Arabidopsis lamin-like proteins CRWN1 and CRWN2 interact with SUPPRESSOR OF NPR1-1 INDUCIBLE 1 and RAD51D to prevent DNA damage.

Plants cope with various recurring stress conditions that often induce DNA damage, ultimately affecting plant genome integrity, growth, and productivity. The CROWDED NUCLEI (CRWN) family comprises lamin-like proteins with multiple functions, such as regulating gene expression, genome organization, and DNA damage repair in Arabidopsis (Arabidopsis thaliana). However, the mechanisms and consequences of CRWNs in DNA damage repair are largely unknown. Here, we reveal that CRWNs maintain genome stability by forming repairing nuclear bodies at DNA double-strand breaks. We demonstrate that CRWN1 and CRWN2 physically associate with the DNA damage repair proteins RAD51D and SUPPRESSOR OF NPR1-1 Inducible 1 (SNI1) and act in the same genetic pathway to mediate this process. Moreover, CRWN1 and CRWN2 partially localize at γ-H2AX foci upon DNA damage. Notably, CRWN1 and CRWN2 undergo liquid-liquid phase separation to form highly dynamic droplet-like structures with RAD51D and SNI1 to promote the DNA damage response (DDR). Collectively, our data shed light on the function of plant lamin-like proteins in the DDR and maintenance of genome stability.

Histone Variant H3.3 Controls Arabidopsis Fertility by Regulating Male Gamete Development.

Reprograming of chromatin structures and changes in gene expression are critical for plant male gamete development, and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3-1 is sterile due to male sterility. Transcriptome analysis of young inflorescence has identified 2348 genes downregulated in h3.3-1 mutant, among which 1087 target genes are directly bound by H3.3, especially at their 3' ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation and pollen tube growth. The function of H3.3 in male gamete development is dependent on the Anti-Silencing Factor 1A/1B (ASF1A/1B)-Histone regulator A (HIRA)-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development.

The N-terminal coiled-coil domain of Arabidopsis CROWDED NUCLEI 1 is required for nuclear morphology maintenance.

The Arabidopsis CROWDED NUCLEI (CRWN) family proteins form a lamina-like meshwork beneath the nuclear envelope with multiple functions, including maintenance of nuclear morphology, genome organization, DNA damage repair and transcriptional regulation. CRWNs can form homodimers/heterodimers through protein‒protein interactions; however, the exact molecular mechanism of CRWN dimer formation and the diverse functions of different CRWN domains are not clear. In this report, we show that the N-terminal coiled-coil domain of CRWN1 facilitates its homodimerization and heterodimerization with the coiled-coil domains of CRWN2-CRWN4. We further demonstrated that the N-terminus but not the C-terminus of CRWN1 is sufficient to rescue the defect in nuclear morphology of the crwn1 crwn2 mutant to the WT phenotype. Moreover, both the N- and C-terminal fragments of CRWN1 are necessary for its normal function in the regulation of plant development. Collectively, our data shed light on the mechanism of plant lamina network formation and the functions of different domains in plant lamin-like proteins.

The dynamics of H2A.Z on SMALL AUXIN UP RNAs regulate abscisic acid-auxin signaling crosstalk in Arabidopsis.

Extreme environmental changes threaten plant survival and worldwide food production. In response to osmotic stress, the plant hormone abscisic acid (ABA) activates stress responses and restricts plant growth. However, the epigenetic regulation of ABA signaling and crosstalk between ABA and auxin are not well known. Here, we report that the histone variant H2A.Z-knockdown mutant in Arabidopsis Col-0, h2a.z-kd, has altered ABA signaling and stress responses. RNA-sequencing data showed that a majority of stress-related genes are activated in h2a.z-kd. In addition, we found that ABA directly promotes the deposition of H2A.Z on SMALL AUXIN UP RNAs (SAURs), and that this is involved in ABA-repression of SAUR expression. Moreover, we found that ABA represses the transcription of H2A.Z genes through suppressing the ARF7/19-HB22/25 module. Our results shed light on a dynamic and reciprocal regulation hub through H2A.Z deposition on SAURs and ARF7/19-HB22/25-mediated H2A.Z transcription to integrate ABA/auxin signaling and regulate stress responses in Arabidopsis.

Feedback regulation of auxin signaling through the transcription of H2A.Z and deposition of H2A.Z to SMALL AUXIN UP RNAs in Arabidopsis.

