic regulation controls multiple aspects of the plant development. The N-terminal tail of histone can be differently modified to regulate various chromatin activities. One of them, the trimethylation of histone H3 lysine 27 (H3K27me3) confers a repressive chromatin state with gene silencing. H3K27me3 is dynamically deposited and removed throughout development. While components of the H3K27me3 writer, Polycomb repressive complex 2 (PRC2), have been reported for almost 2 decades, it is only recently that JUMONJI (JMJ) proteins are reported as H3K27me3 demethylases, affirming the dynamic nature of histone modifications. This review highlights recent progress in plant epigenetic research, focusing on the H3K27me3 demethylases.

Flowering and genome integrity control by a nuclear matrix protein in Arabidopsis.

The matrix attachment regions (MARs) binding proteins could finely orchestrate temporal and spatial gene expression during development. In Arabidopsis, transposable elements (TEs) and TE-like repeat sequences are transcriptionally repressed or attenuated by the coordination of many key players including DNA methyltransferases, histone deacetylases, histone methyltransferases and the siRNA pathway, which help to protect genomic integrity and control multiple developmental processes such as flowering. We have recently reported that an AT-hook nuclear matrix binding protein, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), participates in a histone deacetylation (HDAC) complex to silence TEs and genes containing a TE-like sequence, including AtMu1, FWA and FLOWERING LOCUS C (FLC) in Ler background. We have shown that TEK knockdown causes increased histone acetylation, reduced H3K9me2 and moderate reduction of DNA methylation in the target loci, leading to the de-repression of FLC and FWA, as well as TE reactivation. Here we discuss the role of TEK as a putative MAR binding protein which functions in the maintenance of genome integrity and in flowering control by silencing TEs and repeat-containing genes.

SUPERMAN regulates floral whorl boundaries through control of auxin biosynthesis

Proper floral patterning, including the number and position of floral organs in most plant species, is tightly controlled by the precise regulation of the persistence and size of floral meristems (FMs). In Arabidopsis, two known feedback pathways, one composed of WUSCHEL (WUS) and CLAVATA3 (CLV3) and the other composed of AGAMOUS (AG) and WUS, spatially and temporally control floral stem cells, respectively. However, mounting evidence suggests that other factors, including phytohormones, are also involved in floral meristem regulation. Here, we show that the boundary gene SUPERMAN (SUP) bridges floral organogenesis and floral meristem determinacy in another pathway that involves auxin signaling. SUP interacts with components of polycomb repressive complex 2 (PRC2) and fine‐tunes local auxin signaling by negatively regulating the expression of the auxin biosynthesis genes YUCCA1/4 (YUC1/4). In sup mutants, derepressed local YUC1/4 activity elevates auxin levels at the boundary between whorls 3 and 4, which leads to an increase in the number and the prolonged maintenance of floral stem cells, and consequently an increase in the number of reproductive organs. Our work presents a new floral meristem regulatory mechanism, in which SUP, a boundary gene, coordinates floral organogenesis and floral meristem size through fine‐tuning auxin biosynthesis.

In Situ Proximity Ligation Assay to Detect the Interaction Between Plant Transcription Factors and Other Regulatory Proteins.

In plants, a lot of transcription factors fulfill their roles in gene regulation through the interaction with other regulatory proteins and co-factors. Thus, confirmation of protein-protein interaction is key to understand the precise function of transcription factors. Many methods have been developed to investigate the protein-protein interaction in vivo and in vitro. In situ Proximity Ligation Assay (PLA) is an innovative method to test protein-protein interaction in your tissues or cells of interest in vivo. Furthermore, this method allows us to detect transient interaction and low-abundance protein interaction with single molecule resolution. In this chapter, we describe a detailed protocol for the study of interaction between plant transcription factors and other regulatory proteins, in the scale of single nuclei of plant organ, tissues and cells.