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Five genes of large phenotypic effect known to confer abiotic stress tolerance in rice were selected to characterize allelic variation in commercial Colombian tropical japonica upland rice cultivars adapted to drought‐prone acid soil environments (cv. Llanura11 and Porvenir12). Allelic variants of the genes ART1, DRO1, SUB1A, PSTOL1, and SPDT were characterized by PCR and/or Sanger sequencing in the two upland cultivars and compared with the Nipponbare and other reference genomes. Two genes were identified as possible targets for gene editing: SUB1A (Submergence 1A), to improve tolerance to flooding, and SPDT (SULTR3;4) (SULTR‐like Phosphorus Distribution Transporter), to improve phosphorus utilization efficiency and grain quality. Based on technical and regulatory considerations, SPDT was targeted for editing. The two upland cultivars were shown to carry the SPDT wild‐type (nondesirable) allele based on sequencing, RNA expression, and phenotypic evaluations under hydroponic and greenhouse conditions. A gene deletion was designed using the CRISPR/Cas9 system, and specialized reagents were developed for SPDT editing, including vectors targeting the gene and a protoplast transfection transient assay. The desired edits were confirmed in protoplasts and serve as the basis for ongoing plant transformation experiments aiming to improve the P‐use efficiency of upland rice grown in acidic soils. [ABSTRACT FROM AUTHOR]

Seed dormancy is a very important trait that maximizes the survival of seed in nature, the control of which can have important repercussions on the yield of many crop species. We have used gene expression profiling to identify genes that are involved in dormancy regulation in Arabidopsis thaliana. RNA was isolated from imbibed dormant (D) and after-ripened (AR) ecotype C24 seeds, and then screened by quantitative RT-PCR (qRT-PCR) for differentially expressed transcription factors (TFs) and other regulatory genes. Out of 2207 genes screened, we have identified 39 that were differentially expressed during the first few hours of imbibition. After analyzing T-DNA insertion mutants for 22 of these genes, two displayed altered dormancy compared with the wild type. These mutants are affected in genes that encode a RING finger and an HDZip protein. The first, named DESPIERTO, is involved in ABA sensitivity during seed development, regulates the expression of ABI3, and produces a complete loss of dormancy when mutated. The second, the HDZip ( ATHB20), is expressed during seed germination in the micropylar endosperm and in the root cap, and increases ABA sensitivity and seed dormancy when mutated. [ABSTRACT FROM AUTHOR]

Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs andmiRNAs in EVs,with serumcontributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serumcontamination. [ABSTRACT FROM AUTHOR]

Seed dormancy is an important agronomic trait in crops, and plants with low dormancy are prone to preharvest sprouting (PHS) under high‐temperature and humid conditions. In this study, we report that the GATA transcription factor TaGATA1 is a positive regulator of seed dormancy by regulating TaABI5 expression in wheat. Our results demonstrate that TaGATA1 overexpression significantly enhances seed dormancy and increases resistance to PHS in wheat. Gene expression patterns, abscisic acid (ABA) response assay, and transcriptome analysis all indicate that TaGATA1 functions through the ABA signaling pathway. The transcript abundance of TaABI5, an essential regulator in the ABA signaling pathway, is significantly elevated in plants overexpressing TaGATA1. Chromatin immunoprecipitation assay (ChIP) and transient expression analysis showed that TaGATA1 binds to the GATA motifs at the promoter of TaABI5 and induces its expression. We also demonstrate that TaGATA1 physically interacts with the putative demethylase TaELF6‐A1, the wheat orthologue of Arabidopsis ELF6. ChIP–qPCR analysis showed that H3K27me3 levels significantly decline at the TaABI5 promoter in the TaGATA1‐overexpression wheat line and that transient expression of TaELF6‐A1 reduces methylation levels at the TaABI5 promoter, increasing TaABI5 expression. These findings reveal a new transcription module, including TaGATA1–TaELF6‐A1–TaABI5, which contributes to seed dormancy through the ABA signaling pathway and epigenetic reprogramming at the target site. TaGATA1 could be a candidate gene for improving PHS resistance. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes are pervasively transcribed by RNA polymerase II. Yet, the molecular and biological implications of such a phenomenon are still largely puzzling. Here, we describe noncoding RNA transcription upstream of the Arabidopsis thaliana DOG1 gene, which governs salt stress responses and is a key regulator of seed dormancy. We find that expression of the DOG1 gene is induced by salt stress, thereby causing a delay in seed germination. We uncover extensive transcriptional activity on the promoter of the DOG1 gene, which produces a variety of lncRNAs. These lncRNAs, named PUPPIES, are co‐directionally transcribed and extend into the DOG1 coding region. We show that PUPPIES RNAs respond to salt stress and boost DOG1 expression, resulting in delayed germination. This positive role of pervasive PUPPIES transcription on DOG1 gene expression is associated with augmented pausing of RNA polymerase II, slower transcription and higher transcriptional burst size. These findings highlight the positive role of upstream co‐directional transcription in controlling transcriptional dynamics of downstream genes. Synopsis: DOG1 is a key regulator of plant seed dormancy, whose expression is regulated by multiple mechanisms. Here, salt stress is found to induce pervasive lncRNA transcription within the DOG1 promoter region, resulting in augmented RNA Polymerase II pausing, slower transcription and increased DOG1 expression. The DOG1 gene restricts seed germination under salt stress independently of its function in primary dormancy.The DOG1 promoter region gives rise to salt stress‐responsive PUPPIES lncRNAs, which regulate DOG1 expression in cis to delay germination.Co‐directional pervasive transcription of PUPPIES activates DOG1 expression through augmented RNA polymerase II pausing and enhanced transcriptional bursting.The identified regulatory mechanism involves an increase of RNA molecules produced per DOG1 transcriptional burst, augmented pausing and slower transcription. [ABSTRACT FROM AUTHOR]

Diploid plant genomes typically contain ~35,000 genes, almost all belonging to highly conserved gene families. Only a small fraction are lineage‐specific, which are found in only one or few closely related species. Little is known about how genes arise de novo in plant genomes and how often this occurs; however, they are believed to be important for plants diversification and adaptation. We developed a pipeline to identify lineage‐specific genes in Triticeae, using newly available genome assemblies of wheat, barley, and rye. Applying a set of stringent criteria, we identified 5942 candidate Triticeae‐specific genes (TSGs), of which 2337 were validated as protein‐coding genes in wheat. Differential gene expression analyses revealed that stress‐induced wheat TSGs are strongly enriched in putative secreted proteins. Some were previously described to be involved in Triticeae non‐host resistance and cold response. Additionally, we show that 1079 TSGs have sequence homology to transposable elements (TEs), ~68% of them deriving from regulatory non‐coding regions of Gypsy retrotransposons. Most importantly, we demonstrate that these TSGs are enriched in transmembrane domains and are among the most highly expressed wheat genes overall. To summarize, we conclude that de novo gene formation is relatively rare and that Triticeae probably possess ~779 lineage‐specific genes per haploid genome. TSGs, which respond to pathogen and environmental stresses, may be interesting candidates for future targeted resistance breeding in Triticeae. Finally, we propose that non‐coding regions of TEs might provide important genetic raw material for the functional innovation of TM domains and the evolution of novel secreted proteins. [ABSTRACT FROM AUTHOR]

