R-Loop Mediated trans Action of the APOLO Long Noncoding RNA.

In eukaryotes, three-dimensional genome organization is critical for transcriptional regulation of gene expression. Long noncoding RNAs (lncRNAs) can modulate chromatin conformation of spatially related genomic locations within the nucleus. Here, we show that the lncRNA APOLO (AUXIN-REGULATED PROMOTER LOOP) recognizes multiple distant independent loci in the Arabidopsis thaliana genome. We found that APOLO targets are not spatially associated in the nucleus and that APOLO recognizes its targets by short sequence complementarity and the formation of DNA-RNA duplexes (R-loops). The invasion of APOLO to the target DNA decoys the plant Polycomb Repressive Complex 1 component LHP1, modulating local chromatin 3D conformation. APOLO lncRNA coordinates the expression of distal unrelated auxin-responsive genes during lateral root development in Arabidopsis. Hence, R-loop formation and chromatin protein decoy mediate trans action of lncRNAs on distant loci. VIDEO ABSTRACT.

Microbe-induced drought tolerance by ABA-mediated root architecture and epigenetic reprogramming.

The use of beneficial microbes to mitigate drought stress tolerance of plants is of great potential albeit little understood. We show here that a root endophytic desert bacterium, Pseudomonas argentinensis strain SA190, enhances drought stress tolerance in Arabidopsis. Transcriptome and genetic analysis demonstrate that SA190-induced root morphogenesis and gene expression is mediated via the plant abscisic acid (ABA) pathway. Moreover, we demonstrate that SA190 primes the promoters of target genes in an epigenetic ABA-dependent manner. Application of SA190 priming on crops is demonstrated for alfalfa, showing enhanced performance under drought conditions. In summary, a single beneficial root bacterial strain can help plants to resist drought conditions.

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

GCN5 modulates salicylic acid homeostasis by regulating H3K14ac levels at the 5' and 3' ends of its target genes.

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.

Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1.

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.

INDETERMINATE-DOMAIN 4 (IDD4) coordinates immune responses with plant-growth in Arabidopsis thaliana.

INDETERMINATE DOMAIN (IDD)/ BIRD proteins are a highly conserved plant-specific family of transcription factors which play multiple roles in plant development and physiology. Here, we show that mutation in IDD4/IMPERIAL EAGLE increases resistance to the hemi-biotrophic pathogen Pseudomonas syringae, indicating that IDD4 may act as a repressor of basal immune response and PAMP-triggered immunity. Furthermore, the idd4 mutant exhibits enhanced plant-growth indicating IDD4 as suppressor of growth and development. Transcriptome comparison of idd4 mutants and IDD4ox lines aligned to genome-wide IDD4 DNA-binding studies revealed major target genes related to defense and developmental-biological processes. IDD4 is a phospho-protein that interacts and becomes phosphorylated on two conserved sites by the MAP kinase MPK6. DNA-binding studies of IDD4 after flg22 treatment and with IDD4 phosphosite mutants show enhanced binding affinity to ID1 motif-containing promoters and its function as a transcriptional regulator. In contrast to the IDD4-phospho-dead mutant, the IDD4 phospho-mimicking mutant shows altered susceptibility to PstDC3000, salicylic acid levels and transcriptome reprogramming. In summary, we found that IDD4 regulates various hormonal pathways thereby coordinating growth and development with basal immunity.

Genome wide natural variation of H3K27me3 selectively marks genes predicted to be important for cell differentiation in Phaeodactylum tricornutum.

In multicellular organisms, Polycomb Repressive Complex2 (PRC2) is known to deposit tri-methylation of lysine 27 of histone H3 (H3K27me3) to establish and maintain gene silencing, critical for developmentally regulated processes. The PRC2 complex is absent in both widely studied model yeasts, which initially suggested that PRC2 arose with the emergence of multicellularity. However, its discovery in several unicellular species including microalgae questions its role in unicellular eukaryotes. Here, we use Phaeodactylum tricornutum enhancer of zeste E(z) knockouts and show that P. tricornutum E(z) is responsible for di- and tri-methylation of lysine 27 of histone H3. H3K27me3 depletion abolishes cell morphology in P. tricornutum providing evidence for its role in cell differentiation. Genome-wide profiling of H3K27me3 in fusiform and triradiate cells further revealed genes that may specify cell identity. These results suggest a role for PRC2 and its associated mark in cell differentiation in unicellular species, and highlight their ancestral function in a broader evolutionary context than currently is appreciated.

