In monoecious melon (Cucumis melo), sex is determined by the differential expression of sex determination genes (SDGs) and adoption of sex-specific transcriptional programs. Histone modifications such as H3K27me3 have been previously shown to be a hallmark associated to unisexual flower development in melon; yet, no genetic approaches have been conducted for elucidating the roles of H3K27me3 writers, readers, and erasers in this process. Here we show that melon homologs to Arabidopsis LHP1, CmLHP1A and B, redundantly control several aspects of plant development, including sex expression. Cmlhp1ab double mutants displayed an overall loss and redistribution of H3K27me3, leading to a deregulation of genes involved in hormone responses, plant architecture, and flower development. Consequently, double mutants display pleiotropic phenotypes and, interestingly, a general increase of the male:female ratio. We associated this phenomenon with a general deregulation of some hormonal response genes and a local activation of male-promoting SDGs and MADS-box transcription factors. Altogether, these results reveal a novel function for CmLHP1 proteins in maintenance of monoecy and provide novel insights into the polycomb-mediated epigenomic regulation of sex lability in plants.

Additional file 2: Table S1. Quantification of hypocotyl length of Arabidopsis over-expression or mutant lines grown at 23°C or 29°C. Table S2. Lists of differentially expressed genes in WT (23°C vs 29°C), OE APOLO-1 and vim1-3. Table S3. APOLO targets a subset of genes differentially methylated in the vim1 mutant. Table S4. List of primers used in this study. Table S5. Lists of differentially expressed genes in WT, OE APOLO-1 and OE UPAT-1 plants.

Background RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP ( APOLO ), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Results Here, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO -VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript ( UPAT ), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO . In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner. Conclusions Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.

Characterizing the molecular mechanisms regulating gene expression is crucial for understanding the regulatory processes underlying physiological responses to environmental and developmental signals in eukaryotes. The covalent modification of histones contributes to the compaction levels of chromatin, as well as the recruitment of the transcriptional machinery to specific loci, facilitating metastable changes in gene activity. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) has become the gold standard method for determining histone modification profiles among different organisms, tissues, and genotypes. In the current protocol, we describe a highly robust method for performing ChIP-seq of histone modifications in Arabidopsis thaliana plantlets. Besides its robustness, this method uses in-house-prepared buffers for chromatin extraction, immunoprecipitation, washing, and elusion, making it cost-effective in contrast to commercial kits.

SUMMARY RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP ( APOLO ), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Here, we showed that in addition to the PRC1-component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO -VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrated that the lncRNA UHRF1 Protein Associated Transcript ( UPAT ), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO . In addition, we showed that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter. Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation, and reveal that evolutionary unrelated lncRNAs may exert similar functions across kingdoms.

