Danny W.K. Ng

Coordinated histone modifications are associated with gene expression variation within and between species

Histone modifications regulate gene expression in eukaryotes, but their effects on transcriptomes of a multicellular organism and on transcriptomic divergence between species are poorly understood. Here we present the first nucleotide-resolution maps of histone acetylation, methylation, and core histone in Arabidopsis thaliana and a comprehensive analysis of these and all other available maps with gene expression data in A. thaliana, Arabidopsis arenosa, and allotetraploids. H3K9 acetylation (H3K9ac) and H3K4 trimethylation (H3K4me3) are correlated, and their distribution patterns are associated with Gene Ontology (GO) functional classifications. Highly dense and narrow distributions of these modifications near transcriptional start sites are associated with constitutive expression of genes involved in translation, whereas broad distributions toward coding regions correlate with expression variation of the genes involved in photosynthesis, carbohydrate metabolism, and defense responses. Compared to animal stem cells, dispersed distributions of H3K27me3 without bivalent H3K4me3 and H3K9ac marks correlate with developmentally repressed genes in Arabidopsis. Finally, genes affected by A. thaliana histone deacetylase 1 mutation tend to show high levels of expression variation within and between species. The data suggest that genome-wide coordinated modifications of histone acetylation and methylation provide a general mechanism for gene expression changes within and between species and in allopolyploids.

Ordered Histone Modifications Are Associated with Transcriptional Poising and Activation of the phaseolin Promoter

The phaseolin (phas) promoter drives copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues, in which a nucleosome is positioned over its three-phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis thaliana leaves was accomplished by ectopic expression of the transcription factor Phaseolus vulgaris ABI3-like factor (ALF) and application of abscisic acid (ABA). Placement of hemagglutinin-tagged ALF expression under the control of an estradiol-inducible promoter permitted chromatin immunoprecipitation analysis of chronological changes in histone modifications, notably increased acetylation of H3-K9 and H4-K12, as phas chromatin was remodeled (potentiated). A different array of changes, including acetylation of H3-K14 and methylation of H3-K4, was found to be associated with ABA-mediated activation. Thus, temporal separation of phas potentiation from activation revealed that histone H3 and H4 Lys residues are not globally hyperacetylated during phas expression. Whereas decreases in histone H3 and H4 levels were detected during ALF-mediated remodeling, slight increases occurred after ABA-mediated activation, suggesting the restoration of histone–phas interactions or the replacement of histones in the phas chromatin. The observed histone modifications provide insight into factors involved in the euchromatinization and activation of a plant gene and expand the evidence for histone code conservation among eukaryotes.

Big roles for small RNAs in polyploidy, hybrid vigor, and hybrid incompatibility

Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (ta-siRNAs), mediate gene expression and epigenetic regulation. While siRNAs are highly diverged, miRNAs and ta-siRNAs are generally conserved but many are differentially expressed between related species and in interspecific hybrids and allopolyploids. On one hand, combination of diverged maternal and paternal siRNAs in the same nucleus may exert cis-acting and trans-acting effects on transposable elements (TEs) and TE-associated genes, leading to genomic instability and endosperm and embryo failures, constituting a bottleneck for the evolution of hybrids and polyploids. On the other hand, cis and trans-acting small RNAs induce quantitative and qualitative changes in epigenetic regulation, leading to morphological variation and hybrid vigor in F1 hybrids and stable allopolyploids as well as transgressive phenotypes in the progeny, increasing a potential for adaptive evolution.

cis- and trans-Regulation of miR163 and Target Genes Confers Natural Variation of Secondary Metabolites in Two Arabidopsis Species and Their Allopolyploids

Nonadditive expression of miR163 in allopolyploids is caused largely by cis-acting promoters and by trans-acting factors present in Arabidopsis arenosa and allopolyploids but absent in Arabidopsis thaliana. miR163 negatively regulates secondary metabolite pathways in defense response. This is one example of genome-wide cis- and trans-regulation that shapes interspecific hybrids and allopolyploids. MicroRNAs (miRNAs) play essential roles in plant and animal development, but the cause and effect of miRNA expression divergence between closely related species and in interspecific hybrids or allopolyploids are unknown. Here, we show differential regulation of a miR163-mediated pathway in allotetraploids and their progenitors, Arabidopsis thaliana and Arabidopsis arenosa. miR163 is a recently evolved miRNA in A. thaliana and highly expressed in A. thaliana, but its expression was undetectable in A. arenosa and repressed in resynthesized allotetraploids. Repression of A. arenosa MIR163 (Aa MIR163) is caused by a weak cis-acting promoter and putative trans-acting repressor(s) present in A. arenosa and allotetraploids. Moreover, ectopic Aa MIR163 precursors were processed more efficiently in A. thaliana than in resynthesized allotetraploids, suggesting a role of posttranscriptional regulation in mature miR163 abundance. Target genes of miR163 encode a family of small molecule methyltransferases involved in secondary metabolite biosynthetic pathways that are inducible by a fungal elicitor, alamethicin. Loss of miR163 or overexpression of miR163 in mir163 mutant plants alters target transcript and secondary metabolite profiles. We suggest that cis- and trans-regulation of miRNA and other genes provides a molecular basis for natural variation of biochemical and metabolic pathways that are important to growth vigor and stress responses in Arabidopsis-related species and allopolyploids.

