MediatorWeb: a protein-protein interaction network database for the RNA polymerase II Mediator complex.

The protein-protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue-level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.

Serine hydroxymethyltransferase6 is involved in growth and resistance against pathogens via ethylene and lignin production in Arabidopsis.

Photorespiratory serine hydroxymethyltransferases (SHMTs) are important enzymes of cellular one-carbon metabolism. In this study, we investigated the potential role of SHMT6 in Arabidopsis thaliana. We found that SHMT6 is localized in the nucleus and expressed in different tissues during development. Interestingly SHMT6 is inducible in response to avirulent, virulent Pseudomonas syringae and to Fusarium oxysporum infection. Overexpression of SHMT6 leads to larger flowers, siliques, seeds, roots, and consequently an enhanced overall biomass. This enhanced growth was accompanied by increased stomatal conductance and photosynthetic capacity as well as ATP, protein, and chlorophyll levels. By contrast, a shmt6 knockout mutant displayed reduced growth. When challenged with Pseudomonas syringae pv tomato (Pst) DC3000 expressing AvrRpm1, SHMT6 overexpression lines displayed a clear hypersensitive response which was characterized by enhanced electrolyte leakage and reduced bacterial growth. In response to virulent Pst DC3000, the shmt6 mutant developed severe disease symptoms and becomes very susceptible, whereas SHMT6 overexpression lines showed enhanced resistance with increased expression of defense pathway associated genes. In response to Fusarium oxysporum, overexpression lines showed a reduction in symptoms. Moreover, SHMT6 overexpression lead to enhanced production of ethylene and lignin, which are important components of the defense response. Collectively, our data revealed that SHMT6 plays an important role in development and defense against pathogens.

Mediator complex: an important regulator of root system architecture.

Mediator, a multiprotein complex, is an important component of the transcription machinery. In plants, the latest reports from our group and some other studies have established that Mediator functions as a signal processor that conveys transcriptional signals from transcription factors to RNA polymerase II. It has been found to be involved in different developmental and stress-adaptation conditions ranging from embryo, root, and shoot development to flowering and senescence and also in response to different biotic and abiotic stresses. In the last one decade, significant progress has been made in understanding the role of Mediator subunits in root development. They have been shown to transcriptionally regulate development of almost all the components of root system architecture - primary root, lateral root and root hair. Their role has also been appreciated in nutrient acquisition through root. In this review, we have discussed all the known functions of Mediator subunits during root development. We have also highlighted the role of Mediator as a nodal point for processing different hormone signaling that regulate root morphogenesis and growth.

Co-overexpression of SWEET sucrose transporters modulates sucrose synthesis and defence responses to enhance immunity against bacterial blight in rice.

Enhancing carbohydrate export from source to sink tissues is considered to be a realistic approach for improving photosynthetic efficiency and crop yield. The rice sucrose transporters OsSUT1, OsSWEET11a and OsSWEET14 contribute to sucrose phloem loading and seed filling. Crucially, Xanthomonas oryzae pv. oryzae (Xoo) infection in rice enhances the expression of OsSWEET11a and OsSWEET14 genes, and causes leaf blight. Here we show that co-overexpression of OsSUT1, OsSWEET11a and OsSWEET14 in rice reduced sucrose synthesis and transport leading to lower growth and yield but reduced susceptibility to Xoo relative to controls. The immunity-related hypersensitive response (HR) was enhanced in the transformed lines as indicated by the increased expression of defence genes, higher salicylic acid content and presence of HR lesions on the leaves. The results suggest that the increased expression of OsSWEET11a and OsSWEET14 in rice is perceived as a pathogen (Xoo) attack that triggers HR and results in constitutive activation of plant defences that are related to the signalling pathways of pathogen starvation. These findings provide a mechanistic basis for the trade-off between plant growth and immunity because decreased susceptibility against Xoo compromised plant growth and yield.

DNA satellite and chromatin organization at mouse centromeres and pericentromeres.