The physical accessibility of specific genomic regions is a chromatin property that regulates gene expression and allows the establishment of an appropriate transcriptional landscape in response to environmental and developmental signals. In eukaryotes, ATP-dependent chromatin remodeling complexes use the energy produced via ATP hydrolysis by an ATPase subunit to perform DNA translocation. These complexes are classified into four subfamilies, based on the domain organization of their catalytic ATPases (Clapier et al., 2017Clapier C.R. Iwasa J. Cairns B.R. Peterson C.L. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes.Nat. Rev. Mol. Cell Biol. 2017; 18: 407-422Crossref PubMed Scopus (417) Google Scholar). Within the different remodeling complex subfamilies, SWI/SNF (Switch defective/sucrose non-fermentable) remodelers have been the most exhaustively characterized in organisms from different taxa, including plants (Reyes, 2014Reyes J.C. The many faces of plant SWI/SNF complex.Mol. Plant. 2014; 7: 454-458Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). SWI/SNFs are typically associated with increasing chromatin accessibility, since they have been reported to slide and eject nucleosome from their target regions (Clapier et al., 2017Clapier C.R. Iwasa J. Cairns B.R. Peterson C.L. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes.Nat. Rev. Mol. Cell Biol. 2017; 18: 407-422Crossref PubMed Scopus (417) Google Scholar). The Arabidopsis genome encodes multiple homologs of several SWI/SNF subunits: two canonical ATPase subunits, SPLAYED (SYD) and BRAHMA (BRM); four SWI3 subunits (SWI3A–SWI3D); two actin-related proteins (ARP4 and ARP7); two BRM-associated factors (SWP73A and SWP73B); and BUSHY (BSH, an ortholog of SNF5/INI1) (Thouly et al., 2020Thouly C. Le Masson M. Lai X. Carles C.C. Vachon G. Unwinding BRAHMA functions in plants.Genes (Basel). 2020; 11: 90Crossref Scopus (7) Google Scholar). Their combinational assembly has been associated with the formation of complexes with different functions, some of which have been elucidated in the last two decades. Plant SWI/SNF complexes have been shown to be involved in the regulation of a wide range of developmental processes, including embryo and leaf development, cotyledon separation, juvenile-to-adult transition, flowering time, and flower organ development. They are also crucial for photomorphogenesis, responses to plant hormones, and abiotic stresses (Reyes, 2014Reyes J.C. The many faces of plant SWI/SNF complex.Mol. Plant. 2014; 7: 454-458Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Part of their specificity for their target genomic regions has been attributed to the physical interaction of their subunits with DNA-binding proteins, which have been proposed to recruit them to specific loci. Several examples of these regulatory mechanisms have been described since the beginning of the study of the plant SWI/SNFs (e.g., AN3, REF6 [Thouly et al., 2020Thouly C. Le Masson M. Lai X. Carles C.C. Vachon G. Unwinding BRAHMA functions in plants.Genes (Basel). 2020; 11: 90Crossref Scopus (7) Google Scholar]), highlighting their importance in the regulation of well-studied genes and molecular pathways. Despite these, the general mechanisms by which SWI/SNF complexes bind and regulate the expression of their genomic targets remain elusive. In recent years, various studies with new insights have contributed to the understanding of the composition and mode of action of the Arabidopsis SWI/SNF complexes. By combining classic protein biology approaches with genetics and -omic technologies, Li's group identified and characterized two novel SWI/SNF subunits, BRIP1 and BRIP2 (BRAHMA-interacting proteins 1/2), containing a GLTSCR (glioma tumor suppressor candidate region) domain (Yu et al., 2020Yu Y. Liang Z. Song X. Fu W. Xu J. Lei Y. Yuan L. Ruan J. Chen C. Fu W. et al.BRAHMA-interacting proteins BRIP1 and BRIP2 are core subunits of Arabidopsis SWI/SNF complexes.Nat. Plants. 