Transcriptome wide analysis of MADS box genes in Crocus sativus and interplay of CstMADS19-CstMADS26 in orchestrating apocarotenoid biosynthesis.

Flowers of Crocus sativus L. are immensely important not only for arrangement of floral whorls but more because each floral organ is dominated by a different class of specialized compounds. Dried stigmas of C. sativus flowers form commercial saffron, and are known to accumulate unique apocarotenoids like crocin, picrocrocin and safranal. Inspite of being a high value crop, the molecular mechanism regulating flower development in Crocus remains largely unknown. Moreover, it would be very interesting to explore any co-regulatory mechanism which controls floral architecture and secondary metabolic pathways which exist in specific floral organs. Here we report transcriptome wide identification of MADS box genes in Crocus. A total of 39 full length MADS box genes were identified among which three belonged to type I and 36 to type II class. Phylogeny classified them into 11 sub-clusters. Expression pattern revealed some stigma up-regulated genes among which CstMADS19 encoding an AGAMOUS gene showed high expression. Transient over-expression of CstMADS19 in stigmas of Crocus resulted in increased crocin by enhancing expression of pathway genes. Yeast one hybrid assay demonstrated that CstMADS19 binds to promoters of phytoene synthase and carotenoid cleavage dioxygenase 2 genes. Yeast two hybrid and BiFC assays confirmed interaction of CstMADS19 with CstMADS26 which codes for a SEPALATA gene. Co-overexpression of CstMADS19 and CstMADS26 in Crocus stigmas enhanced crocin content more than was observed when genes were expressed individually. Collectively, these findings indicate that CstMADS19 functions as a positive regulator of stigma based apocarotenoid biosynthesis in Crocus.

Development and Validation of a Proximity Labeling Fusion Protein Construct to Identify the Protein-Protein Interactions of Transcription Factors.

Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.

Prediction of Enhancer-Gene Interactions Using Chromatin-Conformation Capture and Epigenome Data Using STARE.

Disentangling the relationship of enhancers and genes is an ongoing challenge in epigenomics. We present STARE, our software to quantify the strength of enhancer-gene interactions based on enhancer activity and chromatin contact data. It implements the generalized Activity-by-Contact (gABC) score, which allows predicting putative target genes of candidate enhancers over any desired genomic distance. The only requirement for its application is a measurement of enhancer activity. In addition to regulatory interactions, STARE calculates transcription factor (TF) affinities on gene level. We illustrate its usage on a public single-cell data set of the human heart by predicting regulatory interactions on cell type level, by giving examples on how to integrate them with other data modalities, and by constructing TF affinity matrices.

Image Analysis Protocol for DNA/RNA/Immunofluorescence (IF)-seqFISH Data.

Imaging-based spatial multi-omics technologies facilitate the analysis of higher-order genomic structures, gene transcription, and the localization of proteins and posttranslational modifications (PTMs) at the single-allele level, thereby enabling detailed observations of biological phenomena, including transcription machinery within cells and tissues. This chapter details the principles of such technologies, with a focus on DNA/RNA/immunofluorescence (IF) sequential fluorescence in situ hybridization (seqFISH). A comprehensive step-by-step protocol for image analysis is provided, covering image preprocessing, spot detection, and data visualization. For practical application, complete Jupyter Notebook codes are made available on GitHub ( https://github.com/Ochiai-Lab/seqFISH_analysis ).

Engineering artificial cross-species promoters with different transcriptional strengths.

As a fundamental tool in synthetic biology, promoters are pivotal in regulating gene expression, enabling precise genetic control and spurring innovation across diverse biotechnological applications. However, most advances in engineered genetic systems rely on host-specific regulation of the genetic portion. With the burgeoning diversity of synthetic biology chassis cells, there emerges a pressing necessity to broaden the universal promoter toolkit spectrum, ensuring adaptability across various microbial chassis cells for enhanced applicability and customization in the evolving landscape of synthetic biology. In this study, we analyzed and validated the primary structures of natural endogenous promoters from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris, and through strategic integration and rational modification of promoter motifs, we developed a series of cross-species promoters (Psh) with transcriptional activity in five strains (prokaryotic and eukaryotic). This series of cross species promoters can significantly expand the synthetic biology promoter toolkit while providing a foundation and inspiration for standardized development of universal components The combinatorial use of key elements from prokaryotic and eukaryotic promoters presented in this study represents a novel strategy that may offer new insights and methods for future advancements in promoter engineering.