Auxin is a critical phytohormone that is involved in the regulation of most plant growth and developmental responses. In particular, epigenetic mechanisms, like histone modifications and DNA methylation, were reported to affect auxin biosynthesis and transport. However, the involvement of other epigenetic factors, such as histone variant H2A.Z, in the auxin-related developmental regulation remains unclear. We report that the histone variant H2A.Z knockdown mutant in Arabidopsis Col-0 ecotype, h2a.z-kd, has more lateral roots and weak gravitational responses related to auxin-regulated growth performances. Further study revealed that auxin promotes the eviction of H2A.Z from the auxin-responsive genes SMALL AUXIN-UP RNAs (SAURs) to activate their transcriptions. We found that IAA promotes the transcription of H2A.Z genes through HOMEOBOX PROTEIN 22/25 (AtHB22/25) transcription factors which work as downstream targets of ARF7/19 in auxin signaling. Double mutant of hb22 hb25 showed similar lateral root and gravitropism phenotypes to h2a.z-kd. Our results shed light on a reciprocal regulation hub through INOSITOL AUXOTROPHY 80-mediated H2A.Z eviction and ARF7/19-HB22/25-mediated H2A.Z transcription to modulate the activation of SAURs and plant growth in Arabidopsis.

The effects of Arabidopsis genome duplication on the chromatin organization and transcriptional regulation.

Autopolyploidy is widespread in higher plants and important for agricultural yield and quality. However, the effects of genome duplication on the chromatin organization and transcriptional regulation are largely unknown in plants. Using High-throughput Chromosome Conformation Capture (Hi-C), we showed that autotetraploid Arabidopsis presented more inter-chromosomal interactions and fewer short-range chromatin interactions compared with its diploid progenitor. In addition, genome duplication contributed to the switching of some loose and compact structure domains with altered H3K4me3 and H3K27me3 histone modification status. 539 genes were identified with altered transcriptions and chromatin interactions in autotetraploid Arabidopsis. Especially, we found that genome duplication changed chromatin looping and H3K27me3 histone modification in Flowering Locus C. We propose that genome doubling modulates the transcription genome-wide by changed chromatin interactions and at the specific locus by altered chromatin loops and histone modifications.

Coordinated regulation of Arabidopsis microRNA biogenesis and red light signaling through Dicer-like 1 and phytochrome-interacting factor 4.

Light and microRNAs (miRNAs) are key external and internal signals for plant development, respectively. However, the relationship between the light signaling and miRNA biogenesis pathways remains unknown. Here we found that miRNA processer proteins DCL1 and HYL1 interact with a basic helix-loop-helix (bHLH) transcription factor, phytochrome-interacting factor 4 (PIF4), which mediates the destabilization of DCL1 during dark-to-red-light transition. PIF4 acts as a transcription factor for some miRNA genes and is necessary for the proper accumulation of miRNAs. DCL1, HYL1, and mature miRNAs play roles in the regulation of plant hypocotyl growth. These results uncovered a previously unknown crosstalk between miRNA biogenesis and red light signaling through the PIF4-dependent regulation of miRNA transcription and processing to affect red-light-directed plant photomorphogenesis.

Depletion of Arabidopsis SC35 and SC35-like serine/arginine-rich proteins affects the transcription and splicing of a subset of genes.

Serine/arginine-rich (SR) proteins are important splicing factors which play significant roles in spliceosome assembly and splicing regulation. However, little is known regarding their biological functions in plants. Here, we analyzed the phenotypes of mutants upon depleting different subfamilies of Arabidopsis SR proteins. We found that loss of the functions of SC35 and SC35-like (SCL) proteins cause pleiotropic changes in plant morphology and development, including serrated leaves, late flowering, shorter roots and abnormal silique phyllotaxy. Using RNA-seq, we found that SC35 and SCL proteins play roles in the pre-mRNA splicing. Motif analysis revealed that SC35 and SCL proteins preferentially bind to a specific RNA sequence containing the AGAAGA motif. In addition, the transcriptions of a subset of genes are affected by the deletion of SC35 and SCL proteins which interact with NRPB4, a specific subunit of RNA polymerase II. The splicing of FLOWERING LOCUS C (FLC) intron1 and transcription of FLC were significantly regulated by SC35 and SCL proteins to control Arabidopsis flowering. Therefore, our findings provide mechanistic insight into the functions of plant SC35 and SCL proteins in the regulation of splicing and transcription in a direct or indirect manner to maintain the proper expression of genes and development.

Intron Lariat RNA Inhibits MicroRNA Biogenesis by Sequestering the Dicing Complex in Arabidopsis.