Arabidopsis ATRX Modulates H3.3 Occupancy and Fine-Tunes Gene Expression.

Histones are essential components of the nucleosome, the major chromatin subunit that structures linear DNA molecules and regulates access of other proteins to DNA. Specific histone chaperone complexes control the correct deposition of canonical histones and their variants to modulate nucleosome structure and stability. In this study, we characterize the Arabidopsis thaliana Alpha Thalassemia-mental Retardation X-linked (ATRX) ortholog and show that ATRX is involved in histone H3 deposition. Arabidopsis ATRX mutant alleles are viable, but show developmental defects and reduced fertility. Their combination with mutants of the histone H3.3 chaperone HIRA (Histone Regulator A) results in impaired plant survival, suggesting that HIRA and ATRX function in complementary histone deposition pathways. Indeed, ATRX loss of function alters cellular histone H3.3 pools and in consequence modulates the H3.1/H3.3 balance in the cell. H3.3 levels are affected especially at genes characterized by elevated H3.3 occupancy, including the 45S ribosomal DNA (45S rDNA) loci, where loss of ATRX results in altered expression of specific 45S rDNA sequence variants. At the genome-wide scale, our data indicate that ATRX modifies gene expression concomitantly to H3.3 deposition at a set of genes characterized both by elevated H3.3 occupancy and high expression. Together, our results show that ATRX is involved in H3.3 deposition and emphasize the role of histone chaperones in adjusting genome expression.

Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression.

The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.

Insights into global diatom distribution and diversity in the world's ocean.

Diatoms (Bacillariophyta) constitute one of the most diverse and ecologically important groups of phytoplankton. They are considered to be particularly important in nutrient-rich coastal ecosystems and at high latitudes, but considerably less so in the oligotrophic open ocean. The Tara Oceans circumnavigation collected samples from a wide range of oceanic regions using a standardized sampling procedure. Here, a total of ∼12 million diatom V9-18S ribosomal DNA (rDNA) ribotypes, derived from 293 size-fractionated plankton communities collected at 46 sampling sites across the global ocean euphotic zone, have been analyzed to explore diatom global diversity and community composition. We provide a new estimate of diversity of marine planktonic diatoms at 4,748 operational taxonomic units (OTUs). Based on the total assigned ribotypes, Chaetoceros was the most abundant and diverse genus, followed by Fragilariopsis, Thalassiosira, and Corethron We found only a few cosmopolitan ribotypes displaying an even distribution across stations and high abundance, many of which could not be assigned with confidence to any known genus. Three distinct communities from South Pacific, Mediterranean, and Southern Ocean waters were identified that share a substantial percentage of ribotypes within them. Sudden drops in diversity were observed at Cape Agulhas, which separates the Indian and Atlantic Oceans, and across the Drake Passage between the Atlantic and Southern Oceans, indicating the importance of these ocean circulation choke points in constraining diatom distribution and diversity. We also observed high diatom diversity in the open ocean, suggesting that diatoms may be more relevant in these oceanic systems than generally considered.

Thermopriming triggers splicing memory in Arabidopsis.

Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat-shock memory and the role of priming in Arabidopsis thaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat-shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link 'splicing memory' to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat-stress responses in plants and other organisms as many of the key components are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (ß-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

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

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

Microbe-induced coordination of plant iron-sulfur metabolism enhances high light stress tolerance of Arabidopsis.

High light stress in subtropical and tropical regions strongly limits agricultural production due to photo-oxidative damage, decreased growth and yield. Here, we investigated whether beneficial microbes can protect plants under high light stress. We found that Enterobacter sp. SA187 (SA187) supports Arabidopsis thaliana growth under high light stress by reducing the accumulation of reactive oxygen species (ROS) and maintaining photosynthesis. When subjected to high light stress, SA187 triggers dynamic changes in Arabidopsis gene expression related to fortified iron metabolism and redox regulation thereby enhancing the plant anti-oxidative glutathione/glutaredoxin redox system. Genetic analysis shows that SA187-enhanced iron and sulfur metabolism are coordinated by ethylene signaling. In summary, beneficial microbes could be an effective and inexpensive means for enhancing high light stress tolerance in plants.