Summary: One of the strategies that plants adopt to cope with an unfavorable environment is to sacrifice their growth for tolerance. Although moderate salt stress can induce root growth inhibition, the molecular mechanisms regulating this process have yet to be elucidated.Here, we found that overexpression of a zinc finger‐homeodomain family transcription factor, HOMEOBOX PROTEIN 24 (HB24), led to longer primary roots than in the wild‐type in the presence of 125 mM NaCl, whereas this phenotype was reversed for the hb24 loss‐of‐function mutant, indicating a negative impact of HB24 on salt‐induced root growth inhibition. We then found that salt stress triggered the degradation of HB24 via the ubiquitin–proteasome pathway, as mediated by a plant U‐box type E3 ubiquitin ligase 30 (PUB30) that directly targets HB24.We verified that HB24 is able to directly bind to the promoters of Sugars Will Eventually be Exported Transporter 11/12 (SWEET11/12) to regulate their expression in roots. Through genetic and biochemical assays, we further demonstrated that the HB24‐SWEET11 module plays a negative role in salt‐induced root growth inhibition.Therefore, we propose that under salt stress, PUB30 mediates HB24′s degradation, thereby downregulating the expression of SWEET11, resulting in reduced sucrose supply and root growth inhibition. [ABSTRACT FROM AUTHOR]

(−)‐Antrocin (1), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase‐like terpene cyclase AncC that biosynthesizes the drimane‐type sesquiterpene (+)‐albicanol (2) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2. Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)‐trans‐γ‐monocyclofarnesol instead of 2. Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane‐type scaffold. [ABSTRACT FROM AUTHOR]

SUMMARY: In nature, multiple stress factors occur simultaneously. The screening of natural diversity panels and subsequent Genome‐Wide Association Studies (GWAS) is a powerful approach to identify genetic components of various stress responses. Here, the nutritional status variation of a set of 270 natural accessions of Arabidopsis thaliana grown on a natural saline‐carbonated soil is evaluated. We report significant natural variation on leaf Na (LNa) and Fe (LFe) concentrations in the studied accessions. Allelic variation in the NINJA and YUC8 genes is associated with LNa diversity, and variation in the ALA3 is associated with LFe diversity. The allelic variation detected in these three genes leads to changes in their mRNA expression and correlates with plant differential growth performance when plants are exposed to alkaline salinity treatment under hydroponic conditions. We propose that YUC8 and NINJA expression patters regulate auxin and jasmonic signaling pathways affecting plant tolerance to alkaline salinity. Finally, we describe an impairment in growth and leaf Fe acquisition associated with differences in root expression of ALA3, encoding a phospholipid translocase active in plasma membrane and the trans Golgi network which directly interacts with proteins essential for the trafficking of PIN auxin transporters, reinforcing the role of phytohormonal processes in regulating ion homeostasis under alkaline salinity. Significance Statement: Soil salinization is a major constraint affecting crop growth and productivity, especially in arid and semi‐arid regions. Salinity and alkalinity frequently coexist in nature, being more destructive to plants than neutral salts. The exploration of genetic diversity and a comprehensive understanding of the molecular tolerance mechanisms involved in salinity‐alkalinity tolerance are essential for breeding resilient crops and thus facing one of the biggest agricultural challenges posed by climate change and increasing population. [ABSTRACT FROM AUTHOR]

Summary: Divergent selection of populations in contrasting environments leads to functional genomic divergence. However, the genomic architecture underlying heterogeneous genomic differentiation remains poorly understood. Here, we de novo assembled two high‐quality wild barley (Hordeum spontaneum K. Koch) genomes and examined genomic differentiation and gene expression patterns under abiotic stress in two populations. These two populations had a shared ancestry and originated in close geographic proximity but experienced different selective pressures due to their contrasting micro‐environments. We identified structural variants that may have played significant roles in affecting genes potentially associated with well‐differentiated phenotypes such as flowering time and drought response between two wild barley genomes. Among them, a 29‐bp insertion into the promoter region formed a cis‐regulatory element in the HvWRKY45 gene, which may contribute to enhanced tolerance to drought. A single SNP mutation in the promoter region may influence HvCO5 expression and be putatively linked to local flowering time adaptation. We also revealed significant genomic differentiation between the two populations with ongoing gene flow. Our results indicate that SNPs and small SVs link to genetic differentiation at the gene level through local adaptation and are maintained through divergent selection. In contrast, large chromosome inversions may have shaped the heterogeneous pattern of genomic differentiation along the chromosomes by suppressing chromosome recombination and gene flow. Our research offers novel insights into the genomic basis underlying local adaptation and provides valuable resources for the genetic improvement of cultivated barley. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm‐climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE‐RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9‐CIS‐EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR‐induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID‐INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm‐climate crops in horticulture. [ABSTRACT FROM AUTHOR]

SUMMARY: Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long‐day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti‐parallel alpha‐helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene‐edited mutants developed and flowered normally, but an Mting2‐1 Tnt1 insertion mutant and gene‐edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT‐like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2‐1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene‐edited mutants indicated that it was dispensable to MtING2 function in wild‐type plants. RNA sequencing experiments indicated that >7000 genes are mis‐expressed in the Mting2‐1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP‐seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2‐1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism. Significance Statement: The timing of flowering is important for plant adaptation and crop productivity, but knowledge on how this is regulated in legumes is limited. Here, we report the functional characterization of MtING2 and show that it is important in regulating plant architecture and flowering time in the model legume Medicago truncatula, providing evidence of the physiological role of an ING‐like gene in plants. [ABSTRACT FROM AUTHOR]

Malformed fruits depreciate a plant's market value. In tomato (Solanum lycopersicum), fruit malformation is associated with the multi‐locule trait, which involves genes regulating shoot apical meristem (SAM) development. The expression pattern of TOPLESS3 (SlTPL3) throughout SAM development prompted us to investigate its functional significance via RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR‐associated nuclease 9 (Cas9)‐mediated gene editing. Lower SlTPL3 transcript levels resulted in larger fruits with more locules and larger SAMs at the 5 d after germination (DAG5) stage. Differentially expressed genes in the SAM of wild‐type (WT) and SlTPL3‐RNAi plants, identified by transcriptome deep sequencing (RNA‐seq), were enriched in the gibberellin (GA) biosynthesis and plant hormone signaling pathways. Moreover, exogenous auxin and paclobutrazol treatments rescued the multi‐locule phenotype, indicating that SlTPL3 affects SAM size by mediating auxin and GA levels in the SAM. Furthermore, SlTPL3 interacted with WUSCHEL (SlWUS), which plays an important role in SAM size maintenance. We conducted RNA‐seq and DNA affinity purification followed by sequencing (DAP‐seq) analyses to identify the genes regulated by SlTPL3 and SlWUS in the SAM and to determine how they regulate SAM size. We detected 24 overlapping genes regulated by SlTPL3 and SlWUS and harboring an SlWUS‐binding motif in their promoters. Furthermore, functional annotation revealed a notable enrichment for functions in auxin transport, auxin signal transduction, and GA biosynthesis. Dual‐luciferase assays also revealed that SlTPL3 enhances SlWUS‐mediated regulation (repression and activation) of SlPIN3 and SlGA2ox4 transcription, indicating that the SlTPL3–SlWUS module regulates SAM size by mediating auxin distribution and GA levels, and perturbations of this module result in enlarged SAM. These results provide novel insights into the molecular mechanism of SAM maintenance and locule formation in tomato and highlight the SlTPL3–SlWUS module as a key regulator. [ABSTRACT FROM AUTHOR]