BACKGROUND: Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of assembling repetitive genomic regions. RESULTS: Using recently available PacBio long-read sequencing data from the C57BL/6 strain, we find that contrary to the previous reports of their homogeneous nature, both centromeric minor satellites and pericentromeric major satellites exhibit a high degree of variation in sequence and organization within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Using chromatin immunoprecipitation sequencing (ChIP-seq), we find that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence enrichment and homogeneity at these regions. The transposable elements at centromeric regions are not part of functional centromeres as they lack significant CENP-A enrichment. Furthermore, both CENP-A and H3K9me3 nucleosomes occupy minor and major satellites spanning centromeric-pericentric junctions and a low yet significant amount of CENP-A spreads locally at centromere junctions on both pericentric and telocentric sides. Finally, while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays are poorly phased. Interestingly, the homogeneous class of major satellites also phase CENP-A and H3K27me3 nucleosomes, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. CONCLUSIONS: Our findings reveal that mouse centromeres and pericentromeres display a high diversity in satellite sequence, organization, and chromatin structure.

N-acetylglucosamine: a behavioral fate switch in Candida albicans.

Candida albicans, a significant commensal fungus in the human gut, causes a wide spectrum of opportunistic infections. In a recent study, Yang et al. revealed the importance of a host-associated gut signal, GlcNAc, in C. albicans and described its significant role towards achieving a successful commensal-virulence trade-off program in the human body.

Identification of novel inhibitors against Med15a KIX domain of Candida glabrata.

Candida glabrata, the second most common cause of invasive fungal infections, exhibits multi-drug resistance to commonly used antifungal drugs. To counter this resistance, there is a critical need for novel antifungals. This study identifies small molecule inhibitors that target a three-helix bundle KIX domain in the Med15a Mediator subunit of Candida glabrata (CgMed15a KIX). This domain plays a crucial role by interacting with the Pleiotropic Drug Resistance transcription factor Pdr1, a key regulator of the multidrug resistance pathway in Candida glabrata. We performed high throughput computational screening of large chemical datasets against the binding sites of the CgMed15a KIX domain to identify novel inhibitors. We selected six potential candidates with high affinity and confirmed their binding with the CgMed15a KIX domain. A phytochemical compound, Chebulinic acid binds to the CgMed15a KIX domain with a KD value of 0.339 µM and shows significant inhibitory effects on the growth of Candida glabrata. Molecular dynamics simulation studies further revealed the structural stability of the CgMed15a KIX-Chebulinic acid complex. Thus, in conclusion, this study highlights Chebulinic acid as a novel potential antifungal compound against Candida glabrata.

DNA satellite and chromatin organization at house mouse centromeres and pericentromeres.

Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of sequencing and assembling repetitive genomic regions. Using recently available PacBio long-read sequencing data from the C57BL/6 strain and chromatin profiling, we found that contrary to the previous reports of their highly homogeneous nature, centromeric and pericentromeric satellites display varied sequences and organization. We find that both centromeric minor satellites and pericentromeric major satellites exhibited sequence variations within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Additionally, we investigated CENP-A and H3K9me3 chromatin organization at centromeres and pericentromeres using Chromatin immunoprecipitation sequencing (ChIP-seq). We found that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence abundance and homogeneity at these regions. Furthermore, the transposable elements at centromeric regions are not part of functional centromeres as they lack CENP-A enrichment. Finally, we found that while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays lack phased organization. Interestingly, the homogeneous class of major satellites phase CENP-A and H3K27me3 nucleosomes as well, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. Overall, our findings reveal that house mouse centromeres and pericentromeres, which were previously thought to be highly homogenous, display significant diversity in satellite sequence, organization, and chromatin structure.

CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation.

Background: The number and escape levels of genes that escape X chromosome inactivation (XCI) in female somatic cells vary among tissues and cell types, potentially contributing to specific sex differences. Here we investigate the role of CTCF, a master chromatin conformation regulator, in regulating escape from XCI. CTCF binding profiles and epigenetic features were systematically examined at constitutive and facultative escape genes using mouse allelic systems to distinguish the inactive X (Xi) and active X (Xa) chromosomes. Results: We found that escape genes are located inside domains flanked by convergent arrays of CTCF binding sites, consistent with the formation of loops. In addition, strong and divergent CTCF binding sites often located at the boundaries between escape genes and adjacent neighbors subject to XCI would help insulate domains. Facultative escapees show clear differences in CTCF binding dependent on their XCI status in specific cell types/tissues. Concordantly, deletion but not inversion of a CTCF binding site at the boundary between the facultative escape gene Car5b and its silent neighbor Siah1b resulted in loss of Car5b escape. Reduced CTCF binding and enrichment of a repressive mark over Car5b in cells with a boundary deletion indicated loss of looping and insulation. In mutant lines in which either the Xi-specific compact structure or its H3K27me3 enrichment was disrupted, escape genes showed an increase in gene expression and associated active marks, supporting the roles of the 3D Xi structure and heterochromatic marks in constraining levels of escape. Conclusion: Our findings indicate that escape from XCI is modulated both by looping and insulation of chromatin via convergent arrays of CTCF binding sites and by compaction and epigenetic features of the surrounding heterochromatin.