2020; 6: 996-1007Crossref PubMed Scopus (15) Google Scholar) (Figure 1). In agreement with their name, these proteins were found to physically interact with BRM. Due to their high degree of sequence similarity and expression patterns, the authors generated and studied the brip1 brip2 double mutant, which they found to display transcriptome and phenotypes similar to those of the brm-3 mutant (BRM lacking its bromodomain), including short roots, downward-curled leaves, early flowering, and reduced fertility. Furthermore, BRIP1 and BRIP2 were found to be required for BRM binding to its genomic targets, indicating that this is a BRIP1/2-dependent process. Remarkably, the loss of BRIP1 and BRIP2 not only affected BRM binding to chromatin but also decreased its abundance. The chemical inhibition of the proteasome in brip1 brip2 partially complemented BRM levels, suggesting that BRIP1/2 post-translationally contribute to BRM stability. Similarly, the relative abundance of other known SWI/SNF subunits was also altered in the brip1 brip2 background, highlighting the role of these two novel proteins in the maintenance of SWI/SNF complexes. Bromodomains are highly conserved eukaryotic protein domains that recognize and bind to acetylated lysines on histones. In animals and yeast, some SWI/SNF subunits contain bromodomains, which have been associated with the SWI/SNF binding to acetylated nucleosomes in these organisms (Awad and Hassan, 2008Awad S. Hassan A.H. The Swi2/Snf2 bromodomain is important for the full binding and remodeling activity of the SWI/SNF complex on H3- and H4-acetylated nucleosomes.Ann. N. Y. Acad. Sci. 2008; 1138: 366-375Crossref PubMed Scopus (29) Google Scholar). An association between SWI/SNF subunit binding and histone acetylation has been observed in Arabidopsis (Jégu et al., 2017Jégu T. Veluchamy A. Ramirez-Prado J.S. Rizzi-Paillet C. Perez M. Lhomme A. Latrasse D. Coleno E. Vicaire S. Legras S. et al.The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility.Genome Biol. 2017; 18: 114Crossref PubMed Scopus (31) Google Scholar). However, BRM, the—until recently—only known plant SWI/SNF subunit containing a bromodomain, did not show any preferential binding for their acetylated histones, raising the question of whether other bromodomain-containing proteins are involved in the SWI/SNF recruitment to chromatin. When performing immunoprecipitation followed by mass spectrometry of BRM-GFP, Li's team also identified three bromodomain-containing proteins—BRD1, BRD2, and BRD13—that interact with BRM and other core subunits of the Arabidopsis SWI/SNF complexes (Yu et al., 2021Yu Y. Fu W. Xu J. Lei Y. Song X. Liang Z. Zhu T. Liang Y. Hao Y. Yuan L. et al.Bromodomain-containing proteins BRD1, BRD2, and BRD13 are core subunits of SWI/SNF complexes and are vital for their genomic targeting in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.018Abstract Full Text Full Text PDF Scopus (2) Google Scholar) (Figure 1). The transcriptomic and phenotypic characterization of the triple brd1/2/13 mutant suggested that these BRDs act redundantly to control the expression of a common gene set with BRM, since the brd1/2/13 mutant phenocopied the brm-3 mutant. Furthermore, through chromatin immunoprecipitation sequencing analyses, the authors showed that the BRDs co-localize with BRM on H4K5ac- and H4K8ac-rich chromatin, suggesting that they are bona fide SWI/SNF subunits. Additionally, the disruption of the bromodomain in BRD2 was found to significantly reduce its genomic occupancy and the absence of BRDs to negatively influence BRM recruitment, indicating that BRD bromodomains are essential for the recruitment of SWI/SNF complexes to chromatin (Figure 1). Interestingly, and similar to brip1 brip2, the loss of function of brd1/2/13 leads to a dramatic decrease of BRM at the protein level, suggesting that BRDs contribute to maintaining the physiological abundance of BRM (Yu et al., 2021Yu Y. Fu W. Xu J. Lei Y. Song X. Liang Z. Zhu T. Liang Y. Hao Y. Yuan L. et al.Bromodomain-containing proteins BRD1, BRD2, and BRD13 are core subunits of SWI/SNF complexes and are vital for their genomic targeting in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.018Abstract Full Text Full Text PDF Scopus (2) Google Scholar). Jarończyk and collaborators also identified the Arabidopsis BRD1, BRD2, and BRD13 as SWI/SNF components (Jarończyk et al., 2021Jarończyk K. Sosnowska K. Zaborowski A. Pupel P. Bucholc M. Małecka E. Siwirykow N. Stachula P. Iwanicka-Nowicka R. Koblowska M. et al.Bromodomain-containing subunits BRD1, BRD2, and BRD13 are required for proper functioning of SWI/SNF complexes in Arabidopsis.Plant Commun. 