SUMMARY: The calmodulin‐binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C‐REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3334, which includes the N‐terminal CG‐1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN‐LIKE A1 (EXPL1) and NINE‐CIS‐EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold‐treated plants. Rather, domain‐swapping experiments indicated that the CAMTA3 CG‐1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG‐1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C‐terminal calmodulin‐binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature. Significance Statement: Determining how plants alter gene expression in response to environmental stimuli is fundamental to our understanding of how plants cope with environmental stress. Here, we present results indicating that the N‐terminal DNA binding region of CAMTA3 has a role in regulating its ability to induce the expression of target genes in response to cold temperature. [ABSTRACT FROM AUTHOR]

B‐cell progenitor fate determinant interferon regulatory factor 4 (IRF4) exerts key roles in the pathogenesis and progression of multiple myeloma (MM), a currently incurable plasma cell malignancy. Aberrant expression of IRF4 and the establishment of a positive auto‐regulatory loop with oncogene MYC, drives a MM specific gene‐expression program leading to the abnormal expansion of malignant immature plasma cells. Targeting the IRF4‐MYC oncogenic loop has the potential to provide a selective and effective therapy for MM. Here we evaluate the use of bromodomain inhibitors to target the IRF4‐MYC axis through combined inhibition of their known epigenetic regulators, BRD4 and CBP/EP300. Although all inhibitors induced cell death, we found no synergistic effect of targeting both of these regulators on the viability of MM cell‐lines. Importantly, for all inhibitors over a time period up to 72 h, we detected reduced IRF4 mRNA, but a limited decrease in IRF4 protein expression or mRNA levels of downstream target genes. This indicates that inhibitor‐induced loss of cell viability is not mediated through reduced IRF4 protein expression, as previously proposed. Further analysis revealed a long half‐life of IRF4 protein in MM cells. In support of our experimental observations, gene network modeling of MM suggests that bromodomain inhibition is exerted primarily through MYC and not IRF4. These findings suggest that despite the autofeedback positive regulatory loop between IRF4 and MYC, bromodomain inhibitors are not effective at targeting IRF4 in MM and that novel therapeutic strategies should focus on the direct inhibition or degradation of IRF4. [ABSTRACT FROM AUTHOR]

The rapid invasion of the non‐native Phragmites australis (Poaceae, subfamily Arundinoideae) is a major threat to native wetland ecosystems in North America and elsewhere. We describe the first reference genome for P. australis and compare invasive (ssp. australis) and native (ssp. americanus) genotypes collected from replicated populations across the Laurentian Great Lakes to deduce genomic bases driving its invasive success. Here, we report novel genomic features including a Phragmites lineage‐specific whole genome duplication, followed by gene loss and preferential retention of genes associated with transcription factors and regulatory functions in the remaining duplicates. Comparative transcriptomic analyses revealed that genes associated with biotic stress and defence responses were expressed at a higher basal level in invasive genotypes, but native genotypes showed a stronger induction of defence responses when challenged by a fungal endophyte. The reference genome and transcriptomes, combined with previous ecological and environmental data, add to our understanding of mechanisms leading to invasiveness and support the development of novel, genomics‐assisted management approaches for invasive Phragmites. [ABSTRACT FROM AUTHOR]

Introduction:Vascular contributions to cognitive impairment and dementia (VCID) are a leading cause of dementia. An underappreciated, modifiable risk factor for VCID is hyperhomocysteinemia (HHcy), defined by elevated levels of plasma homocysteine, most often due to impaired B vitamin absorption in aged persons. Studies aimed at identifying neuropathologic features and gene expression profiles associated with HHcy have been lacking. Methods:Asubset of research volunteers from the University of Kentucky Alzheimer's Disease Research Center longitudinal cohort came to autopsy and had ante mortem plasma homocysteine levels available. Brain tissue and blood plasma drawn closest to deathwere used tomeasure homocysteine and relatedmetabolites in the current pilot study. Genetic expression profiles of inflammatory markers were evaluated using the HumanNeuroinflammation NanoString panel. Further analyses included an evaluation of plasma homocysteine effects on amyloid beta, tau, ionized calcium-binding adaptor molecule 1, and glial fibrillary acidic protein immunohistochemistry in the frontal and occipital cortices. Analytes and other study outcomes were evaluated in relation to ante mortem HHcy status:We identified 13 persons with normal ante mortem plasma homocysteine levels (<14 µmol/L) and 18 who had high plasma homocysteine levels (=14 µmol/L). Results: Participants with HHcy demonstrated increased levels of several plasma homocysteine cycle metabolites such as total cysteine, S-adenosyl-homocysteine, cystathionine, and choline. Inflammatory gene expression profiles showed a general downregulation in the setting of elevated plasma homocysteine. HHcy was associated with more and longer microglial processes, but smaller and fewer astrocytes, especially in participants of older age at death. HHcy in older participants was also associated with occipital cortex microhemorrhages and increased severity of atherosclerosis throughout the cerebral vasculature. Conclusions: Increased plasma homocysteine and older age were associated with the downregulation of inflammatory gene expression markers in association with significant glial and vascular pathology changes. Impaired immune function is a plausible mechanism by which HHcy increases cerebrovascular damage leading to impaired cognitive function. [ABSTRACT FROM AUTHOR]

Objective: Genetic variants spanning UBE2L3 are associated with increased expression of the UBE2L3‐encoded E2 ubiquitin‐conjugating enzyme H7 (UbcH7), which facilitates activation of proinflammatory NF‐κB signaling and susceptibility to autoimmune diseases. We undertook this study to delineate how genetic variants carried on the UBE2L3/YDJC autoimmune risk haplotype function to drive hypermorphic UBE2L3 expression. Methods: We used bioinformatic analyses, electrophoretic mobility shift assays, and luciferase reporter assays to identify and functionally characterize allele‐specific effects of risk variants positioned in chromatin accessible regions of immune cells. Chromatin conformation capture with quantitative polymerase chain reaction (3C‐qPCR), chromatin immunoprecipitation (ChIP)–qPCR, and small interfering RNA (siRNA) knockdown assays were performed on patient‐derived Epstein‐Barr virus–transformed B cells homozygous for the UBE2L3/YDJC nonrisk or risk haplotype to determine if the risk haplotype increases UBE2L3 expression by altering the regulatory chromatin architecture in the region. Results: Of the 7 prioritized variants, 5 demonstrated allele‐specific increases in nuclear protein binding affinity and regulatory activity. High‐throughput sequencing of chromosome conformation capture coupled with ChIP (HiChIP) and 3C‐qPCR uncovered a long‐range interaction between the UBE2L3 promoter (rs140490, rs140491, rs11089620) and the downstream YDJC promoter (rs3747093) that was strengthened in the presence of the UBE2L3/YDJC risk haplotype, and correlated with the loss of CCCTC‐binding factor (CTCF) and gain of YY1 binding at the risk alleles. Depleting YY1 by siRNA disrupted the long‐range interaction between the 2 promoters and reduced UBE2L3 expression. Conclusion: The UBE2L3/YDJC autoimmune risk haplotype increases UBE2L3 expression through strengthening a YY1‐mediated interaction between the UBE2L3 and YDJC promoters. [ABSTRACT FROM AUTHOR]

