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DNA supercoiling, the level of under- or overwinding of the DNA polymer around itself, is widely recognized as an ancestral regulation mechanism of gene expression in bacteria. Higher levels of negative supercoiling facilitate the opening of the DNA double helix at gene promoters and thereby increase gene transcription rates. Different levels of supercoiling have been measured in bacteria exposed to different environments, leading to the hypothesis that variations in supercoiling could be a response to changes in the environment. Moreover, DNA transcription has been shown to generate local variations in the supercoiling level and, therefore, to impact the transcription rate of neighboring genes. In this work, we study the coupled dynamics of DNA supercoiling and transcription at the genome scale. We implement a genome-wide model of gene expression based on the transcription-supercoiling coupling. We show that, in this model, a simple change in global DNA supercoiling is sufficient to trigger differentiated responses in gene expression levels via the transcription-supercoiling coupling. Then, studying our model in the light of evolution, we demonstrate that this non-linear response to different environments, mediated by the transcription-supercoiling coupling, can serve as the basis for the evolution of specialized phenotypes.

Circular RNAs (circRNAs) are a class of single-stranded closed non-coding RNA molecules (ncRNAs), which are formed as a result of reverse splicing of mRNAs. Despite their relative abundance, an interest in understanding their regulatory importance is rather recent. High stability, abundance and evolutionary conservation among species underline some of their important traits. CircRNAs perform a variety of cellular functions ranging from miRNA and proteins sponges to transcriptional modulation and splicing. Additionally, most circRNAs are expressed aberrantly in pathological conditions suggesting their possible exploitation as diagnostic biomarkers. Their covalent closed cyclic structure resulting in resistance to RNases further makes them suitable as cancer biomarkers. Studies involving human tumors have verified differences in the expression profiles of circRNAs, indicating a regulatory role in cancer pathogenesis and metastasis. As endogenous competitive RNA, circRNAs can regulate tumor proliferation and invasion. Further, some circRNAs located in the nucleus can regulate transcription of genes by binding to RNA polymerase II. In this review, we elaborate the characteristics, functions and mechanisms of action of circRNAs in cancer. We also discuss the possibility of using circRNAs as potential therapeutic targets and biomarkers for cancer.

The underlying mechanisms of E2F5 upregulation and its pro-tumor functions have not been elucidated in gastric cancer (GC). Here, the expression, prognostic value, mutation status, and promoter methylation of E2F5 were evaluated. The effects of E2F5 depletion on cell proliferation and invasion in GC, were also assessed through in vitro experiments. Additionally, gene set enrichment analysis (GSEA) was applied to analyze the potential downstream regulator of E2F5. The study also assessed the correlation and transcription regulation between E2F5 and UBE2T. Finally, the roles of UBE2T in E2F5-related pro-tumor functions were examined. The findings revealed that E2F5 was upregulated and showed remarkable association with pathological variables and prognosis. Hypomethylation of the E2F5 promoter predicted poor prognosis and partially caused E2F5 upregulation in GC. E2F5 knockdown significantly inhibited the proliferation and invasion of GC cells. E2F5 had a significant positive correlation with UBE2T in GC. Mechanistically, E2F5 promoted UBE2T transcription and UBE2T overexpression reversed the effects of E2F5 depletion on the proliferation and invasion of cells in GC. Taken together, this study originally confirmed the upregulation of E2F5 in GC, revealed that E2F5 can directly upregulate UBE2T transcription, and subsequently promote the malignant progression, which highlights that the E2F5/UBE2T axis can potentially be used in the diagnosis and treatment of GC.

Promoters and enhancers are sites of transcription initiation (TSSs) and carry active histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval from the TSS to the first upstream Alu accommodates essentially all H3K4me3 marks, while excluding DNA methylation. In contrast, enhancer-enriched H3K4me1 and H3K27ac marks eventually cross the first-Alu limit, consistent with enhancer-annotation occasionally overlapping with Alu elements. Remarkably, the average length of TSS-to-first Alu intervals greatly varies in-between tissues, being longer in stem- and shorter in immune-cells. Shortest TSS-to-Alu intervals were observed at promoters active in T cells, particularly at immune genes, correlating with serendipitous RNA polymerase II transcription and accumulation of H3K4me1 signal at the first-Alu. At several T-cell first-Alus, the DNA methylation was further found to evolved with age, regressing from young to middle-aged, then recovering later in life. Thus, the first-Alu upstream of TSSs functions as a dynamic boundary for regulatory elements, initiating the upstream DNA-methylation landscape, while also participating in the recording of immune gene transcriptional events.