2021; https://doi.org/10.1016/j.xplc.2021.100174Abstract Full Text Full Text PDF Scopus (2) Google Scholar). In addition to showing that BRDs interact with BRM and other SWI/SNF subunits, they provided evidence that SWI3C and SWP73B (BAF60) enable the simultaneous incorporation of two BRDs to the SWI/SNF complexes in vivo, contributing to the understanding of the assembly of these complexes in plants (Figure 1). Aside from the developmental characterization of brd1/2/13, they found that, as for other previously described SWI/SNF subunit mutants (Sarnowska et al., 2016Sarnowska E. Gratkowska D.M. Sacharowski S.P. Cwiek P. Tohge T. Fernie A.R. Siedlecki J.A. Koncz C. Sarnowski T.J. The role of SWI/SNF chromatin remodeling complexes in hormone crosstalk.Trends Plant Sci. 2016; 21: 594-608Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), brd1/2/13 displays increased abscisic acid and paclobutrazol (a gibberellin biosynthesis inhibitor) sensitivity. Moreover, the quadruple brm-3 brd1/2/13 mutant was shown to phenocopy the brm-1 null mutant. These findings provide strong genetic evidence that the studied BRDs are SWI/SNF components that act in the same complex as BRM for the regulation of previously known SWI/SNF-regulated pathways. Remarkably, the fact that the loss of three BRDs and the BRM bromodomain in brm-3 brd1/2/13 enhances the brm-3 phenotype suggests that the BRD subunits act in cooperation with the bromodomain of BRM, whereas the underlying molecular mechanisms remain unclear so far. Even though these studies represent important milestones in the characterization of plant SWI/SNFs, they also raise numerous fundamental questions about the understanding of the molecular function of these complexes in the green lineage. For instance, although the presence of bromodomain-containing proteins in SWI/SNFs suggests that they direct the complexes to acetylated nucleosomes (Figure 1), this remains to be experimentally validated, as they could also exert their main molecular function via other mechanisms, e.g., interacting with other proteins. On the other hand, the specific role of SWI/SNF complexes in transcription remains to be deciphered. Unlike SAGA or polycomb complexes, which mediate the deposition of H3K14ac and H3K27me3, respectively, SWI/SNFs are not specifically associated with either active or repressive transcriptional states. In fact, their effect on transcription appears to be context dependent, a phenomenon that could be associated with the combinational assembly of different subunits for the formation of SWI/SNFs with different molecular properties. For instance, previous co-immunoprecipitation of the histone deacetylase HD2C interactors identified various SWI/SNF subunits (BRM, SWI3A, SWI3B, SWP73B, and BSH) (Buszewicz et al., 2016Buszewicz D. Archacki R. Palusiński A. Kotliński M. Fogtman A. Iwanicka-Nowicka R. Sosnowska K. Kuciński J. Pupel P. Olędzki J. et al.HD2C histone deacetylase and a SWI/SNF chromatin remodelling complex interact and both are involved in mediating the heat stress response in Arabidopsis.Plant Cell Environ. 2016; 39: 2108-2122Crossref PubMed Scopus (63) Google Scholar); however, no BRD was detected in this complex, which may suggest the existence of BRD-free SWI/SNFs with affinity for genomic regions different from those of their BRD-containing counterparts. This hypothesis is supported by the fact that from the known four Arabidopsis SWI3 and two SWP73 SWI/SNF subunits, only SWI3B, SWI3C, and SWP73B physically interact with the described BRDs (Jarończyk et al., 2021Jarończyk K. Sosnowska K. Zaborowski A. Pupel P. Bucholc M. Małecka E. Siwirykow N. Stachula P. Iwanicka-Nowicka R. Koblowska M. et al.Bromodomain-containing subunits BRD1, BRD2, and BRD13 are required for proper functioning of SWI/SNF complexes in Arabidopsis.Plant Commun. 2021; https://doi.org/10.1016/j.xplc.2021.100174Abstract Full Text Full Text PDF Scopus (2) Google Scholar; Yu et al., 2021Yu Y. Fu W. Xu J. Lei Y. Song X. Liang Z. Zhu T. Liang Y. Hao Y. Yuan L. et al.Bromodomain-containing proteins BRD1, BRD2, and BRD13 are core subunits of SWI/SNF complexes and are vital for their genomic targeting in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.018Abstract Full Text Full Text PDF Scopus (2) Google Scholar) (Figure 1). This suggests that the incorporation of different SWI3 and SWP73 isoforms may lead to or inhibit the recruitment of a diverse number of BRDs into SWI/SNF complexes. SWI3B has also been recently reported to interact with the HDA6 histone deacetylase to co-repress a subset of transposable elements (Yang et al., 2020Yang J. Yuan L. Yen M. Zheng F. Ji R. Peng T. Gu D. Yang S. Cui Y. Chen P. et al.SWI3B and HDA6 interact and are required for transposon silencing in Arabidopsis.Plant J. 2020; 102: 809-822Crossref PubMed Scopus (18) Google Scholar), suggesting that the differential incorporation of SWI3 and SWP73 isoforms with different interacting partners leads to the formation of SWI/SNFs with dissimilar molecular roles. The further detailed identification of the SWI/SNF subunit interactome, combined with genetic, transcriptomic and epigenomic methods, will allow the identification of a divergent set of plant SWI/SNFs and their specific biological function in the future. No conflict of interest declared.