A genus-specific nsp12 region impacts polymerase assembly in Alpha- and Gammacoronaviruses.

Coronavirus relevancy for human health has surged over the past 20 years as they have a propensity for spillover into humans from animal reservoirs resulting in pandemics such as COVID-19. The diversity within the Coronavirinae subfamily and high infection frequency in animal species worldwide creates a looming threat that calls for research across all genera within the Coronavirinae subfamily. We sought to contribute to the limited structural knowledge within the Gammacoronavirus genera and determined the structure of the viral core replication-transcription complex (RTC) from Infectious Bronchitis Virus (IBV) using single-particle cryo-EM. Comparison between our IBV structure with published RTC structures from other Coronavirinae genera reveals structural differences across genera. Using in vitro biochemical assays, we characterized these differences and revealed their differing involvement in core RTC formation across different genera. Our findings highlight the value of cross-genera Coronavirinae studies, as they show genera specific features in coronavirus genome replication. A broader knowledge of coronavirus replication will better prepare us for future coronavirus spillovers.

Pathway Gene Paralogues and Transcription Factors Differentially Associate with Contents of Picrosides in Tissues and Populations of a Medicinal Herb, Picrorhiza kurroa.

Picrorhiza kurroa is a valuable medicinal herb of Himalayan region, containing two major pharmacological iridoid glycosides: Picroside-I and Picroside-II, in addition to several other secondary metabolites. The metabolic diversity of P. kurroa may stem from the evolutionary processes attributed to pathway genes family expansion via gene duplication or splicing giving rise to paralogues which are further controlled by regulatory components. Occurrence of multiple pathway gene paralogues coupled with which TFs associate with paralogues in different genetic backgrounds (populations) in tissue-specific manner are still unresolved. Here, we unravelled possible correlations between TFs and gene paralogues across a range of P. kurroa accessions which might be contributing to differential contents of Picroside-I and Picroside-II in different tissues/organs. Characterization of shoots, roots, and stolons of eighty-five accessions of P. kurroa revealed significant variations for Picroside-I and Picroside-II contents. Comparative transcriptome analysis of shoot-derived transcriptome (PKSS), and root-derived transcriptome (PKSR) followed by their expression analysis in different P. kurroa accessions revealed TFs; PkWRKY71, PkWRKY12, PkNAC25, and PkMyb46 possibly regulate different gene paralogues. Genes encoding these putative TFs and pathway gene paralogues were further used to generate a robust co-expression network, thereby, uncovering their coordinated behaviour in association with Picroside-I and Picroside-II contents in shoots and roots, respectively. The outcome has provided potential leads, which through further functional validation can provide suitable targets, either for pathway engineering or as gene markers for selection of genetically superior populations of P. kurroa.

[Identification and expression analysis of members of R2R3-MYB transcription factors family in Artemisia argyi].

The MYB(v-myb avian myeloblastosis viral oncogene homolog) family of transcription factors is the largest class of genes among higher plant transcription factors, which can be divided into four subfamilies, with the R2R3-MYB being the most common subfamily type. R2R3-MYB transcription factors are widely involved in the regulation of organ development and secondary metabolite biosynthesis in plants. To investigate the role of R2R3-MYB family transcription factors in the synthesis of flavonoids and glandular trichome development in Artemisia argyi, this study screened and identified 92 R2R3-MYB transcription factors based on the whole genome data of A. argyi, and predicted their potential functions based on bioinformatics. The results showed that the amino acid lengths of the 92 transcription factors ranged from 168 to 547 aa, with relative molecular weights ranging from 19. 6 to 60. 5 kDa, all of which were hydrophilic proteins. Subcellular localization analysis showed that 89 AaMYB proteins were located in the nucleus, while three proteins were simultaneously located in the nucleus and cytoplasm. According to the classification of Arabidopsis R2R3-MYB family, the 92 A. argyi R2R3-MYB proteins were divided into 26 subfamilies, with similar gene structures within the same subfamily.Cis-acting element prediction results showed that light-responsive elements, methyl jasmonate elements, and abscisic acid elements were widely distributed in the promoter regions of R2R3-MYB genes. Transcriptome expression analysis results showed that the expression of AaMYB60, AaMYB63, and AaMYB86 in leaves was higher than that in stems and roots, indicating that these three transcription factors mainly function in leaves. Additionally, five candidate R2R3-MYB transcription factors involved in A. argyi flavonoid biosynthesis or glandular trichome development were selected through phylogenetic analysis. This study provides important genetic resources for the breeding of superior varieties and germplasm innovation of A. argyi in the future.