Lariat RNAs formed as by-products of splicing are quickly degraded by the RNA debranching enzyme 1 (DBR1), leading to their turnover. Null dbr1 mutants in both animals and plants are embryo lethal, but the mechanism underlying the lethality remains unclear. Here we characterized a weak mutant allele of DBR1 in Arabidopsis, dbr1-2, and showed that a global increase in lariat RNAs was unexpectedly accompanied by a genome-wide reduction in miRNA accumulation. The dbr1-2 mutation had no effects on expression of miRNA biogenesis genes or primary miRNAs (pri-miRNAs), but the association of pri-miRNAs with the DCL1/HYL1 dicing complex was impaired. Lariat RNAs were associated with the DCL1/HYL1 dicing complex in vivo and competitively inhibited the binding of HYL1 with pri-miRNA. Consistent with the impacts of lariat RNAs on miRNA biogenesis, over-expression of lariat RNAs reduced miRNA accumulation. Lariat RNAs localized in nuclear bodies, and partially co-localize with HYL1, and both DCL1 and HYL1 were mis-localized in dbr1-2. Together with our findings that nearly four hundred lariat RNAs exist in wild type plants and that these lariat RNAs also associate with the DCL1/HYL1 dicing complex in vivo, we thus propose that lariat RNAs, as decoys, inhibit miRNA processing, suggesting a hitherto unknown layer of regulation in miRNA biogenesis.

The Roles of Arabidopsis CDF2 in Transcriptional and Posttranscriptional Regulation of Primary MicroRNAs.

The precise regulation of microRNA (miRNA) transcription and processing is important for eukaryotic development. Plant miRNAs are first transcribed as stem-loop primary miRNAs (pri-miRNAs) by RNA polymerase II,then cleaved in the nucleus into mature miRNAs by Dicer-like 1 (DCL1). We identified a cycling DOF transcription factor, CDF2, which interacts with DCL1 and regulates the accumulation of a population of miRNAs. CDF2 binds directly to the promoters of some miRNAs and works as a transcription activator or repressor for these miRNA genes. CDF2 binds preferentially to the pri-miRNAs regulated by itself and affects DCL1-mediated processing of these pri-miRNAs. Genetically, CDF2 works in the same pathway as miR156 or miR172 to control flowering. We conclude that CDF2 regulates a group of pri-miRNAs at both the transcriptional and posttranscriptional levels to maintain proper levels of their mature miRNAs to control plant development.

Complementation of HYPONASTIC LEAVES1 by double-strand RNA-binding domains of DICER-LIKE1 in nuclear dicing bodies.

MicroRNAs (miRNAs) are a class of small regulatory RNAs that are found in almost all of the eukaryotes. Arabidopsis (Arabidopsis thaliana) miRNAs are processed from primary miRNAs (pri-miRNAs), mainly by the ribonuclease III-like enzyme DICER-LIKE1 (DCL1) and its specific partner, HYPONASTIC LEAVES1 (HYL1), a double-strand RNA-binding protein, both of which contain two double-strand RNA-binding domains (dsRBDs). These dsRBDs are essential for miRNA processing, but the functions of them are not clear. Here, we report that the two dsRBDs of DCL1 (DCL1-D1D2), and to some extent the second dsRBD (DCL1-D2), complement the hyl1 mutant, but not the first dsRBD of DCL1 (DCL1-D1). DCL1-D1 is diffusely distributed throughout the nucleoplasm, whereas DCL1-D2 and DCL1-D1D2 concentrate in nuclear dicing bodies in which DCL1 and HYL1 colocalize. We show further that protein-protein interaction is mainly mediated by DCL1-D2, while DCL1-D1 plays a major role in binding of pri-miRNAs. These results suggest parallel roles between C-terminal dsRBDs of DCL1 and N-terminal dsRBDs of HYL1 and support a model in which Arabidopsis pri-miRNAs are recruited to dicing bodies through functionally divergent dsRBDs of microprocessor for accurate processing of plant pri-miRNAs.

Four amino acids guide the assembly or disassembly of Arabidopsis histone H3.3-containing nucleosomes.