In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.

Nucleosomal histone tails are subject to a vast repertoire of covalent modifications that alter the physical accessibility of their associated DNA, impacting gene expression. Specifically, the acetylation of histones is associated with transcriptional activation, as it has been shown to reduce their affinity for DNA (Grant, 2001Grant P.A. A tale of histone modifications.Genome Biol. 2001; 2: 1-5Crossref Google Scholar). The dynamic changes on histone acetylation levels throughout development are the result of active acetylation and deacetylation processes, mediated by histone acetyltransferases (HATs) and deacetylases, respectively. The first identified eukaryotic HAT was the budding yeast GCN5, which is one of the catalytic subunits of two multi-proteic complexes, the SAGA and ADA complexes (Vlachonasios et al., 2021Vlachonasios K. Poulios S. Mougiou N. The histone acetyltransferase GCN5 and the associated coactivators ADA2: from evolution of the SAGA complex to the biological roles in plants.Plants. 2021; 10: 308Crossref Scopus (9) Google Scholar). The SAGA complex has been characterized in detail in yeast, in which it is organized into four modules: the HAT, the deubiquitinase (DUB), and the CORE modules (TAF and SPT modules) (Papai et al., 2020Papai G. Frechard A. Kolesnikova O. Crucifix C. Schultz P. Ben-Shem A. Structure of SAGA and mechanism of TBP deposition on gene promoters.Nature. 2020; 577: 711-716Crossref PubMed Scopus (52) Google Scholar). Several of the components of the SAGA complex have been found to be conserved among different eukaryotic lineages, including metazoans and plants; however, lineage-specific particularities have been also identified. For instance, in Arabidopsis thaliana, which has been used as a reference for the characterization of the plant SAGA complex, homologs of certain CORE and DUB subunits appear to be absent (Vlachonasios et al., 2021Vlachonasios K. Poulios S. Mougiou N. The histone acetyltransferase GCN5 and the associated coactivators ADA2: from evolution of the SAGA complex to the biological roles in plants.Plants. 2021; 10: 308Crossref Scopus (9) Google Scholar). Until recently, the existence of plant-specific SAGA subunits remained undetermined; however, through an affinity purification-mass spectrometry screening, using several conserved Arabidopsis SAGA proteins as baits, Wu et al., 2021Wu C.-J. Liu Z.-Z. Wei L. Zhou J.-X. Cai X.-W. Su Y.-N. Li L. Chen S. He X.-J. Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.014Abstract Full Text Full Text PDF Scopus (7) Google Scholar identified four previously uncharacterized proteins that co-purified with various SAGA components (Figure 1). Further efforts for the characterization of these newly identified proteins revealed that three of these are paralogs, from which one (SAGA complex subunit 1 [SCS1]) is distantly related to the other two (SCS2A, SCS2B). The high sequence similarity and the absence of physical interaction between these three proteins led the authors to hypothesize their redundant role in SAGA complexes. To prove this hypothesis, they studied the scs1, scs2a, and scs2b mutants and their respective crosses. Since the single mutants did not display any developmental phenotype, but the double and heterozygous triple mutants phenocopied the mutants of the core SAGA subunit hag1 and ada2b, they were able to conclude that these newly discovered proteins are SAGA components with a redundant function, functionally related to HAG1 and ADA2B. While HAG1/GCN5 is an acetyltransferase of H3K14 and H3K36 in Arabidopsis (Earley et al., 2007Earley K.W. Shook M.S. Brower-Toland B. Hicks L. Pikaard C.S. In vitro specificities of Arabidopsis co-activator histone acetyltransferases: implications for histone hyperacetylation in gene activation.Plant J. 2007; 52: 615-626Crossref PubMed Scopus (136) Google Scholar; Mahrez et al., 2016Mahrez W. Arellano M.S.T. Moreno-Romero J. Nakamura M. Shu H. Nanni P. Köhler C. Gruissem W. Hennig L. H3K36ac is an evolutionary conserved plant histone modification that marks active genes.Plant Physiol. 2016; 170: 1566-1577Crossref PubMed Scopus (50) Google Scholar), ADA2B has been shown to enhance HAG1 activity (Mao et al., 2006Mao Y. Pavangadkar K.A. Thomashow M.F. Triezenberg S.J. Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1.Biochim. Biophys. Acta. 2006; 1759: 69-79Crossref PubMed Scopus (85) Google Scholar). Mutations in HAG1 and ADA2B induce a series of pleiotropic developmental defects, ranging from dwarfism, loss of apical dominance, and aberrant flower development to reduced fertility (Vlachonasios et al., 2003Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression.Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (239) Google Scholar). The in-detail characterization of the diverse generated scs mutants indicated that, together with HAG1 and ADA2B, SCS1/2A/2B regulate juvenile-to-adult phase transition, trichome development and flowering time (Wu et al., 2021Wu C.-J. Liu Z.-Z. Wei L. Zhou J.-X. Cai X.-W. Su Y.-N. Li L. Chen S. He X.-J. Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.014Abstract Full Text Full Text PDF Scopus (7) Google Scholar). To explore the molecular mechanisms by which they regulate these processes, the authors used a combination of transcriptomic and epigenomic methods, which they applied to scs1/2a, since the homozygous triple scs1/2a/2b mutant was not viable. By comparing the transcriptome of scs1/2a with those of hag1 and ada2b, they demonstrated that SCSs co-regulate with HAG1 and ADA2B the expression of a significantly overlapping set of genes, including SPL3 and SOC1, known regulators of juvenile-to-adult phase transition and flowering time, respectively. On the other hand, the three mutants also displayed a downregulation in the expression of various well-known negative regulators of trichome development (including TCL1, TCL2, TRY, ETC1, ETC2, and ETC3) and the consequent upregulation of their trichome-promoting target, GL2 (Wu et al., 2021Wu C.-J. Liu Z.-Z. Wei L. Zhou J.-X. Cai X.-W. Su Y.-N. Li L. Chen S. He X.-J. Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.014Abstract Full Text Full Text PDF Scopus (7) Google Scholar). Overall, these results confirmed that the newly described SCS proteins are SAGA subunits and regulate common developmental processes with HAG1 and ADA2B, through the promotion of the expression of a common subset of genomic targets. On the other hand, since SAGA activity is associated with histone acetylation, the authors assessed the effect of the scs1/2a mutation on the levels and distribution of this histone modification (Wu et al., 2021Wu C.