Astragaloside IV ameliorates pressure overload-induced heart failure by enhancing angiogenesis through HSF1/VEGF pathway.

Astragaloside IV(AS-IV), the main active ingredient of Astragalus, has been used as a treatment for heart failure with favorable effects, but its molecular mechanism has not been fully elucidated. Network pharmacological analysis and molecular docking revealed that Heat shock transcription factor 1 (HSF1) is a potential target of AS-IV. We designed cellular and animal experiments to investigate the role and intrinsic molecular mechanisms of AS-IV in ameliorating pressure overload-induced heart failure. In cellular experiments, Myocardial microvascular endothelial cells (MMVECs) were cultured in isolation and stimulated by adding high and low concentrations of AS-IV, and a cell model with down-regulation of HSF1 expression was constructed by using siRNA technology. Changes in the expression of key molecules of HSF1/VEGF signaling pathway and differences in tube-forming ability were detected in different groups of cells using PCR, WB and tube-forming assay. In animal experiments, TAC technology was applied to establish a pressure overload-induced heart failure model in C57 mice, postoperative mice were ingested AS-IV by gavage, and adenoviral transfection technology was applied to construct a mouse model with down-regulation of HSF1 expression.Small animal ultrasound for cardiac function assessment, MASSON staining, CD31 immunohistochemistry, and Western blotting (WB) were performed on the mice. The results showed that AS-IV could promote the expression of key molecules of HSF1/VEGF signaling pathway, enhance the tube-forming ability of MMVECs, increase the density of myocardial capillaries, reduce myocardial fibrosis, and improve the cardiac function of mice with TAC.AS-IV could modulate the HSF1/VEGF signaling pathway to promote the angiogenesis and improve the pressure overload-induced heart failure.

First reported case of primary small cell cancer of the pancreas with positive TTF-1 tumor marker.

Small cell carcinomas are very aggressive malignancies that are most often linked with lung cancer, although they may also develop in the pancreas, colon, rectum, skin, and cervix. SCC of the pancreas accounts for about 1% of these neoplasms. An 88-year-old male with several comorbidities who presented with significant weight loss was diagnosed with metastatic pancreatic neuroendocrine carcinoma after complaining of persistent epistaxis and back pain. This case underscores the significance of using atypical tumor markers, such as thyroid transcription factor 1, to diagnose small-cell pancreatic cancer. It also emphasizes the importance of a multidisciplinary, patient-centered approach to managing these aggressive tumors.

Fine genetic mapping and transcriptomic analysis revealed major gene modulating the clear stripe margin pattern of watermelon peel.