The histone variant H3.3 and the canonical histone H3.1, which differ in only 4- to 5-aa positions, are coexpressed in complex multicellular eukaryotes from fly to human and plant. H3.3 is mainly associated with active chromatin by replacing H3.1 through chaperones such as histone regulator A, death domain associated protein DAXX, thalassemia/mental retardation syndrome X-linked homolog ATRX, or proto-oncogene protein DEK and plays important roles in the germline, epigenetic memory, and reprogramming. However, the signals within H3.3 that serve as a guide for its dynamic deposition or depletion in plant chromatin are not clear. Here, we show that Arabidopsis histone H3.3 differs from H3.1 by 4-aa sites: amino acids 31, 41, 87, and 90. Although histone H3.1 is highly enriched in chromocenters, H3.3 is present in nucleolar foci in addition to being diffusely distributed in the nucleoplasm. We have evaluated the function of the 4 aa that differ between H3.1 and H3.3. We show that amino acid residue 87, and to some extent residue 90, of Arabidopsis histone H3.3 are critical for its deposition into rDNA arrays. When RNA polymerase I-directed nucleolar transcription is inhibited, wild type H3.3, but not H3.3 containing mutations at residues 31 and 41, is depleted from the rDNA arrays. Together, our results are consistent with a model in which amino acids 87 and 90 in the core domain of H3.3 guide nucleosome assembly, whereas amino acids 31 and 41 in the N-terminal tail of Arabidopsis H3.3 guide nucleosome disassembly in nucleolar rDNA.

Histone H2A monoubiquitination marks are targeted to specific sites by cohesin subunits in Arabidopsis.

Histone H2A monoubiquitination (H2Aub1) functions as a conserved posttranslational modification in eukaryotes to maintain gene expression and guarantee cellular identity. Arabidopsis H2Aub1 is catalyzed by the core components AtRING1s and AtBMI1s of polycomb repressive complex 1 (PRC1). Because PRC1 components lack known DNA binding domains, it is unclear how H2Aub1 is established at specific genomic locations. Here, we show that the Arabidopsis cohesin subunits AtSYN4 and AtSCC3 interact with each other, and AtSCC3 binds to AtBMI1s. H2Aub1 levels are reduced in atsyn4 mutant or AtSCC3 artificial microRNA knockdown plants. ChIP-seq assays indicate that most binding events of AtSYN4 and AtSCC3 are associated with H2Aub1 along the genome where transcription is activated independently of H3K27me3. Finally, we show that AtSYN4 binds directly to the G-box motif and directs H2Aub1 to these sites. Our study thus reveals a mechanism for cohesin-mediated recruitment of AtBMI1s to specific genomic loci to mediate H2Aub1.

DNA methylation in transposable elements buffers the connection between three-dimensional chromatin organization and gene transcription upon rice genome duplication.

INTRODUCTION: Polyploidy is a major force in plant evolution and the domestication of cultivated crops. OBJECTIVES: The study aimed to explore the relationship and underlying mechanism between three-dimensional (3D) chromatin organization and gene transcription upon rice genome duplication. METHODS: The 3D chromatin structures between diploid (2C) and autotetraploid (4C) rice were compared using high-throughput chromosome conformation capture (Hi-C) analysis. The study combined genetics, transcriptomics, whole-genome bisulfite sequencing (WGBS-seq) and 3D genomics approaches to uncover the mechanism for DNA methylation in modulating gene transcription through 3D chromatin architectures upon rice genome duplication. RESULTS: We found that 4C rice presents weakened intra-chromosomal interactions compared to its 2C progenitor in some chromosomes. In addition, we found that changes of 3D chromatin organizations including chromatin compartments, topologically associating domains (TADs), and loops, are uncorrelated with gene transcription. Moreover, DNA methylations in the regulatory sequences of genes in compartment A/B switched regions and TAD boundaries are unrelated to their expression. Importantly, although there was no significant difference in the methylation levels in transposable elements (TEs) in differentially expressed gene (DEG) and non-DEG promoters between 2C and 4C rice, we found that the hypermethylated TEs across genes in compartment A/B switched regions and TAD boundaries may suppress the expression of these genes. CONCLUSION: The study proposed that the rice genome doubling might modulate TE methylation to buffer the effects of chromatin architecture on gene transcription in compartment A/B switched regions and TAD boundaries, resulting in the disconnection between 3D chromatin structure alteration and gene transcription upon rice genome duplication.

Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants.