-J. Liu Z.-Z. Wei L. Zhou J.-X. Cai X.-W. Su Y.-N. Li L. Chen S. He X.-J. Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.014Abstract Full Text Full Text PDF Scopus (7) Google Scholar). Through western blot and chromatin immunoprecipitation sequencing assays, they demonstrated that, similar to hag1 and ada2b, the scs1/2a mutation significantly reduced H3K9ac and H3K14ac levels, which could be associated with the considerably larger sets of downregulated than upregulated genes in the three mutants. As expected, the analyzed mutants displayed reduction in histone acetylation on an overlapping gene set, including the development-related downregulated genes SPL3, SCO1, TCL1, and TCL2, indicating that the reduction on acetylation levels on these loci contributes to their downregulation in the studied mutants. Altogether, these results suggest that SCS1/2A/2B participate in the fine-tuning of gene expression in plants through the regulation of histone acetylation. In fact, HAG1 binding to its target loci seems to be reduced in the absence of scs1/2a, which could suggest that SCS1/2A/2B are necessary for the recruitment of this acetyltransferase for histone acetylation (Wu et al., 2021Wu C.-J. Liu Z.-Z. Wei L. Zhou J.-X. Cai X.-W. Su Y.-N. Li L. Chen S. He X.-J. Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.014Abstract Full Text Full Text PDF Scopus (7) Google Scholar). On the other hand, even when SAGA activity has been associated with H3K9ac, as various SAGA subunit mutants display a significant reduction on this histone mark (Kim et al., 2020Kim S. Piquerez S.J.M. Ramirez-Prado J.S. Mastorakis E. Veluchamy A. Latrasse D. Manza-Mianza D. Brik-Chaouche R. Huang Y. Rodriguez-Granados N.Y. et al.GCN5 modulates salicylic acid homeostasis by regulating H3K14ac levels at the 5′ and 3′ ends of its target genes.Nucleic Acids Res. 2020; 48: 5953-5966Crossref PubMed Google Scholar), the process by which this complex promotes this specific histone modification remains unknown, since HAG1 has been shown to specifically acetylate H3K14 and H3K36 in vitro (Earley et al., 2007Earley K.W. Shook M.S. Brower-Toland B. Hicks L. Pikaard C.S. In vitro specificities of Arabidopsis co-activator histone acetyltransferases: implications for histone hyperacetylation in gene activation.Plant J. 2007; 52: 615-626Crossref PubMed Scopus (136) Google Scholar; Mahrez et al., 2016Mahrez W. Arellano M.S.T. Moreno-Romero J. Nakamura M. Shu H. Nanni P. Köhler C. Gruissem W. Hennig L. H3K36ac is an evolutionary conserved plant histone modification that marks active genes.Plant Physiol. 2016; 170: 1566-1577Crossref PubMed Scopus (50) Google Scholar). Furthermore, the hag1 mutation has also been shown to induce an increase in the H3K14ac levels on the 3′ of some HAG1 targets, a phenomenon that has been associated with the upregulation of these loci in this mutant (Kim et al., 2020Kim S. Piquerez S.J.M. Ramirez-Prado J.S. Mastorakis E. Veluchamy A. Latrasse D. Manza-Mianza D. Brik-Chaouche R. Huang Y. Rodriguez-Granados N.Y. et al.GCN5 modulates salicylic acid homeostasis by regulating H3K14ac levels at the 5′ and 3′ ends of its target genes.Nucleic Acids Res. 2020; 48: 5953-5966Crossref PubMed Google Scholar). This result seems counterintuitive, taking into account that HAG1 is an H3K14ac HAT; however, it may indicate that GCN5 prevents the 3′ deposition of this histone mark by other HATs on its targets. Together, these phenomena highlight our current limited understanding on the molecular mechanisms governing histone acetylation homeostasis in plants. Using several baits for the identification of novel plant SAGA subunits, the authors were also able to re-construct a high-resolution protein interaction network. Based on this, they concluded that SCS1/2A/2B physically connect the HAT and the CORE SAGA modules (Figure 1), representing functional analogs of the yeast ADA3, the ortholog of which is absent in the Arabidopsis genome (Wu et al., 2021Wu C.-J. Liu Z.-Z. Wei L. Zhou J.-X. Cai X.-W. Su Y.-N. Li L. Chen S. He X.-J. Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.014Abstract Full Text Full Text PDF Scopus (7) Google Scholar). Furthermore, and opposite to the previous theories, they observed that proteins from the DUB module barely interact with subunits of the SAGA complex, which may indicate that the DUB module is not part of SAGA in Arabidopsis and functions independently of the latter (Figure 1). This result is in contrast to observations in yeast, where the DUB module is connected to SAGA via the SGF73 subunit (Papai et al., 2020Papai G. Frechard A. Kolesnikova O. Crucifix C. Schultz P. Ben-Shem A. Structure of SAGA and mechanism of TBP deposition on gene promoters.Nature. 2020; 577: 711-716Crossref PubMed Scopus (52) Google Scholar). The absence of SGF73 orthologs in Arabidopsis may contribute to the lack of interaction between SAGA and the DUB module in this organism. Interestingly, an ortholog of SGF73 has been identified in non-vascular land plants, as well as in gymnosperms and early angiosperms, while this gene appears to be absent in higher monocot and dicot species (Moraga and Aquea, 2015Moraga F. Aquea F. Composition of the SAGA complex in plants and its role in controlling gene expression in response to abiotic stresses.Front. Plant Sci. 2015; 6: 1-9Crossref PubMed Scopus (43) Google Scholar; Vlachonasios et al., 2021Vlachonasios K. Poulios S. Mougiou N. The histone acetyltransferase GCN5 and the associated coactivators ADA2: from evolution of the SAGA complex to the biological roles in plants.Plants. 2021; 10: 308Crossref Scopus (9) Google Scholar), which could indicate that this gene was lost during angiosperm evolution. However, the functional implications of the physical de-coupling of histone deubiquitination and acetylation in higher plants remains to be explored. Finally, the fourth plant-specific SAGA subunit identified by Wu et al., 2021Wu C.-J. Liu Z.-Z. Wei L. Zhou J.-X. Cai X.-W. Su Y.-N. Li L. Chen S. He X.-J. Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.014Abstract Full Text Full Text PDF Scopus (7) Google Scholar, a protein containing a histone-fold domain (named TAFL, for TAF-like protein) appears to be a subunit of the TAF module of SAGA, as it interacts with the subunits TAF10 and TAF12B (Figure 1). However, this protein remains to be functionally characterized for its regulatory function as a SAGA subunit. No conflict of interest declared.