Cis- and trans -regulatory divergence between progenitor species determines gene-expression novelty in Arabidopsis allopolyploids

Gene-expression divergence between species shapes morphological evolution, but the molecular basis is largely unknown. Here we show cis- and trans-regulatory elements and chromatin modifications on gene-expression diversity in genetically tractable Arabidopsis allotetraploids. In Arabidopsis thaliana and Arabidopsis arenosa, both cis and trans with predominant cis-regulatory effects mediate gene-expression divergence. The majority of genes with both cis- and trans-effects are subjected to compensating interactions and stabilizing selection. Interestingly, cis- and trans-regulation is associated with chromatin modifications. In F1 allotetraploids, Arabidopsis arenosa trans factors predominately affect allelic expression divergence. Arabidopsis arenosa trans factors tend to upregulate Arabidopsis thaliana alleles, whereas Arabidopsis thaliana trans factors up- or down-regulate Arabidopsis arenosa alleles. In resynthesized and natural allotetraploids, trans effects drive expression of both homoeologous loci into the same direction. We provide evidence for natural selection and chromatin regulation in shaping gene-expression diversity during plant evolution and speciation.

Proteomic divergence in Arabidopsis autopolyploids and allopolyploids and their progenitors

Autopolyploidy and allopolyploidy are common in many plants and some animals. Rapid changes in genomic composition and gene expression have been observed in both autopolyploids and allopolyploids, but the effects of polyploidy on proteomic divergence are poorly understood. Here, we report quantitative analysis of protein changes in leaves of Arabidopsis autopolyploids and allotetraploids and their progenitors using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. In more than 1000 proteins analyzed, the levels of protein divergence were relatively high (∼18%) between Arabidopsis thaliana and Arabidopsis arenosa, relatively low (∼6.8%) between an A. thaliana diploid and autotetraploid and intermediate (∼8.3 and 8.2%) in F1- and F8-resynthesized allotetraploids relative to mid-parent values, respectively. This pattern of proteomic divergence was consistent with the previously reported gene expression data. In particular, many non-additively accumulated proteins (61–62%) in the F1 and F8 allotetraploids were also differentially expressed between the parents. The differentially accumulated proteins in functional categories of abiotic and biotic stresses were overrepresented between an A. thaliana autotetraploid and diploid and between two Arabidopsis species, but not significantly different between allotetraploids and their progenitors. Although the trend of changes is similar, the percentage of differentially accumulated proteins that matched previously reported differentially expressed genes was relatively low. Western blot analysis confirmed several selected proteins with isoforms the cumulative levels of which were differentially expressed. These data suggest high protein divergence between species and rapid changes in post-transcriptional regulation and translational modifications of proteins during polyploidization.

The 5 UTR negatively regulates quantitative and spatial expression from the ABI3 promoter

The involvement of transcription factors Arabidopsis abscisic acid-insensitive3 (ABI3), maize viviparous1 (VP1) and Phaseolus vulgaris ABI3-like factor (PvALF) in the spatial control of storage protein gene expression is well established. However, little insight exists as to how they are themselves regulated. To address this, a 5.15 kb ABI3 upstream sequence including a 4.6 kb full-length promoter and 519 bp of 5′-untranslated region (UTR) was used to drive either β-glucuronidase (GUS) or green fluorescent protein (GFP) expression in Arabidopsis. Expression from the full-length (−4630/+519ABI3) and various 5′-truncated promoters was detected during embryogenesis in all lines, except those transgenic for promoter elements shorter than 364 bp. Two upstream activating regions, −3600 to −2033 and −2033 to −882, enhanced GUS expression in seeds. The −882 to −364 region was sufficient to confer seed-specific expression of GUS when fused to a −64/+6CaMV 35S minimal promoter. Expression from the ABI3 promoter constructs was seed-specific, except in the presence of exogenous abscisic acid (ABA) (>0.3 μM), when GUS expression was detected in seedling roots. Excision of a 405 bp region containing three upstream open reading frames (uORFs) from the 5′-UTR dramatically increased GUS expression and debilitated constraint of reporter expression in roots. Negative regulation of ABI3 expression by the 5′-UTR may involve a post-transcriptional mechanism analogous to that of tumor suppressor genes which also bear long, uORF-containing, 5′-UTRs, or through interactions with RNA-binding proteins.