MicroRNAs (miRNAs) are important for regulating gene expression in muticellular organisms. MiRNA processing is a two-step process. In animal cells, the first step is nuclear and the second step cytoplasmic, whereas in plant cells, both steps occur in the nucleus via the enzyme Dicer-like1 (DCL1) and other proteins including the zinc-finger-domain protein Serrate (SE) and a double-stranded RNA (dsRNA) binding-domain protein, Hyponastic Leaves1 (HYL1). Furthermore, plant miRNAs are methylated by Hua Enhancer (HEN1) at their 3' ends and loaded onto Argonaute1 (AGO1). However, little is known about the cellular basis of miRNA biogenesis. Using live-cell imaging, we show here that DCL1 and HYL1 colocalize in discrete nuclear bodies in addition to being present in a low-level diffuse nucleoplasmic distribution. These bodies, which we refer to as nuclear dicing bodies (D-bodies), differ from Cajal bodies. A mutated DCL1 with impaired function in miRNA processing fails to target to D-bodies, and an introduced primary (pri)-miRNA transcript is recruited to D-bodies. Furthermore, bimolecular fluorescence complementation (BiFC) shows that DCL1, HYL1, and SE interact in D-bodies. On the basis of these data, we propose that D-bodies are crucial for orchestrating pri-miRNA processing and/or storage/assembly of miRNA-processing complexes in the nuclei of plant cells.

B-box transcription factor 28 regulates flowering by interacting with constans.

B-box transcription factors (BBXs) are important regulators of flowering, photomorphogenesis, shade-avoidance, abiotic and biotic stresses and plant hormonal pathways. In Arabidopsis, 32 BBX proteins have been identified and classified into five groups based on their structural domains. Little is known about the fifth group members (BBX26-BBX32) and the detailed molecular mechanisms relevant to their functions. Here we identified B-box transcription factor 28 (BBX28) that interacts with Constans (CO), a transcriptional activator of Flowering Locus T (FT). Overexpressing BBX28 leads to late flowering with dramatically decreased FT transcription, and bbx28 deficient mutant displays a weak early flowering phenotype under long days (LD), indicating that BBX28 plays a negative and redundant role in flowering under LD. Additionally, the interaction between BBX28 and CO decreases the recruitment of CO to FT locus without affecting the transcriptional activation activity of CO. Moreover, the N-terminal cysteines, especially those within the B-box domain, are indispensable for the heterodimerization between BBX28 and CO and activation of CO on FT transcription. Genetic evidences show that the later flowering caused by BBX28 overexpression is compromised by CO ectopic expression. Collectively, these results supported that BBX28 functions with CO and FT to negatively regulate Arabidopsis flowering, in which the N-terminal conserved cysteines of BBX28 might play a central role.

Genome-wide identification of Arabidopsis long noncoding RNAs in response to the blue light.

Long non-coding RNAs (lncRNAs) have been shown in animals to play roles in a wide range of biological processes. In plant, light modulates the growth and development as a key external signal. However, little is known about the role of plant lncRNA in response to light. In this study, we sequenced the messenger RNAs (mRNAs), lncRNAs and microRNAs (miRNAs) in Arabidopsis seedlings under blue light for 2 h and 8 h. Compared to dark, we identified 4197 mRNAs, 375 miRNAs and 481 lncRNAs, or 5207 mRNAs, 286 miRNAs and 545 lncRNAs of differential expressions under blue light treatments for 2 h or 8 h respectively. Subsequently, a total of 407 competing endogenous RNA (ceRNA) pairs (lncRNA-mRNA-miRNA) were constructed. We identified a blue light-induced lncRNA which plays roles in blue light-directed plant photomorphogenesis and response to mannitol stress by serving as a ceRNA to sequester miR167 in a type of target mimicry. These results revealed previously unknown roles of the lncRNA in blue light signaling and mannitol stress, and provided useful resources of lncRNAs associated with miRNAs in response to blue light.

Author Correction: Genome-wide identification of Arabidopsis long noncoding RNAs in response to the blue light.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-wide transposon tagging reveals location-dependent effects on transcription and chromatin organization in Arabidopsis.

The interphase nucleus exists as a highly dynamic system, the physical properties of which have functional importance in gene regulation. Not only can gene expression be influenced by the local sequence context, but also by the architecture of the nucleus in three-dimensions (3D), and by the interactions between these levels via chromatin modifications. A challenging task is to resolve the complex interplay between sequence- and genome structure-based control mechanisms. Here, we created a collection of 277 Arabidopsis lines that allow the visual tracking of individual loci in living plants while comparing gene expression potential at these locations, via an identical reporter cassette. Our studies revealed regional gene silencing near a heterochromatin island, via DNA methylation, that is correlated with mobility constraint and nucleolar association. We also found an example of nucleolar association that does not correlate with gene suppression, suggesting that distinct mechanisms exist that can mediate interactions between chromatin and the nucleolus. These studies demonstrate the utility of this novel resource in unifying structural and functional studies towards a more comprehensive model of how global chromatin organization may coordinate gene expression over large scales.