In recent years, we have witnessed a significant increase in studies addressing the three-dimensional (3D) chromatin organization of the plant nucleus. Important advances in chromatin conformation capture (3C)-derived and related techniques have allowed the exploration of the nuclear topology of plants with large and complex genomes, including various crops. In addition, the increase in their resolution has permitted the depiction of chromatin compartmentalization and interactions at the gene scale. These studies have revealed the highly complex mechanisms governing plant nuclear architecture and the remarkable knowledge gaps in this field. Here we discuss the state-of-the-art in plant chromosome architecture, including our knowledge of the hierarchical organization of the genome in 3D space and regarding other nuclear components. Furthermore, we highlight the existence in plants of topologically associated domain (TAD)-like structures that display striking differences from their mammalian counterparts, proposing the concept of ICONS—intergenic condensed spacers. Similarly, we explore recent advances in the study of chromatin loops and R-loops, and their implication in the regulation of gene activity. Finally, we address the impact that polyploidization has had on the chromatin topology of modern crops, and how this is related to phenomena such as subgenome dominance and biased gene retention in these organisms.

The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5' and 3' ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5' end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3' ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5' and 3' ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions.

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development, and adaptation to environmental fluctuations of most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the transgenerational inheritance of histone marks, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases play a dual role in plants by helping to maintain transcriptional states through development and safeguard genome integrity during sexual reproduction.

BACKGROUND:Polyploidy is ubiquitous in eukaryotic plant and fungal lineages, and it leads to the co-existence of several copies of similar or related genomes in one nucleus. In plants, polyploidy is considered a major factor in successful domestication. However, polyploidy challenges chromosome folding architecture in the nucleus to establish functional structures.RESULTS:We examine the hexaploid wheat nuclear architecture by integrating RNA-seq, ChIP-seq, ATAC-seq, Hi-C, and Hi-ChIP data. Our results highlight the presence of three levels of large-scale spatial organization: the arrangement into genome territories, the diametrical separation between facultative and constitutive heterochromatin, and the organization of RNA polymerase II around transcription factories. We demonstrate the micro-compartmentalization of transcriptionally active genes determined by physical interactions between genes with specific euchromatic histone modifications. Both intra- and interchromosomal RNA polymerase-associated contacts involve multiple genes displaying similar expression levels.CONCLUSIONS:Our results provide new insights into the physical chromosome organization of a polyploid genome, as well as on the relationship between epigenetic marks and chromosome conformation to determine a 3D spatial organization of gene expression, a key factor governing gene transcription in polyploids.

Polycomb repressive complexes (PRCs) have been traditionally associated with the regulation of developmental processes in various organisms, including higher plants. However, similar to other epigenetic regulators, there is accumulating evidence for their role in the regulation of stress and immune-related pathways. In the current study we show that the PRC1 protein LHP1 is required for the repression of the MYC2 branch of jasmonic acid (JA)/ethylene (ET) pathway of immunity. Loss of LHP1 induces the reduction in H3K27me3 levels in the gene bodies of ANAC019 and ANAC055, as well as some of their targets, leading to their transcriptional upregulation. Consistently, increased expression of these two transcription factors leads to the misregulation of several of their genomic targets. The lhp1 mutant mimics the MYC2, ANAC019, and ANAC055 overexpressers in several of their phenotypes, including increased aphid resistance, abscisic acid (ABA) sensitivity and drought tolerance. In addition, like the MYC2 and ANAC overexpressers, lhp1 displays reduced salicylic acid (SA) content caused by a deregulation of ICS1 and BSMT1, as well as increased susceptibility to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. Together, our results indicate that LHP1 regulates the expression of stress-responsive genes as well as the homeostasis and responses to the stress hormones SA and ABA. This protein emerges as a key chromatin player fine tuning the complex balance between developmental and stress-responsive processes.