CRISPR-broad: combined design of multi-targeting gRNAs and broad, multiplex target finding.

In CRISPR-Cas and related nuclease-mediated genome editing, target recognition is based on guide RNAs (gRNAs) that are complementary to selected DNA regions. While single site targeting is fundamental for localized genome editing, targeting to expanded and multiple chromosome elements is desirable for various biological applications such as genome mapping and epigenome editing that make use of different fusion proteins with enzymatically dead Cas9. The current gRNA design tools are not suitable for this task, as these are optimized for defining single gRNAs for unique loci. Here, we introduce CRISPR-broad, a standalone, open-source application that defines gRNAs with multiple but specific targets in large continuous or spread regions of the genome, as defined by the user. This ability to identify multi-targeting gRNAs and corresponding multiple targetable regions in genomes is based on a novel aggregate gRNA scoring derived from on-target windows and off-target sites. Applying the new tool to the genomes of two model species, C. elegans and H. sapiens, we verified its efficiency in determining multi-targeting gRNAs and ranking potential target regions optimized for broad targeting. Further, we demonstrated the general usability of CRISPR-broad by cellular mapping of a large human genome element using dCas9 fused to green fluorescent protein.

PhaeoEpiView: an epigenome browser of the newly assembled genome of the model diatom Phaeodactylum tricornutum.

Recent advances in DNA sequencing technologies particularly long-read sequencing, greatly improved genomes assembly. However, this has created discrepancies between published annotations and epigenome tracks, which have not been updated to keep pace with the new assemblies. Here, we used the latest improved telomere-to-telomere assembly of the model pennate diatom Phaeodactylum tricornutum to lift over the gene models from Phatr3, a previously annotated reference genome. We used the lifted genes annotation and newly published transposable elements to map the epigenome landscape, namely DNA methylation and post-translational modifications of histones. This provides the community with PhaeoEpiView, a browser that allows the visualization of epigenome data and transcripts on an updated and contiguous reference genome, to better understand the biological significance of the mapped data. We updated previously published histone marks with a more accurate peak calling using mono instead of poly(clonal) antibodies and deeper sequencing. PhaeoEpiView ( https://PhaeoEpiView.univ-nantes.fr ) will be continuously updated with the newly published epigenomic data, making it the largest and richest epigenome browser of any stramenopile. In the upcoming era of molecular environmental studies, where epigenetics plays a significant role, we anticipate that PhaeoEpiView will become a widely used tool.

The Promising Epigenetic Regulators for Refractory Epilepsy: An Adventurous Road Ahead.

The attribution of seizure freedom is yet to be achieved for patients suffering from refractory epilepsy, e.g. Dravet Syndrome (DS). The confined ability of mono-chemical entity-based antiseizure drugs (ASDs) to act directly at genomic level is one of the factors, combined with undetermined seizure triggers lead to recurrent seizure (RS) in DS, abominably affecting the sub-genomic architecture of neural cells. Thus, the RS and ASD appear to be responsible for the spectrum of exorbitant clinical pathology. The RS distresses the 5-HT-serotonin pathway, hypomethylates genes of CNS, and modulates the microRNA (miRNA)/long non-coding RNA (lncRNA), eventually leading to frozen molecular alterations. These changes shall be reverted by compatible epigenetic regulators (EGR) like, miRNA and lncRNA from Breast milk (BML) and Bacopa monnieri (BMI). The absence of studious seizure in SCN1A mutation-positive babies for the first 6 months raises the possibility that the consequences of mutation in SCN1A are subsidized by EGRs from BML. EGR-dependent-modifier gene effect is likely imposed by the other members of the SCN family. Therefore, we advocate that miRNA/lncRNA from BML and bacosides/miRNA from BMI buffer the effect of SCN1A mutation by sustainably maintaining modifier gene effect in the aberrant neurons. The presence of miRNA-155-5p, -30b-5p, and -30c-5p family in BML and miR857, miR168, miR156, and miR158 in BMI target at regulating SCN family and CLCN5 as visualized by Cystoscope. Thus, we envisage that the possible effects of EGR might include (a) upregulating the haploinsufficient SCN1A strand, (b) down-regulating seizure-elevated miRNA, (c) suppressing the seizure-induced methyltransferases, and (d) enhancing the GluN2A subunit of NMDA receptor to improve cognition. The potential of these EGRs from BML and BML is to further experimentally strengthen, long-haul step forward in molecular therapeutics.