Genomic amplification of OTUD7B is frequently found across human cancers. But its role in tumorigenesis is poorly understood. Lysine‐specific demethylase 1 (LSD1) is known to execute epigenetic regulation by forming corepressor complex with CoREST/histone deacetylases (HDACs). However, the molecular mechanisms by which cells maintain LSD1/CoREST complex integrity are unknown. Here, it is reported that LSD1 protein undergoes K63‐linked polyubiquitination. OTUD7B is responsible for LSD1 deubiquitination at K226/277 residues, resulting in dynamic control of LSD1 binding partner specificity and cellular homeostasis. OTUD7B deficiency increases K63‐linked ubiquitination of LSD1, which disrupts LSD1/CoREST complex formation and targets LSD1 for p62‐mediated proteolysis. Consequently, OTUD7B deficiency impairs genome‐wide LSD1 occupancy and enhances the methylation of H3K4/H3K9, therefore profoundly impacting global gene expression and abrogating breast cancer metastasis. Moreover, physiological fluctuation of OTUD7B modulates cell cycle‐dependent LSD1 oscillation, ensuring the G1/S transition. Both OTUD7B and LSD1 proteins are overpresented in high‐grade or metastatic human breast cancer, while dysregulation of either protein is associated with poor survival and metastasis. Thus, OTUD7B plays a unique partner‐switching role in maintaining the integrity of LSD1/CoREST corepressor complex, LSD1 turnover, and breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Transcriptional dysregulation is central to many diseases including cancer. Mutation or deregulated expression of proteins involved in transcriptional machinery leads to aberrant gene expression that disturbs intricate cellular processes of division and differentiation. The subunits of the mediator complex are master regulators of stimuli‐derived transcription and are essential for transcription by RNA polymerase II. MED12 is a part of the CDK8 kinase module of the mediator complex and is essential for kinase assembly and function. Other than its function in activation of the kinase activity of CDK8 mediator, it also brings about transcription repression or activation, in response to several signalling pathways, a function that is independent of its role as a part of kinase assembly. Accumulating evidence suggests that MED12 controls complex transcription programs that are defining in cell fate determination, differentiation, and carcinogenesis. Mutations or differential expression of MED12 manifest in several human disorders and diseases. For instance, MED12 mutations are the gold standard for the diagnosis of several X‐linked intellectual disability syndromes. Further, certain MED12 mutations are categorised as driver mutations in carcinogenesis as well. This is a timely review that provides for the first time a wholesome view on the critical roles and pathways regulated by MED12, its interactions along with the implications of MED12 alterations/mutations in various cancers and nonneoplastic disorders. Based on the preclinical studies, MED12 indeed emerges as an attractive novel therapeutic target for various diseases and intellectual disorders. [ABSTRACT FROM AUTHOR]

Summary: The timing of reproduction is a critical developmental decision in the life cycle of many plant species.Fine mapping of a rapid‐flowering mutant was done using whole‐genome sequence data from bulked DNA from a segregating F2 mapping populations. The causative mutation maps to a gene orthologous with the third subunit of DNA polymerase δ (POLD3), a previously uncharacterized gene in plants. Expression analyses of POLD3 were conducted via real time qPCR to determine when and in what tissues the gene is expressed.To better understand the molecular basis of the rapid‐flowering phenotype, transcriptomic analyses were conducted in the mutant vs wild‐type. Consistent with the rapid‐flowering mutant phenotype, a range of genes involved in floral induction and flower development are upregulated in the mutant.Our results provide the first characterization of the developmental and gene expression phenotypes that result from a lesion in POLD3 in plants. See also the Commentary on this article by Catalán & Vogel, 227: 1587–1590. [ABSTRACT FROM AUTHOR]

Premise: Despite myriad examples of local adaptation, the phenotypes and genetic variants underlying such adaptive differentiation are seldom known. Recent work on freezing tolerance and local adaptation in ecotypes of Arabidopsis thaliana from Italy and Sweden provides an essential foundation for uncovering the genotype–phenotype–fitness map for an adaptive response to a key environmental stress. Methods: We examined the consequences of a naturally occurring loss‐of‐function (LOF) mutation in an Italian allele of the gene that encodes the transcription factor CBF2, which underlies a major freezing‐tolerance locus. We used four lines with a Swedish genetic background, each containing a LOF CBF2 allele. Two lines had introgression segments containing the Italian CBF2 allele, and two contained deletions created using CRISPR‐Cas9. We used a growth chamber experiment to quantify freezing tolerance and gene expression before and after cold acclimation. Results: Freezing tolerance was lower in the Italian (11%) compared to the Swedish (72%) ecotype, and all four experimental CBF2 LOF lines had reduced freezing tolerance compared to the Swedish ecotype. Differential expression analyses identified 10 genes for which all CBF2 LOF lines, and the IT ecotype had similar patterns of reduced cold responsive expression compared to the SW ecotype. Conclusions: We identified 10 genes that are at least partially regulated by CBF2 that may contribute to the differences in cold‐acclimated freezing tolerance between the Italian and Swedish ecotypes. These results provide novel insight into the molecular and physiological mechanisms connecting a naturally occurring sequence polymorphism to an adaptive response to freezing conditions. [ABSTRACT FROM AUTHOR]

Background and Aims: Alcoholic liver disease (ALD) is the most common liver disease and a severe mortality burden in the world. However, ALD is rarely detected at its early stages. Thus, exploration of an early event for ALD may help the prognosis and further therapy of ALD. Several circRNAs were proven as novel molecular biomarkers for the progression of chronic nonalcoholic fatty liver disease. Whether circRNAs are involved explicitly in ALD remains unknown. Methods: The expression profile of circRNAs in an ALD mouse model was depicted by circRNA sequencing. The dysregulated circRNAs were verified by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Bioinformatics and circRNA/microRNA (miRNA) crosstalk analyses were applied to predict the potential functions of circRNAs. Finally, the miRNA expression was confirmed by miRNA sequencing. Results: Compared with the control group, 6 members of circRNAs were up‐regulated, and 4 were down‐regulated in the ALD model. GO enrichment analyses revealed these circRNAs were predominantly enriched in the endoplasmic reticulum, arachidonic acid metabolism, and cytochrome P450 metabolism pathway. Among these circRNAs, the differential expression of 5 circRNAs was validated and consistent with qRT‐PCR, and only the up‐regulated mou_circ_1657 is included in the circBase. Further, the crosstalk analysis of circRNA–miRNA revealed 7 miRNAs were targeted by mou_circ_1657, of which miR‐19‐5b was the only miRNA that was down‐regulated in the ALD mice according to the miRNA sequencing data, suggesting it needs further attention in ALD. Conclusions: This study demonstrates that a cluster of circRNAs is aberrantly expressed in the livers of ALD mice. mou_circ_1657/miR‐19‐5b may play a critical role in the development of ALD. Our study provides new insight into the future investigation and therapy on ALD. [ABSTRACT FROM AUTHOR]

Summary: The distinct functions of individual cell types require cells to express specific sets of genes. The germinating seed is an excellent model to study genome regulation between cell types since the majority of the transcriptome is differentially expressed in a short period, beginning from a uniform, metabolically inactive state. In this study, we applied laser‐capture microdissection RNA‐sequencing to small numbers of cells from the plumule, radicle tip and scutellum of germinating barley seeds every 8 h, over a 48 h time course. Tissue‐specific gene expression was notably common; 25% (910) of differentially expressed transcripts in plumule, 34% (1876) in radicle tip and 41% (2562) in scutellum were exclusive to that organ. We also determined that tissue‐specific storage of transcripts occurs during seed development and maturation. Co‐expression of genes had strong spatiotemporal structure, with most co‐expression occurring within one organ and at a subset of specific time points during germination. Overlapping and distinct enrichment of functional categories were observed in the tissue‐specific profiles. We identified candidate transcription factors amongst these that may be regulators of spatiotemporal gene expression programs. Our findings contribute to the broader goal of generating an integrative model that describes the structure and function of individual cells within seeds during germination. Significance Statement: The different tissues and cells of germinating seeds have distinct functions that require tight spatial and temporal control of gene expression. How such control is achieved is poorly understood in crop species. Here we characterized the transcriptomes of hundreds of cells from the radicle, plumule, and scutellum in barley seeds at high temporal resolution across germination, defined gene networks, then identified candidate tissue‐specific regulatory transcription factors. [ABSTRACT FROM AUTHOR]