PvALF and FUS3 activate expression from the phaseolin promoter by different mechanisms

Transcription from the phaseolin (phas) promoter requires two major events: chromatin remodeling, mediated by PvALF, a B3 domain factor, and activation by an ABA-induced signal transduction cascade. Expression from phas is normally seed-specific, but high levels of expression in leaves can be obtained by ectopic expression of PvALF. Here, the system was used to compare the ability of PvALF and Arabidopsis FUS3, another B3 domain transcription factor that lacks the N-terminal activation and B1 domain present in PvALF, to activate phas expression in vegetative tissues. When compared to PvALF-mediated phas activation in the presence of ABA, a delay in phas activation was observed in the presence of both FUS3 and ABA in vegetative tissue. Significant differences in histone modifications at the phas promoter were mediated by FUS3 and PvALF, suggesting that they function through different epigenetic mechanisms. The relationship between PvALF and ABI5, a bZIP transcription factor, in mediating phas expression was also evaluated. Interestingly, over-expression of ABI5 rendered phas expression ABA-independent in the presence of PvALF. Changes in phas activity in different regions within seed embryos were demonstrated using abi5 mutants. Our results show that (1) redundant factors, such as PvALF and FUS3, employ different mechanisms to regulate their common target gene (phas); (2) ABI5, and possibly other redundant bZIP factors, act downstream of ABA in modulating phas expression in the presence of PvALF.

A Role for CHH Methylation in the Parent-of-Origin Effect on Altered Circadian Rhythms and Biomass Heterosis in Arabidopsis Intraspecific Hybrids

This work describes a unique role for the RNA-directed DNA methylation pathway (mainly CHH methylation, where H = A, T, or C) in mediating the parent-of-origin effect on the expression of the circadian clock gene CCA1 in Arabidopsis intraspecific hybrids. Altered CCA1 expression amplitudes are associated with heterosis of embryo growth and biomass accumulation in the reciprocal hybrids. Hybrid plants and animals often show increased levels of growth and fitness, a phenomenon known as hybrid vigor or heterosis. Circadian rhythms optimize physiology and metabolism in plants and animals. In plant hybrids and polyploids, expression changes of the genes within the circadian regulatory network, such as CIRCADIAN CLOCK ASSOCIATED1 (CCA1), lead to heterosis. However, the relationship between allelic CCA1 expression and heterosis has remained elusive. Here, we show a parent-of-origin effect on altered circadian rhythms and heterosis in Arabidopsis thaliana F1 hybrids. This parent-of-origin effect on biomass heterosis correlates with altered CCA1 expression amplitudes, which are associated with methylation levels of CHH (where H = A, T, or C) sites in the promoter region. The direction of rhythmic expression and hybrid vigor is reversed in reciprocal F1 crosses involving mutants that are defective in the RNA-directed DNA methylation pathway (argonaute4 and nuclear RNA polymerase D1a) but not in the maintenance methylation pathway (methyltransferase1 and decrease in DNA methylation1). This parent-of-origin effect on circadian regulation and heterosis is established during early embryogenesis and maintained throughout growth and development.

Regulation of miR163 and its targets in defense against Pseudomonas syringae in Arabidopsis thaliana

Small RNAs are important regulators for a variety of biological processes, including leaf development, flowering-time, embryogenesis and defense responses. miR163 is a non-conserved miRNA and its locus has evolved recently through inverted duplication of its target genes to which they belong to the SABATH family of related small-molecule methyltransferases (MTs). In Arabidopsis thaliana, previous study demonstrated that miR163 accumulation was induced by alamethicin treatment, suggesting its roles in defense response pathways. Enhanced resistance against Pseudomonas syringae pv. tomato (Pst) was observed in the mir163 mutant, whereas transgenic lines overexpressing miR163 showed increase sensitivity to Pst, suggesting that miR163 is a negative regulator of defense response. Elevated level of miR163 and its targets in A. thaliana were observed upon Pst treatment, suggesting a modulating relationship between miR163 and its targets. In addition, miR163 and histone deacetylase were found to act cooperatively in mediating defense against Pst. Transgenic plants overexpressing miR163-resistant targets suggested their different contributions in defense. Results from this study revealed that the stress-inducible miR163 and its targets act in concert to modulate defense responses against bacterial pathogen in A. thaliana.