The peel stripe margin pattern is one of the most important quality traits of watermelon. In this study, two contrasted watermelon lines [slb line (P1) with a clear peel stripe margin pattern and GWAS-38 line (P2) with a blurred peel stripe margin pattern] were crossed, and biparental F2 mapping populations were developed. Genetic segregation analysis revealed that a single recessive gene is modulating the main-effect genetic locus (Clcsm) of the clear stripe margin pattern of peel. Bulked segregant analysis-based sequencing (BSA-Seq) and fine genetic mapping exposed the delimited Clcsm locus to a 19.686-kb interval on chromosome 6, and the Cla97C06G126680 gene encoding the MYB transcription factor family was identified. The gene mutation analysis showed that two non-synonymous single-nucleotide polymorphism (nsSNP) sites [Chr6:28438793 (A-T) and Chr6:28438845 (A-C)] contribute to the clear peel stripe margin pattern, and quantitative real-time polymerase chain reaction (qRT-PCR) also showed a higher expression trend in the slb line than in the GWAS-38 line. Further, comparative transcriptomic analysis identified major differentially expressed genes (DEGs) in three developmental periods [4, 12, and 20 days after pollination (DAP)] of both parental lines. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses indicated highly enriched DEGs involved in metabolic processes and catalytic activity. A total of 44 transcription factor families and candidate genes belonging to the ARR-B transcription factor family are believed to regulate the clear stripe margin trait of watermelon peel. The gene structure, sequence polymorphism, and expression trends depicted significant differences in the peel stripe margin pattern of both parental lines. The ClMYB36 gene showed a higher expression trend for regulating the clear peel stripe margin of the slb line, and the ClAPRR5 gene depicted a higher expression for modulating the blurred peel stripe margin in the GWAS-38 line. Overall, our fine genetic mapping and transcriptomic analysis revealed candidate genes differentiating the clear and blurred peel stripe patterns of watermelon fruit.

Context-Dependent and Locus-Specific Role of H3K36 Methylation in Transcriptional Regulation.

H3K36 methylation is a critical histone modification involved in transcription regulation. It involves the mono (H3K36me1), di (H3K36me2), and/or tri-methylation (H3K36me3) of lysine 36 on histone H3 by methyltransferases. In yeast, Set2 catalyzes all three methylation states. By contrast, in higher eukaryotes, at least eight methyltransferases catalyze different methylation states, including SETD2 for H3K36me3 and the NSD family for H3K36me2 in vivo. Both Set2 and SETD2 interact with the phosphorylated CTD of RNA Pol II, which links H3K36 methylation to transcription. In yeast, H3K36me3 and H3K36me2 peak at the 3' ends of genes. In higher eukaryotes, this is also true for H3K36me3 but not for H3K36me2, which is enriched at the 5' ends of genes and intergenic regions, suggesting that H3K36me2 and H3K36me3 may play different regulatory roles. Whether H3K36me1 demonstrates preferential distribution remains unclear. H3K36me3 is essential for inhibiting transcription elongation. It also suppresses cryptic transcription by promoting histone deacetylation by the histone deacetylases Rpd3S (yeast) and variant NuRD (higher eukaryotes). H3K36me3 also facilitates DNA methylation by DNMT3B, thereby preventing spurious transcription initiation. H3K36me3 not only represses transcription since it promotes the activation of mRNA and cryptic promoters in response to environmental changes by targeting the histone acetyltransferase NuA3 in yeast. Further research is needed to elucidate the methylation state- and locus-specific functions of H3K36me1 and the mechanisms that regulate it.

IGFBP2 Functions as an Endogenous Protector against Hepatic Steatosis via Suppression of the EGFR-STAT3 Pathway.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is deemed as an emerging global epidemic, whereas the underlying pathogenic mechanism remains to be clarified. We aimed to systemically analyze all the NAFLD-related gene expression datasets from published human-based studies, by which exploring potential key factors and mechanisms accounting for the pathogenesis of NAFLD. APPROACH & RESULTS: By using Robust rank aggregation method to integrate all public datasets of human NAFLD transcriptome, the present study identified IGFBP2 (Insulin-like growth factor binding protein 2) being the most significantly down-regulated gene in all NAFLD subjects. The decreased IGFBP2 expression was further confirmed in the liver tissues from patients and animal models of NAFLD. IGFBP2 deficiency aggravated hepatic steatosis and NASH phenotypes and promoted lipogenic gene expression both in vivo and in vitro. Mechanistically, IGFBP2 directly binds to and regulates EGFR, whereas blockage of the IGFBP2-EGFR complex by knockdown of IGFBP2 resulted in the EGFR-STAT3 pathway activation, which in turn promoted the promoter activity of Srebf1. By using molecular docking simulation and protein-protein interaction analysis, the sequence of 233-257 amino acids in IGFBP2 was characterized as a key motif responding for its specific binding to EGFR and the protective effect against hepatic steatosis. CONCLUSIONS: The current study has, for the first time, identified IGFBP2 as a novel protector against hepatosteatosis. The protective effect is mediated by its specific interaction with EGFR and thereby suppressing the EGFR-STAT3 pathway. Therefore, pharmaceutically targeting the IGFBP2-EGFR-STAT3 axis may provide a theoretical basis for for the treatment of NAFLD/NASH and the associated diseases.