Summary: Appropriate regulation of crop seed germination is of significance for agriculture production. In this study, we show that TaJAZ1, most closely related to Arabidopsis JAZ3, negatively modulates abscisic acid (ABA)‐inhibited seed germination and ABA‐responsive gene expression in bread wheat. Biochemical interaction assays demonstrate that the C‐terminal part containing the Jas domain of TaJAZ1 physically interacts with TaABI5.Similarly, Arabidopsis jasmonate‐ZIM domain (JAZ) proteins also negatively modulate ABA responses. Further we find that a subset of JAZ proteins could interact with ABI5 using the luciferase complementation imaging assays.Choosing JAZ3 as a representative, we demonstrate that JAZ3 interacts with ABI5 in vivo and represses the transcriptional activation activity of ABI5. ABA application could abolish the enrichment of JAZ proteins in the ABA‐responsive gene promoter. Furthermore, we find that ABA application could induce the expression of jasmonate (JA) biosynthetic genes and then increase the JA concentrations partially dependent on the function of ABI5, consequently leading to the degradation of JAZ proteins.This study sheds new light on the crosstalk between JA and ABA in modulating seed germination in bread wheat and Arabidopsis. [ABSTRACT FROM AUTHOR]

Plants need to cope with changing environmental conditions, be it variable light or temperature, different availability of water or nutrients, or attack by pathogens or insects. Some of these changing conditions can become stressful and require strong countermeasures to ensure plant survival. Plants have evolved numerous distinct sensing and signalling mechanisms to perceive and respond appropriately to a variety of stresses. Because of the unpredictable nature of numerous stresses, resource‐saving stress response mechanisms are inducible and become activated only upon a stress experience. Furthermore, plants have evolved mechanisms by which they can remember past stress events and prime their responses in order to react more rapidly or more strongly to recurrent stress. Research over the last decade has revealed mechanisms of this information storage and retrieval, which include epigenetic regulation, transcriptional priming, primed conformation of proteins, or specific hormonal or metabolic signatures. There is also increasing understanding of the ecological constraints and relevance of stress priming and memory. This special issue presents research articles and reviews addressing various aspects of this exciting and growing field of research. Here, we introduce the topic by referring to the articles published in this issue, and we outline open questions and future directions of research. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) improves endoplasmic reticulum (ER) protein folding in order to alleviate stress. Yet it is becoming increasingly clear that the UPR regulates processes well beyond those directly involved in protein folding, in some cases by mechanisms that fall outside the realm of canonical UPR signaling. These pathways are highly specific from one cell type to another, implying that ER stress signaling affects each tissue in a unique way. Perhaps nowhere is this more evident than in the liver, which—beyond being a highly secretory tissue—is a key regulator of peripheral metabolism and a uniquely proliferative organ upon damage. The liver provides a powerful model system for exploring how and why the UPR extends its reach into physiological processes that occur outside the ER, and how ER stress contributes to the many systemic diseases that involve liver dysfunction. This review will highlight the ways in which the study of ER stress in the liver has expanded the view of the UPR to a response that is a key guardian of cellular homeostasis outside of just the narrow realm of ER protein folding. The unfolded protein response (UPR) restores endoplasmic reticulum homeostasis during stress. In the liver, the UPR is regulated by—and in turn contributes to—both metabolism and liver damage. This review highlights these connections and what they tell us about both the physiology and pathophysiology of the liver and the cell biology of the UPR. [ABSTRACT FROM AUTHOR]

Ticks are obligatorily hematophagous but spend the majority of their lives off host in an unfed state where they must resist starvation between bouts of blood feeding. Survival during these extended off‐host periods is critical to the success of these arthropods as vectors of disease; however, little is known about the underlying physiological and molecular mechanisms of starvation tolerance in ticks. We examined the bioenergetic, transcriptomic and behavioural changes of female American dog ticks, Dermacentor variabilis, throughout starvation (up to nine months post‐bloodmeal). As starvation progressed, ticks utilized glycogen and lipid, and later protein as energy reserves with proteolysis and autophagy facilitating the mobilization of endogenous nutrients. The metabolic rate of the ticks was expectedly low, but showed a slight increase as starvation progressed possibly reflecting the upregulation of several energetically costly processes such as transcription/translation and/or increases in host‐seeking behaviours. Starved ticks had higher activity levels, increased questing behaviour and augmented expression of genes related to chemosensing, immunity and salivary gland proteins. The shifts in gene expression and associated behavioural and physiological processes are critical to allowing these parasites to exploit their ecological niche as extreme sit‐and‐wait parasites. The overall responses of ticks to starvation were similar to other blood‐feeding arthropods, but we identified unique responses that could have epidemiological and ecological significance for ticks as ectoparasites that must be tolerant of sporadic feeding. see also the Perspective by Gibbs [ABSTRACT FROM AUTHOR]

Different culture‐systems for derivation of induced pluripotent stem cells (iPSC) in vitro from human fibroblasts have been established. Here, we compared the efficacy of two different feeder‐free culture‐systems; Matrigel‐coated surfaces in combination with mTeSR1 medium versus Vitronectin‐coated surfaces in combination with Essential 8 (E8) medium. The comparison was performed by counting the number of emerging iPSC‐looking colonies of re‐programmed fibroblasts. The fibroblasts were re‐programmed using episomal plasmids expressing OCT3/4, SOX2, KLF4, L‐MYC, LIN28, and a p53 knock down shP53. Three different fibroblast lines, K40 and K48 from healthy controls and BBS1 from a patient with Bardet–Biedl syndrome, were used in two independent setups. The BBS1 line was used in both setups in combination with K40 and K48 respectively. In all four re‐programming experiments, we observed a significantly higher number of emerging colonies with the combination Matrigel/mTeSR1 as compared to the combination Vitronectin/E8. The presence of iPSC was verified by alkaline phosphatase and Tra‐1‐60 staining. Furthermore, a higher expression of the pluripotency‐associated markers NANOG and SOX2 in cells under Matrigel/mTeSR1 conditions compared with Vitronectin/E8 supported the higher proportion of iPSC on Matrigel/mTeSR1 plates. In conclusion, the combination Matrigel/mTeSR1 is more efficient for derivation of iPSC compared to the Vitronectin/E8 combination. [ABSTRACT FROM AUTHOR]

Summary: We recently developed a postharvest low‐temperature‐conditioning (LTC) treatment for ‘Wonderful’ pomegranate fruits that involve prestorage exposure to 15 °C for 10 days, and it markedly enhanced chilling tolerance. To elucidate the molecular mechanisms involved in the fruit responses to LTC, we conducted RNA‐Seq analysis of ‘Wonderful’ pomegranate inner membrane tissue at four different time points: (i) immediately after harvest; (ii) after the LTC treatment; (iii) after 2 weeks of cold storage at 1 °C; and (iv) after LTC + 2 weeks at 1 °C. Pairwise comparisons revealed that 7798 transcripts were significantly (P ≤ 0.05) induced or repressed by a factor of at least four in the chilling treatment (2422 upregulated and 5376 downregulated) as compared with just 1997 transcripts in the LTC + chilling treatment (974 upregulated and 1023 downregulated). Thus, application of LTC mitigated the overall changes in transcript levels associated with cold storage. Furthermore, the LTC + chilling affected gene expression patterns related to regulatory and stress mechanisms, including modification of specific transcription factor, signalling and stress‐related genes. [ABSTRACT FROM AUTHOR]