MEDIATOR SUBUNIT17 is required for transcriptional optimization of root system architecture in Arabidopsis.

Sucrose and auxin are well-known determinants of root system architecture (RSA). However, the factors that connect the signaling pathways evoked by these two critical factors during root development are poorly understood. In this study, we report the role of MEDIATOR SUBUNIT17 (MED17) in RSA and its involvement in the transcriptional integration of sugar and auxin signaling pathways in Arabidopsis (Arabidopsis thaliana). Sucrose regulates root meristem activation through the TARGET OF RAPAMYCIN-E2 PROMOTER BINDING FACTOR A (TOR-E2FA) pathway, and auxin regulates lateral root (LR) development through AUXIN RESPONSE FACTOR-LATERAL ORGAN BOUNDARIES DOMAIN (ARF-LBDs). Both sucrose and auxin play a vital role during primary and LR development. However, there is no clarity on how sucrose is involved in the ARF-dependent regulation of auxin-responsive genes. This study establishes MED17 as a nodal point to connect sucrose and auxin signaling. Transcription of MED17 was induced by sucrose in an E2FA/B-dependent manner. Moreover, E2FA/B interacted with MED17, which can aid in the recruitment of the Mediator complex on the target promoters. Interestingly, E2FA/B and MED17 also occupied the promoter of ARF7, but not ARF19, leading to ARF7 expression, which then activates auxin signaling and thus initiates LR development. MED17 also activated cell division in the root meristem by occupying the promoters of cell-cycle genes, thus regulating their transcription. Thus, MED17 plays an important role in relaying the transcriptional signal from sucrose to auxin-responsive and cell-cycle genes to regulate primary and lateral root development, highlighting the role of the Mediator as the transcriptional processor for optimal root system architecture in Arabidopsis.

Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries.

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF and MYC TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer-promoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

Transcriptome analysis and differential expression in Arabidopsis thaliana in response to rohitukine (a chromone alkaloid) treatment.

Rohitukine is a chromone alkaloid and precursor of potent anticancer drugs flavopiridol, P-276-00, and 2,6-dichloro-styryl derivative (11d) (IIIM-290). The metabolite is reported to possess anticancer, anti-inflammatory, antiadipogenic, immunomodulatory, gastroprotective, anti-implantation, antidyslipidemic, anti-arthritic, and anti-fertility properties. However, the physiological role of rohitukine in plant system is yet to be explored. Here, we studied the effect of rohitukine isolated from Dysoxylum gotadhora on Arabidopsis thaliana. The A. thaliana plants grown on a medium fortified with different rohitukine concentrations showed a significant effect on the growth and development. The root growth of A. thaliana seedlings showed considerable inhibition when grown on medium containing 1.0 mM of rohitukine. Transcriptomic analysis indicated the expression of 895 and 932 genes in control and treated samples respectively at a cut-off of FPKM ≥ 1 and P-value < 0.05. Gene ontology (GO) analysis revealed the upregulation of genes related to photosynthesis, membrane transport, antioxidation, xenobiotic degradation, and some transcription factors (TFs) in response to rohitukine. Conversely, rohitukine downregulated several genes including RNA helicases and those involved in nitrogen compound metabolism. The RNA-seq result was also validated by real-time qRT-PCR analysis. In light of these results, we discuss (i) likely ecological importance of rohitukine in parent plant as well as (ii) comparison between responses to rohitukine treatment in plants and mammals.

Physiological implications of SWEETs in plants and their potential applications in improving source-sink relationships for enhanced yield.