Astrocyte allocation during brain development is controlled by Tcf4-mediated fate restriction.

Astrocytes in the brain exhibit regional heterogeneity contributing to regional circuits involved in higher-order brain functions, yet the mechanisms controlling their distribution remain unclear. Here, we show that the precise allocation of astrocytes to specific brain regions during development is achieved through transcription factor 4 (Tcf4)-mediated fate restriction based on their embryonic origin. Loss of Tcf4 in ventral telencephalic neural progenitor cells alters the fate of oligodendrocyte precursor cells to transient intermediate astrocyte precursor cells, resulting in mislocalized astrocytes in the dorsal neocortex. These ectopic astrocytes engage with neocortical neurons and acquire features reminiscent of dorsal neocortical astrocytes. Furthermore, Tcf4 functions as a suppressor of astrocyte fate during the differentiation of oligodendrocyte precursor cells derived from the ventral telencephalon, thereby restricting the fate to the oligodendrocyte lineage in the dorsal neocortex. Together, our findings highlight a previously unappreciated role for Tcf4 in regulating astrocyte allocation, offering additional insights into the mechanisms underlying neurodevelopmental disorders linked to Tcf4 mutations.

The role of transcriptional and epigenetic modifications in astrogliogenesis.

Astrocytes are widely distributed and play a critical role in the central nervous system (CNS) of the human brain. During the development of CNS, astrocytes provide essential nutritional and supportive functions for neural cells and are involved in their metabolism and pathological processes. Despite the numerous studies that have reported on the regulation of astrogliogenesis at the transcriptional and epigenetic levels, there is a paucity of literature that provides a comprehensive summary of the key factors influencing this process. In this review, we analyzed the impact of transcription factors (e.g., NFI, JAK/STAT, BMP, and Ngn2), DNA methylation, histone acetylation, and noncoding RNA on astrocyte behavior and the regulation of astrogliogenesis, hope it enhances our comprehension of the mechanisms underlying astrogliogenesis and offers a theoretical foundation for the treatment of patients with neurological diseases.

Genome-wide identification of nuclear factor -Y (NF-Y) transcription factor family in finger millet reveals structural and functional diversity.

The Nuclear Factor Y (NF-Y) is one of the widely explored transcription factors (TFs) family for its potential role in regulating molecular mechanisms related to stress response and developmental processes. Finger millet (Eleusine coracana (L.) Gaertn) is a hardy and stress-tolerant crop where partial efforts have been made to characterize a few transcription factors. However, the NF-Y TF is still poorly explored and not well documented. The present study aims to identify and characterize NF-Y genes of finger millet using a bioinformatics approach. Genome mining revealed 57 EcNF-Y (Eleusine coracana Nuclear Factor-Y) genes in finger millet, comprising 18 NF-YA, 23 NF-YB, and 16 NF-YC genes. The gene organization, conserved motif, cis-regulatory elements, miRNA target sites, and three-dimensional structures of these NF-Ys were analyzed. The nucleotide substitution rate and gene duplication analysis showed the presence of 7 EcNF-YA, 10 EcNF-YB, and 8 EcNF-YC paralogous genes and revealed the possibilities of synonymous substitution and stabilizing selection during evolution. The role of NF-Ys of finger millet in abiotic stress tolerance was evident by the presence of relevant cis-elements such as ABRE (abscisic acid-responsive elements), DRE (dehydration-responsive element), MYB (myeloblastosis) or MYC (myelocytomatosis). Twenty-three isoforms of miR169, mainly targeting a single NF-Y gene, i.e., the EcNF-YA13 gene, were observed. This interaction could be targeted for finger millet improvement against Magnaporthe oryzae (blast fungus). Therefore, by this study, the putative functions related to biotic and abiotic stress tolerance for many of the EcNF-Y genes could be explored in finger millet.