Objective: The goal of this study was to refine our understanding of disease risk attributable to common genetic variation in SNCA, a major locus in Parkinson disease, with potential implications for clinical trials targeting α-synuclein. We aimed to dissect the multiple independent association signals, stratify individuals by SNCA-specific risk profiles, and explore expression quantitative trait loci.Methods: We analyzed participant-level data from 12,503 patients and 12,502 controls, optimizing a risk model and assessing SNCA-specific risk scores and haplotypes as predictors of individual risk. We also explored hypotheses about functional mechanisms and correlated risk variants to gene expression in human brain and protein levels in cerebrospinal fluid.Results: We report and replicate a novel, third independent association signal at genome-wide significance level downstream of SNCA (rs2870004, p = 3.0*10-8 , odds ratio [OR] = 0.88, 95% confidence interval [CI] = 0.84-0.92). SNCA risk score stratification showed a 2-fold difference in disease susceptibility between top and bottom quintiles (OR = 1.99, 95% CI = 1.78-2.23). Contrary to previous reports, we provide evidence supporting top variant rs356182 as functional in itself and associated with a specific SNCA 5' untranslated region transcript isoform in frontal cortex.Interpretation: The SNCA locus harbors a minimum of 3 independent association signals for Parkinson disease. We demonstrate a fine-grained stratification of α-synuclein-related genetic burden in individual patients of potential future clinical relevance. Further efforts to pinpoint the functional mechanisms are warranted, including studies of the likely causal top variant rs356182 and its role in regulating levels of specific SNCA mRNA transcript variants. Ann Neurol 2018;83:117-129. [ABSTRACT FROM AUTHOR]

Abstract: Seed germination is a complex process regulated by intrinsic hormonal cues such as abscisic acid (ABA) and gibberellin (GA), and environmental signals including temperature. Using pharmacological, molecular and metabolomics approaches, we show that supraoptimal temperature delays wheat seed germination through maintaining elevated embryonic ABA level via increased expression of ABA biosynthetic genes (TaNCED1 and TaNCED2), increasing embryo ABA sensitivity through upregulation of genes regulating ABA signalling positively (TaPYL5, TaSnRK2, ABI3 and ABI5) and decreasing embryo GA sensitivity via induction of TaRHT1 that regulates GA signalling negatively. Endospermic ABA and GA appeared to have minimal roles in regulating germination at supraoptimal temperature. Germination inhibition by suboptimal temperature is associated with elevated ABA level in the embryo and endosperm tissues, mediated by induction of TaNCEDs and decreased expression of endospermic ABA catabolic genes (TaCYP707As), and increased ABA sensitivity in both tissues via upregulation of TaPYL5, TaSnRK2, ABI3 and ABI5 in the embryo and TaSnRK2 and ABI5 in the endosperm. Furthermore, suboptimal temperature suppresses GA synthesis in both tissues and GA sensitivity in the embryo via repressing GA biosynthetic genes (TaGA20ox and TaGA3ox2) and inducing TaRHT1, respectively. These results highlight that spatiotemporal modulation of ABA and GA metabolism and signalling in wheat seeds underlies germination response to temperature. [ABSTRACT FROM AUTHOR]

Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the humanRPEcells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cellswere infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to dedifferentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells. [ABSTRACT FROM AUTHOR]

The complex interactions between transcription factors ( TFs) and their target genes in a spatially and temporally specific manner are crucial to all cellular processes. Reconstruction of gene regulatory networks ( GRNs) from gene expression profiles can help to decipher TF-gene regulations in a variety of contexts; however, the inevitable prediction errors of GRNs hinder optimal data mining of RNA-Seq transcriptome profiles. Here we perform an integrative study of Zea mays (maize) seed development in order to identify key genes in a complex developmental process. First, we reverse engineered a GRN from 78 maize seed transcriptome profiles. Then, we studied collective gene interaction patterns and uncovered highly interwoven network communities as the building blocks of the GRN. One community, composed of mostly unknown genes interacting with opaque2, brittle endosperm1 and shrunken2, contributes to seed phenotypes. Another community, composed mostly of genes expressed in the basal endosperm transfer layer, is responsible for nutrient transport. We further integrated our inferred GRN with gene expression patterns in different seed compartments and at various developmental stages and pathways. The integration facilitated a biological interpretation of the GRN. Our yeast one-hybrid assays verified six out of eight TF-promoter bindings in the reconstructed GRN. This study identified topologically important genes in interwoven network communities that may be crucial to maize seed development. [ABSTRACT FROM AUTHOR]

The plant hormone abscisic acid ( ABA) is a key player in responses to abiotic stress. ABA regulates a plant's water status and mediates drought tolerance by controlling stomatal gas exchange, water conductance and differential gene expression. ABA is recognized and bound by the Regulatory Component of ABA Receptors ( RCARs)/ PYR1/ PYL (Pyrabactin Resistance 1/ PYR1-like). Ligand binding stabilizes the interaction of RCARs with type 2C protein phosphatases ( PP2C), which are ABA co-receptors. While the core pathway of ABA signalling has been elucidated, the large number of different ABA receptors and co-receptors within a plant species generates a complexity of heteromeric receptor complexes that has not functionally been resolved in any plant species to date. In this study, we characterized ABA receptors and co-receptors of grey poplar ( Populus x canescens [Ait.] Sm.) and their capacity to regulate ABA responses. We observed a high number of regulatory combinations of holo-receptor complexes, but also some preferential and selective RCAR- PP2C interactions. Poplar and Arabidopsis ABA receptor components revealed a strong structural and functional conservation. Heterologous receptor complexes of poplar and Arabidopsis components showed functionality in vitro and regulated ABA-responsive gene expression in cells of both species. ABA-responsive promoters of Arabidopsis were also active in poplar, which was explored to generate poplar reporter lines expressing green fluorescent protein in response to ABA. The study presents a detailed analysis of receptor complexes of a tree species and shows high conservation of ABA receptor components between an annual and a perennial plant. [ABSTRACT FROM AUTHOR]

An endoplasmic reticulum (ER)-resident protein that regulates cytosolic and ER free-Ca2+ concentration by induction of store-operated calcium entry: that is the original definition of STIM2 and its function. While its activity strongly depends on the amount of calcium stored in the ER, its function goes further, to intracellular signalling and gene expression. Initially under-studied owing to the prominent function of STIM1, STIM2 came to be regarded as vital in mice, gradually emerging as an important player in the nervous system, and cooperating with STIM1 in the immune system. STIM2 has also been proposed as a relevant player in pathological conditions related to ageing, Alzheimer's and Huntington's diseases, autoimmune disorders and cancer. The discovery of additional functions, together with new splicing forms with opposite roles, has clarified existing controversies about STIM2 function in SOCE. With STIM2 being essential for life, but apparently not for development, newly available data demonstrate a complex and still intriguing behaviour that this review summarizes, updating current knowledge of STIM2 function. [ABSTRACT FROM AUTHOR]