Pathogen recognition by plants results in the activation of signaling pathways that induce defense reactions. There is growing evidence indicating that epigenetic mechanisms directly participate in plant immune memory. Here, we discuss current knowledge of diverse epigenomic processes and elements, such as noncoding RNAs, DNA and RNA methylation, histone post-translational modifications, and chromatin remodeling, that have been associated with the regulation of immune responses in plants. Furthermore, we discuss the currently limited evidence of transgenerational inheritance of pathogen-induced defense priming, together with its potentials, challenges, and limitations for crop improvement and biotechnological applications.

Relying on an immune system comes with a high energetic cost for plants. Defense responses in these organisms are therefore highly regulated and fine-tuned, permitting them to respond pertinently to the attack of a microbial pathogen. In recent years, the importance of the physical modification of chromatin, a highly organized structure composed of genomic DNA and its interacting proteins, has become evident in the research field of plant-pathogen interactions. Several processes, including DNA methylation, changes in histone density and variants, and various histone modifications, have been described as regulators of various developmental and defense responses. Herein, we review the state of the art in the epigenomic aspects of plant immunity, focusing on chromatin modifications, chromatin modifiers, and their physiological consequences. In addition, we explore the exciting field of understanding how plant pathogens have adapted to manipulate the plant epigenomic regulation in order to weaken their immune system and thrive in their host, as well as how histone modifications in eukaryotic pathogens are involved in the regulation of their virulence.

The term non-coding RNA (ncRNA) refers to functional RNA molecules that, despite being transcribed from DNA, are not translated into proteins. These molecules can play an important role in the regulation of gene expression in the eukaryotic cell, and they can act either as long ncRNAs or being processed into small RNAs, being globally classified by their size, function, or genomic origin. In recent years, it has been found that diverse ncRNAs participate directly or indirectly in several epigenetic phenomena controlling different phenotypes within clonal cells, and in the specificity determination of various physiological processes. Although some of their mechanisms of action have been characterized, much remains to be known to understand the highly complex processes in which most of these molecules are involved. In this chapter, we discuss and illustrate examples of different ncRNAs that can interact with the plant epigenomic machinery or intervene in its function, leading to specific epigenetic, transcriptional, and physiological states. We explore the link between chromatin compaction, histone modifications, DNA methylation, gene silencing, and these molecules, which represent a high proportion of the cellular transcriptome.

BACKGROUND:Plant adaptive responses to changing environments involve complex molecular interplays between intrinsic and external signals. Whilst much is known on the signaling components mediating diurnal, light, and temperature controls on plant development, their influence on chromatin-based transcriptional controls remains poorly explored.RESULTS:In this study we show that a SWI/SNF chromatin remodeler subunit, BAF60, represses seedling growth by modulating DNA accessibility of hypocotyl cell size regulatory genes. BAF60 binds nucleosome-free regions of multiple G box-containing genes, opposing in cis the promoting effect of the photomorphogenic and thermomorphogenic regulator Phytochrome Interacting Factor 4 (PIF4) on hypocotyl elongation. Furthermore, BAF60 expression level is regulated in response to light and daily rhythms.CONCLUSIONS:These results unveil a short path between a chromatin remodeler and a signaling component to fine-tune plant morphogenesis in response to environmental conditions.

Micrococcus luteus strain K39 is an endophyte bacterium isolated from roots of the desert plant Cyperus conglomeratus collected from the Red Sea shore, Thuwal, Saudi Arabia. The draft genome sequence of strain K39 revealed a number of enzymes involved in salinity and oxidative stress tolerance or having herbicide-resistance activity.

Background: Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.Results: Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.Conclusions: By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

As the most recent evidence of eukaryotic cell complexity, genome architecture has astounded the scientific community and prompted a variety of technical and cognitive challenges. Several technologies have emerged and evidenced the integration of chromatin packaging and topology, epigenetic processes, and transcription for the pertinent regulation of gene expression. In the present addendum we present and discuss some of our recent research, directed toward the holistic comprehension of the processes by which plants respond to environmental and developmental stimuli. We propose that the study of genome topology and genomic interactions is essential for the understanding of the molecular mechanisms behind a phenotype. Even though our knowledge and understanding of genome architecture and hierarchy has improved substantially in the last few years -in Arabidopsis and other eukaryotes -, there is still a long way ahead in this relatively new field of study. For this, it is necessary to take advantage of the high resolution of the emerging available techniques, and perform integrative approaches with which it will be possible to depict the role of chromatin architecture in the regulation of transcription and ultimately, physiological processes.