The sugars will eventually be exported transporters (SWEET) family of transporters in plants is identified as a novel class of sugar carriers capable of transporting sugars, sugar alcohols and hormones. Functioning in intercellular sugar transport, SWEETs influence a wide range of physiologically important processes. SWEETs regulate the development of sink organs by providing nutritional support from source leaves, responses to abiotic stresses by maintaining intracellular sugar concentrations, and host-pathogen interactions through the modulation of apoplastic sugar levels. Many bacterial and fungal pathogens activate the expression of SWEET genes in species such as rice and Arabidopsis to gain access to the nutrients that support virulence. The genetic manipulation of SWEETs has led to the generation of bacterial blight (BB)-resistant rice varieties. Similarly, while the overexpression of the SWEETs involved in sucrose export from leaves and pathogenesis led to growth retardation and yield penalties, plants overexpressing SWEETs show improved disease resistance. Such findings demonstrate the complex functions of SWEETs in growth and stress tolerance. Here, we review the importance of SWEETs in plant-pathogen and source-sink interactions and abiotic stress resistance. We highlight the possible applications of SWEETs in crop improvement programmes aimed at improving sink and source strengths important for enhancing the sustainability of yield. We discuss how the adverse effects of the overexpression of SWEETs on plant growth may be overcome.

Prediction of novel and potent inhibitors of lanosterol 14-α demethylase.

Lanosterol 14-α demethylase (LDM) is one of the promising drug targets of azoles antifungal. In this study, we have screened a large number of small molecules from different chemical databases (ZINC, DrugBank, ChEMBL, and ChemDiv) to find out novel and potential inhibitors of LDM. As a result, from more than a hundred thousand molecules, the two best candidates, C1 (ZINC000299817826) and C3 (ZINC000095786149), were selected from the top-scoring compounds and further validated in Molecular Dynamic (MD) simulation. The Glide scores of C1 and C3 were -19.33 kcal/mol and -19.13 kcal/mol, suggesting that these compounds bind with LDM with higher binding affinity than the benchmark compound (itraconazole), which has a Glide score of -6.85 kcal/mol. Docking poses reveal that the compounds C1 and C3 bind to the outermost region of the LDM binding site, which can prevent the lanosterol from getting into the catalytic pocket. Furthermore, MD simulation studies were performed to assess the stability of C1 and C3 in complex with LDM and were found to be stable over the 100 nanosecond simulation time. Binding free energy calculated by the MMPBSA method suggested that the C3 forms a more stable complex with the LDM as close to the benchmark compounds. Among the top selected molecules, C1 and C3 were predicted to be the significant inhibitors of LDM.Communicated by Ramaswamy H. Sarma.

Root hair-specific transcriptome reveals response to low phosphorus in Cicer arietinum.

Root hairs (RH) are a single-cell extension of root epidermal cells. In low phosphorus (LP) availability, RH length and density increase thus expanding the total root surface area for phosphate (Pi) acquisition. However, details on genes involved in RH development and response to LP are missing in an agronomically important leguminous crop, chickpea. To elucidate this response in chickpea, we performed tissue-specific RNA-sequencing and analyzed the transcriptome modulation for RH and root without RH (Root-RH) under LP. Root hair initiation and cellular differentiation genes like RSL TFs and ROPGEFs are upregulated in Root-RH, explaining denser, and ectopic RH in LP. In RH, genes involved in tip growth processes and phytohormonal biosynthesis like cell wall synthesis and loosening (cellulose synthase A catalytic subunit, CaEXPA2, CaGRP2, and CaXTH2), cytoskeleton/vesicle transport, and ethylene biosynthesis are upregulated. Besides RH development, genes involved in LP responses like lipid and/or pectin P remobilization and acid phosphatases are induced in these tissues summarizing a complete molecular response to LP. Further, RH displayed preferential enrichment of processes involved in symbiotic interactions, which provide an additional benefit during LP. In conclusion, RH shows a multi-faceted response that starts with molecular changes for epidermal cell differentiation and RH initiation in Root-RH and later induction of tip growth and various LP responses in elongated RH.

Role of C4 photosynthetic enzyme isoforms in C3 plants and their potential applications in improving agronomic traits in crops.