DNA-binding with one finger (DOF)-type transcription factors are involved in many fundamental processes in higher plants, from responses to light and phytohormones to flowering time and seed maturation, but their relation with abiotic stress tolerance is largely unknown. Here, we identify the roles of CDF3, an Arabidopsis DOF gene in abiotic stress responses and developmental processes like flowering time. CDF3 is highly induced by drought, extreme temperatures and abscisic acid treatment. The CDF3 T-DNA insertion mutant cdf3-1 is much more sensitive to drought and low temperature stress, whereas CDF3 overexpression enhances the tolerance of transgenic plants to drought, cold and osmotic stress and promotes late flowering. Transcriptome analysis revealed that CDF3 regulates a set of genes involved in cellular osmoprotection and oxidative stress, including the stress tolerance transcription factors CBFs, DREB2A and ZAT12, which involve both gigantea-dependent and independent pathways. Consistently, metabolite profiling disclosed that the total amount of some protective metabolites including γ-aminobutyric acid, proline, glutamine and sucrose were higher in CDF3-overexpressing plants. Taken together, these results indicate that CDF3 plays a multifaceted role acting on both flowering time and abiotic stress tolerance, in part by controlling the CBF/DREB2A-CRT/DRE and ZAT10/12 modules. [ABSTRACT FROM AUTHOR]

Identification of cellular proteins, in addition to already known transcription factors such as NF-κB, Sp1, C-EBPβ, NFAT, ATF/CREB, and LEF-1, which interact with the HIV-1 LTR, is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells, a macrophage model of latency. We observed that HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA-induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA-induced U1 cells in comparison to PMA-induced U937 cells. We focused on understanding the potential role of PKM2 in HIV-1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA-activated U1 and TZM-bl cells demonstrated the interaction of PKM2 with the HIV-1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV-1 LTR-luciferase reporter in U937, U-87 MG, and TZM-bl cells. Using various truncated constructs of the HIV-1 LTR, we mapped the region spanning −120 bp to −80 bp to be essential for PKM2-mediated transactivation. This region contains the NF-κB binding site and deletion of this site attenuated PKM2-mediated activation of HIV-1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF-κB. These observations demonstrate for the first time that PKM2 is a transcriptional co-activator of HIV-1 LTR. J. Cell. Physiol. 232: 517-525, 2017. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression through post-transcriptional regulation. Here, we report the identification and characterization of miRNAs in two closely related screwworm flies with different feeding habits: Cochliomyia hominivorax and Cochliomyia macellaria. The New World screwworm, C. hominivorax, is an obligatory parasite of warm-blooded vertebrates, whereas the secondary screwworm, C. macellaria, is a free-living organism that feeds on decaying organic matter. Here, the small RNA transcriptomes of adults and third-instar larvae of both species were sequenced. A total of 110 evolutionarily conserved miRNAs were identified, and 10 putative precursor miRNAs (pre-miRNAs) were predicted. The relative expression of six selected miRNAs was further investigated, including miRNAs that are related to reproduction and neural processes in other insects. Mature miRNAs were also characterized across an evolutionary time scale, suggesting that the majority of them have been conserved since the emergence of the Arthropoda [540 million years ago (Ma)], Hexapoda (488 Ma) and Brachycera (195 Ma) lineages. This study is the first report of miRNAs for screwworm flies. We also performed a comparative analysis with the hereby predicted miRNAs from the sheep blowfly, Lucilia cuprina. The results presented may advance our understanding of parasitic habits within Calliphoridae and assist further functional studies in blowflies. [ABSTRACT FROM AUTHOR]

VP1, the major structural protein of the mouse polyomavirus ( MPyV), is the major architectural component of the viral capsid. Its pentamers are able to self-assemble into capsid-like particles and to non-specifically bind DNA. Surface loops of the protein interact with sialic acid of ganglioside receptors. Although the replication cycle of the virus, including virion morphogenesis, proceeds in the cell nucleus, a substantial fraction of the protein is detected in the cytoplasm of late-phase MPyV-infected cells. In this work, we detected VP1 mainly in the cytoplasm of mammalian cells transfected with plasmid expressing VP1. In the cytoplasm, VP1-bound microtubules, including the mitotic spindle, and the interaction of VP1 with microtubules resulted in cell cycle block at the G2/M phase. Furthermore, in the late phase of MPyV infection and in cells expressing VP1, microtubules were found to be hyperacetylated. We then sought to understand how VP1 interacts with microtubules. Dynein is not responsible for the VP1-microtubule association, as neither overexpression of p53/dynamitin nor treatment with ciliobrevin-D (an inhibitor of dynein activity) prevented binding of VP1 to microtubules. A pull-down assay for VP1-interacting proteins identified the heat shock protein 90 (Hsp90) chaperone, and Hsp90 was also detected in the VP1-microtubule complexes. Although Hsp90 is known to be associated with acetylated microtubules, it does not mediate the interaction between VP1 and microtubules. Our study provides insight into the role of the major structural protein in MPyV replication, indicating that VP1 is a multifunctional protein that participates in the regulation of cell cycle progression in MPyV-infected cells. [ABSTRACT FROM AUTHOR]

The NGATHA ( NGA) clade of transcription factors (TFs) forms a small subfamily of four members in Arabidopsis thaliana. NGA genes act redundantly to direct the development of apical tissues in the gynoecium, where they have been shown to be essential for style and stigma specification. In addition, NGA genes have a more general role in controlling lateral organ growth. The four NGA genes in Arabidopsis are expressed in very similar domains, although little is known about the nature of their putative regulators. Here, we have identified a conserved region within the four NGA promoters that we have used as a bait to screen a yeast library, aiming to identify such NGA regulators. Three members of the TCP family of TFs, named after the founding factors TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING CELL FACTOR 1 AND 2), were recovered from this screening, of which two [ TCP2 and TCP3, members of the CINCINNATA (CIN) family of TCP genes (CIN-TCP) subclade] were shown to activate the NGA3 promoter in planta. We provide evidence that support that CIN-TCP genes are true regulators of NGA gene expression, and that part of the CIN-TCP role in leaf development is mediated by NGA upregulation. Moreover, we have found that this TCP-NGA regulatory interaction is likely conserved in angiosperms, including important crop species, for which the regulation of leaf development is a target for biotechnological improvement. [ABSTRACT FROM AUTHOR]

Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus ( Boophilus) microplus, an important vector of bovine babesiosis and anaplasmosis. Commercial promoters were evaluated for transcriptional activity driving luciferase expression in the tick cell lines. The human phosphoglycerate kinase (PGK) promoter resulted in detectable firefly luciferase activity within 2 days post-transfection of the R. microplus cell line BME26, with maximal activity at 5 days post-transfection. Several other promoters were weaker or inactive in the tick cells, prompting identification and assessment of transcriptional activity of the homologous ribosomal protein L4 (rpL4, GenBank accession no.: KM516205) and elongation factor 1α (EF-1α, GenBank accession no.: KM516204) promoters cloned from R. microplus. Evaluation of luciferase expression driven by various promoters in tick cell culture resulted in selection of the R. microplus rpL4 promoter and the human PGK promoter driving transcription of sequences encoding modified firefly and NanoLuc® luciferases for construction of a dual luciferase reporter system for use in tick cell culture. [ABSTRACT FROM AUTHOR]

Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9low cluster of differentiation (CD)44lowCD140low prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9low and Sox9high populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. [ABSTRACT FROM AUTHOR]