Exploring the biological behavior of Heat shock protein (HSPs) for understanding the Anti-ischemic stroke in humans.

BACKGROUND: An ischemic stroke can be caused by thrombosis and ischemia, which is a major public health problem around the world, resulting in severe disability and a high death rate. The goal of this work is to examine and target various heat shock proteins (HSPs) via their interacting partners, which may have an anti-ischemic stroke impact. METHODS: Various heat shock proteins are identified and used for construction of PPI network through STRING webserver. Networks are analysed and visualized using the cytoscape for checking the protein-protein interactions. Along with this, multiple cytoscape based modules are integrated for the analysis of results, and Gene Ontology results are analysed using GOView. RESULTS: The core PPI network was revealed with 129 nodes and 1174 edges. Through Gene ontology (GO) and KEGG enrichment analysis the promising function of HSPs in two important signaling pathways were mainly recorded, representing the HSPs are necessary for repair and activations of brain cells during ischemic stroke. In addition, the study is revelation for targeting multiple HSPs via their interacting partners, which can provide anti-ischemic stroke effect. CONCLUSION: Overall, this finding provides a network-based framework for future research on HSP as therapeutic molecules for anti-ischemic stroke related applications.

Evaluating the structural and immune mechanism of Interleukin-6 for the investigation of goat milk peptides as potential treatments for COVID-19.

The function of Immune control, haematopoiesis, and inflammation all depend on the cytokine Interleukin 6 (IL-6), and higher expression of IL-6 is seen in COVID-19 and other diseases. The immune protein IL-6 activation is dependent on binding interactions with IL-6Rα, mIL-6R, and sIL-6R for its cellular function. Termination of these reaction could benefit for controlling the over-expression in COVID-19 patients and that may arise as inhibitors for controlling COVID-19. Traditionally, the goat milk has been prescribed as medicine in ayurvedic practice and through this work, we have explored the benefits of peptides from goat milk as IL-6 inhibitors, and it have the potential of inhibiting the over expression of IL-6 and control the COVID-19 disease. Computational experiments have shown that goat peptides had strong interactions with IL-6, with higher scoring profiles and energy efficiency ranging from -6.00 kcal/mol to -9.00 kcal/mol in docking score and -39.00 kcal/mol in binding energy. Especially the YLGYLEQLLR, VLVLDTDYK and AMKPWIQPK peptides from goat milk holds better scoring and shows strong interactions were identified as the most potential IL-6 inhibitor candidates in this study. Peptides from Goat proteins, which are capable of binding to the IL-6 receptor with strong binding conformations, have no negative effects on other immune system proteins.

Information theoretic perspective on genome clustering.

Shannon's information theoretic perspective of communication helps one to understand the storage and processing of information in one-dimensional sequences. An information theoretic analysis of 937 available completely sequenced prokaryotic genomes and 238 eukaryotic chromosomes is presented. Information content (Id) values were used to cluster these chromosomes. Chargaff's second parity rule i.e compositional self-complementarity, an empirical fact is observed in all the genomes, except for the proteobacteria Candidatus Hodgkinia cicadicola. High information content, arising out of biased base composition in all the 14 chromosomes of Plasmodium falciparum is found among two other genomes of prokaryotes viz. Buchnera aphidicola str. Cc (Cinara cedri) and Candidatus Carsonella ruddii PV. Despite size and compositional variations, both prokaryotic and eukaryotic genomes do not deviate significantly from an equiprobable and random situation. Eukaryotic chromosomes of an organism tend to have similar informational restraints as seen when a simple distance based method is used to cluster them. In eukaryotes, in certain cases, Id values are also similar for the two arms (p and q arm) of the chromosomes. The results of this current study confirm that the information content can provide insights into the clustering of genomes and the evolution of messaging strategies of the genomes. An efficient and robust Perl CGI standalone tool is created based on this information theory algorithm for the analysis of the whole genomes and is made available at https://github.com/AlagurajVeluchamy/InformationTheory.