As compared to C3, C4 plants have higher photosynthetic rates and better tolerance to high temperature and drought. These traits are highly beneficial in the current scenario of global warming. Interestingly, all the genes of the C4 photosynthetic pathway are present in C3 plants, although they are involved in diverse non-photosynthetic functions. Non-photosynthetic isoforms of carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), the decarboxylating enzymes NAD/NADP-malic enzyme (NAD/NADP-ME), and phosphoenolpyruvate carboxykinase (PEPCK), and finally pyruvate orthophosphate dikinase (PPDK) catalyze reactions that are essential for major plant metabolism pathways, such as the tricarboxylic acid (TCA) cycle, maintenance of cellular pH, uptake of nutrients and their assimilation. Consistent with this view differential expression pattern of these non-photosynthetic C3 isoforms has been observed in different tissues across the plant developmental stages, such as germination, grain filling, and leaf senescence. Also abundance of these C3 isoforms is increased considerably in response to environmental fluctuations particularly during abiotic stress. Here we review the vital roles played by C3 isoforms of C4 enzymes and the probable mechanisms by which they help plants in acclimation to adverse growth conditions. Further, their potential applications to increase the agronomic trait value of C3 crops is discussed.

MEDIATOR SUBUNIT17 integrates jasmonate and auxin signaling pathways to regulate thermomorphogenesis.

Plant adjustment to environmental changes involves complex crosstalk between extrinsic and intrinsic cues. In the past two decades, extensive research has elucidated the key roles of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and the phytohormone auxin in thermomorphogenesis. In this study, we identified a previously unexplored role of jasmonate (JA) signaling components, the Mediator complex, and their integration with auxin signaling during thermomorphogenesis in Arabidopsis (Arabidopsis thaliana). Warm temperature induces expression of JA signaling genes including MYC2, but, surprisingly, this transcriptional activation is not JA dependent. Warm temperature also promotes accumulation of the JA signaling receptor CORONATINE INSENSITIVE1 (COI1) and degradation of the JA signaling repressor JASMONATE-ZIM-DOMAIN PROTEIN9, which probably leads to de-repression of MYC2, enabling it to contribute to the expression of MEDIATOR SUBUNIT17 (MED17). In response to warm temperature, MED17 occupies the promoters of thermosensory genes including PIF4, YUCCA8 (YUC8), INDOLE-3-ACETIC ACID INDUCIBLE19 (IAA19), and IAA29. Moreover, MED17 facilitates enrichment of H3K4me3 on the promoters of PIF4, YUC8, IAA19, and IAA29 genes. Interestingly, both occupancy of MED17 and enrichment of H3K4me3 on these thermomorphogenesis-related promoters are dependent on PIF4 (or PIFs). Altered accumulation of COI1 under warm temperature in the med17 mutant suggests the possibility of a feedback mechanism. Overall, this study reveals the role of the Mediator complex as an integrator of JA and auxin signaling pathways during thermomorphogenesis.

Gestational age specific anthropometric postnatal percentile charts for neonates born at tertiary hospital in Eastern Nepal.

BACKGROUND AND OBJECTIVES: Birth weight, Head circumference (HC), and Length are important clinical indicators for evaluation of prenatal growth and identification of neonates requiring detail assessment and monitoring. Gestational age-specific percentile charts are essential tool for both obstetricians and pediatricians in their day to day practice. This study aimed to develop gestational age specific percentile chart of Birth weight, Length and HC for neonates. METHODS: In this Cross sectional observational study, HC, Birth weight and Length of live singleton neonates from 28 to 42 weeks of gestation fulfilling the inclusion criteria were measured over a period of one year. Mean, standard deviation, and percentiles values for different gestational age were calculated. Graphs were constructed using two way graph and Lowess smoothening method. RESULTS: Of total 2662 neonates, male: female ratio was 1.3:1 with maximum neonates in 40 weeks of gestation. The mean Birth weight, HC and Length was 2852.02 gm, 33.6 and 48.42 cm respectively. Overall males have more mean weight than females by 46.35gms. However, mean HC of male and female were similar 33.6 and 33.61 cm respectively and on average males were 0.27 cm longer compared to female. The mean Birth weight, HC and Length at 40 weeks was 3123.43gm (± 427.82), 34.249 cm (± 0.87) and 49.61 cm(± 1.85) respectively. The 10th, 50th and 90th percentile at 40 weeks for Birth weight being 2550gm, 3100gm and 3750gm respectively. The gestational age specific percentile chart and growth curve are appropriately placed in the manuscript. CONCLUSIONS: The percentile charts in this study may be used as reference for local population and similar data from various parts of the nation can provide a national reference curve for healthy neonates.