Cell identity is a reflection of a cell type-specific gene expression profile, and consequently, cell type-specific transcription factor networks are considered to be at the heart of a given cellular phenotype. Although generally stable, cell identity can be reprogrammed in vitro by forced changes to the transcriptional network, the most dramatic example of which was shown by the induction of pluripotency in somatic cells by the ectopic expression of defined transcription factors alone. Although changes to cell fate can be achieved in this way, the efficiency of such conversion remains very low, in large part due to specific chromatin signatures constituting an epigenetic barrier to the transcription factor-mediated reprogramming processes. Here we discuss the two-way relationship between transcription factor binding and chromatin structure during cell fate reprogramming. We additionally explore the potential roles and mechanisms by which histone variants, chromatin remodelling enzymes, and histone and DNA modifications contribute to the stability of cell identity and/or provide a permissive environment for cell fate change during cellular reprogramming. [ABSTRACT FROM AUTHOR]

Leaf trichome density in Mimulus guttatus can be altered by the parental environment. In this study, we compared global gene expression patterns in progeny of damaged and control plants. Significant differences in gene expression probably explain the observed trichome response, and identify additional responsive pathways., Using whole transcriptome RNA sequencing, we estimated differential gene expression between isogenic seedlings whose parents had, or had not, been subject to leaf damage., We identified over 900 genes that were differentially expressed in response to parental wounding. These genes clustered into groups involved in cell wall and cell membrane development, stress response pathways, and secondary metabolism., Gene expression is modified as a consequence of the parental environment in a targeted way that probably alters multiple developmental pathways, and may increase progeny fitness if they experience environments similar to that of their parents. [ABSTRACT FROM AUTHOR]

Generating of induced pluripotent stem cells (iPSCs) can be achieved by ectopic expression of defined transcription factor sets. However, most instances of iPSC induction have been achieved using viral vectors, which carry the risk of unpredictable genetic dysfunction. Here, for the first time, a non-viral vector based on calcium phosphate nanoparticles for the generation of virus-free iPSCs from human umbilical cord mesenchymal stem cells (HUMSCs) via co-delivery of the four plasmids (Oct4, Sox2, Klf4, and c-Myc) is reported. As a result, a total of 98 colonies from 200 000 cells have been obtained, with a reprogramming efficiency of 0.049%. The iPSCs shows positive expression of pluripotency markers, including OCT4, SSEA-3, SSEA-4, NANOG, and TRA-1-81. Moreover, the iPSCs are able to differentiate into all three germ layers in vitro. Subcutaneous injection of the iPSCs into immunocompromised mice results in the formation of teratomas containing a variety of tissues from all three germ layers. These findings indicate that co-delivery of the four Yamanaka factors via plasmid-encapsulated calcium phosphate nanoparticles can provide a simple, safe, and efficient method for the generation of virus-free iPSCs, which is crucial for their future clinical applications in the field of regenerative medicine. [ABSTRACT FROM AUTHOR]

It is becoming increasingly clear that the functionalities of an organism are mostly derived from regulation of its gene repertoire. Specialized cell types are created from pluripotent stem cells by regulating expression of genes. In eukaryotes, genes are primarily regulated by gene regulatory networks consisting of highly sequence-specific transcription factors and epigenetic modifications. The former mode of regulation is more readily reversible and non-heritable across cell generations, whereas the latter mode is less reversible and heritable. In this article, we explore the relationship between cell differentiation and the two modes of regulation of gene expression, focusing primarily on pluripotent and multipotent stem cells. Recent studies suggest that stem cells execute different gene expression programs, probably driven by one or more gene regulatory network(s). It is now also evident that as stem cells differentiate to more specialized progeny cells, rewriting of epigenetic marks occurs in parallel with the change in the pattern of gene expression. A conceptual framework is put forward in which it is proposed that the cell fate determining gene regulatory network in a pluripotent or multipotent cell has the capability to exist in multiple stationary states with each stationary state dictating a particular pattern of gene expression. We also propose that the broad pattern of gene expression in each stationary state, termed the lineage biased state or LIBS, resembles that of a more differentiated progeny cell. The differentiation process leading to a particular progeny cell involves rewriting of epigenetic marks that result in upregulation of genes in a LIBS and silencing of genes involved in alternative LIBS; thus selecting a particular pattern of gene expression and making a lineage commitment. © 2014 IUBMB Life, 66(2):100-109, 2014 [ABSTRACT FROM AUTHOR]

Sucrose synthase (Sus, EC 2.4.1.13) is widely recognized as a key enzyme in sucrose metabolism in plants. However, nothing is known about this gene family in Hevea brasiliensis (para rubber tree). Here, we identified six Sus genes in H. brasiliensis that comprise the entire Sus family in this species. Analysis of the gene structure and phylogeny of the Sus genes demonstrates evolutionary conservation in the Sus families across Hevea and other plant species. The expression of Sus genes was investigated via Solexa sequencing and quantitative PCR in various tissues, at various phases of leaf development, and under abiotic stresses and ethylene treatment. The Sus genes exhibited distinct but partially redundant expression profiles. Each tissue has one abundant Sus isoform, with HbSus3, 4 and 5 being the predominant isoforms in latex (cytoplasm of rubber-producing laticifers), bark and root, respectively. HbSus1 and 6 were barely expressed in any tissue examined. In mature leaves (source), all HbSus genes were expressed at low levels, but HbSus3 and 4 were abundantly expressed in immature leaves (sink). Low temperature and drought treatments conspicuously induced HbSus5 expression in root and leaf, suggesting a role in stress responses. HbSus2 and 3 transcripts were decreased by ethylene treatment, consistent with the reduced sucrose-synthesizing activity of Sus enzymes in the latex in response to ethylene stimulation. Our results are beneficial to further determination of functions for the Sus genes in Hevea trees, especially roles in regulating latex regeneration. [ABSTRACT FROM AUTHOR]

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer. J. Cell. Physiol. 228: 1174-1188, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Summary Seed dormancy is an important agronomic trait in wheat ( Trticum aestivum). Seeds can be released from a physiologically dormant state by after-ripening. To understand the molecular mechanisms underlying the role of after-ripening in conferring developmental switches from dormancy to germination in wheat seeds, we performed comparative transcriptomic analyses between dormant (D) and after-ripened (AR) seeds in both dry and imbibed states. Transcriptional activation of genes represented by a core of 22 and 435 probesets was evident in the dry and imbibed states of D seeds, respectively. Furthermore, two-way ANOVA analysis identified 36 probesets as specifically regulated by dormancy. These data suggest that biological functions associated with these genes are involved in the maintenance of seed dormancy. Expression of genes encoding protein synthesis/activity inhibitors was significantly repressed during after-ripening, leading to dormancy decay. Imbibing AR seeds led to transcriptional activation of distinct biological processes, including those related to DNA replication, nitrogen metabolism, cytoplasmic membrane-bound vesicle, jasmonate biosynthesis and cell wall modification. These after-ripening-mediated transcriptional programs appear to be regulated by epigenetic mechanisms. Clustering of our microarray data produced 16 gene clusters; dormancy-specific probesets and abscisic acid (ABA)-responsive elements were significantly overrepresented in two clusters, indicating the linkage of dormancy in wheat with that of seed sensitivity to ABA. The role of ABA signalling in regulating wheat seed dormancy was further supported by the down-regulation of ABA response-related probesets in AR seeds and absence of differential expression of ABA metabolic genes between D and AR seeds. [ABSTRACT FROM AUTHOR]