Biochemical characterization of Mycobacterial RNA polymerases.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, Mycobacterium smegmatis (Msm), perform the essential function of RNA synthesis, we performed a series of in vitro transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme.IMPORTANCETuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie, et al). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.

Translation of HIV-1 unspliced RNA is regulated by 5' untranslated region structure.

HIV-1 must generate infectious virions to spread to new hosts and HIV-1 unspliced RNA (HIV-1 RNA) plays two central roles in this process. HIV-1 RNA serves as an mRNA that is translated to generate proteins essential for particle production and replication, and it is packaged into particles as the viral genome. HIV-1 uses several transcription start sites to generate multiple RNAs that differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. The virus relies on host machinery to translate its RNAs in a cap-dependent manner. Here, we demonstrate that the 5' context of HIV-1 RNA affects the efficiency of translation both in vitro and in cells. Although both RNAs are competent for translation, 3G RNA is translated more efficiently than 1G RNA. The 5' untranslated region (UTR) of 1G and 3G RNAs has previously been shown to fold into distinct structural ensembles. We show that HIV-1 mutants in which the 5' UTR of 1G and 3G RNAs fold into similar structures were translated at similar efficiencies. Thus, the host machinery translates two 99.9% identical HIV-1 RNAs with different efficiencies, and the translation efficiency is regulated by the 5' UTR structure.IMPORTANCEHIV-1 unspliced RNA contains all the viral genetic information and encodes virion structural proteins and enzymes. Thus, the unspliced RNA serves distinct roles as viral genome and translation template, both critical for viral replication. HIV-1 generates two major unspliced RNAs with a 2-nt difference at the 5' end (3G RNA and 1G RNA). The 1G transcript is known to be preferentially packaged over the 3G transcript. Here, we showed that 3G RNA is favorably translated over 1G RNA based on its 5' untranslated region (UTR) RNA structure. In HIV-1 mutants in which the two major transcripts have similar 5' UTR structures, 1G and 3G RNAs are translated similarly. Therefore, HIV-1 generates two 9-kb RNAs with a 2-nt difference, each serving a distinct role dictated by differential 5' UTR structures.

Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7.

Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses.IMPORTANCECells rapidly induce changes in the transcription of RNA in response to stress and pathogens. Herpes simplex virus (HSV) disrupts many processes of host mRNA transcription, and it is necessary to separate the actions of viral proteins from cellular responses. Here, we demonstrate that viral proteins inhibit two key phosphorylation patterns on the C-terminal domain (CTD) of cellular RNA polymerase II and that this is separate from the degradation of polymerases later in infection. Furthermore, we show that viral genes do not require the full "CTD code." Together, these data distinguish multiple steps in the remodeling of RNA polymerase during infection and suggest that shared transcriptional phenotypes during stress responses do not revolve around a core disruption of CTD modifications.

Pleiotropically activation of azaphilone biosynthesis by overexpressing a pathway-specific transcription factor in marine-derived Aspergillus terreus RA2905.

The genome sequencing of Aspergillus terreus reveals that the vast number of predicted biosynthetic gene clusters have not reflected by the metabolic profile observed under conventional culture conditions. In this study, a silent azaphilone biosynthetic gene cluster was activated by overexpressing a pathway-specific transcription factor gene2642 in marine-derived fungus A. terreus RA2905. Consequently, twenty azaphilone compounds were identified from the OE2642 mutant, including 11 new azaphilones and their precursors, azasperones C-J (1-5, 7-9) and preazasperones A-C (15-17). The structures of those new compounds were unambiguously determined on the basis of NMR and HRESIMS spectra analysis, and the absolute configurations were established depending on ECD calculations. Compounds 1 and 2 were the rarely reported naturally occurring azaphilones with 2-N coupled phenyl-derivative. The bioactivity assay revealed that compounds 18-20 exhibited significant anti-inflammatory activity. Based on the occurrence of diverse intermediates and the putative gene functions, a plausible biosynthetic pathway of these compounds was proposed. The above results demonstrated that overexpression of the pathway-specific transcription factor presents a promising approach for enriching fungal secondary metabolites and accelerating the targeted discovery of novel biosynthetic products.