Bacterial mRNAs have short life cycles, in which transcription is rapidly followed by translation and degradation within seconds to minutes. The resulting diversity of mRNA molecules across different life-cycle stages impacts their functionality but has remained unresolved. Here we quantitatively map the 3’ status of cellular RNAs in Escherichia coli during steady-state growth and report a large fraction of molecules (median>60%) that are fragments of canonical full-length mRNAs. The majority of RNA fragments are decay intermediates, whereas nascent RNAs contribute to a smaller fraction. Despite the prevalence of decay intermediates in total cellular RNA, these intermediates are underrepresented in the pool of ribosome-associated transcripts and can thus distort quantifications and differential expression analyses for the abundance of full-length, functional mRNAs. The large heterogeneity within mRNA molecules in vivo highlights the importance in discerning functional transcripts and provides a lens for studying the dynamic life cycle of mRNAs.

Cells must reliably respond to changes in transcription factor levels in order to execute cell state transitions in the correct time and place. These transitions are typically thought to be triggered by changes in the absolute nuclear concentrations of relevant transcription factors. We have identified a developmental context in which cell fate transitions depend on changes in the relative concentrations of two transcription factors. Here, we quantify the in vivo expression dynamics of Yan and Pointed, two essential E-twenty-six (ETS) proteins that regulate transcription during eye development in Drosophila. These two factors exert opposing influences; one impedes transcription of gene targets required for differentiation while the other promotes it. We show that both proteins are transiently co-expressed in eye progenitor cells and also during photoreceptor specification. To decide whether to undergo state transitions, cells respond to the ratio of the two protein concentrations rather than changes in the absolute abundance of either transcription factor. We show that a simple model based on the statistical physics of protein-DNA binding illustrates how this ratiometric sensing of transcription factor concentrations could occur. Gene dosage experiments reveal that progenitor cells stabilize the ratio against fluctuations in the absolute concentration of either protein. We further show that signaling inputs via the Notch and Receptor Tyrosine Kinase (RTK) pathways set the ratio in progenitor cells, priming them for either transit to differentiation or for continued multipotency. A sustained change in the ratio accompanies the transit to differentiation This novel mechanism allows for distributed control of developmental transitions by multiple transcription factors, making the system robust to fluctuating genetic or environmental conditions.

Cell identity is tightly controlled by specific transcriptional programs which require post-translational modifications of histones. These histone modifications allow the establishment and maintenance of active and repressed chromatin domains. Histone H2A lysine 119 ubiquitination (H2AK119ub1) has an essential role in building repressive chromatin domains during development. It is regulated by the counteracting activities of the Polycomb repressive complex 1 (PRC1) and the Polycomb repressive-deubiquitinase (PR-DUB) complexes, two multi-subunit ensembles that write and erase this modification, respectively. We have catalogued the recurrent genetic alterations in subunits of the PRC1 and PR-DUB complexes in both neurodevelopmental disorders and cancer. These genetic lesions are often shared across disorders, and we highlight common mechanisms of H2AK119ub1 dysregulation and how they affect development in multiple disease contexts.

Gibberellic acid (GA), a ubiquitous phytohormone, has various effects on regulators of plant growth and development. GAs promote growth by overcoming growth restraint mediated by DELLA proteins (DELLAs). DELLAs, in the GRAS family of plant-specific nuclear proteins, are nuclear transcriptional regulators harboring a unique N-terminal GA perception region for binding the GA receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1) and a C-terminal GRAS domain necessary for GA repression activity via interaction with multiple regulatory proteins. The N-terminal conserved region of DELLAs evolved to form a mode of GID1/DELLA-mediated GA signaling originating in bryophytes and ferns. Binding of GA to GID1 increases the affinity between DELLAs and a SCF E3 ubiquitin–ligase complex, thus promoting the eventual destruction of DELLAs by the 26S proteasome. DELLAs negatively regulate GA response by releasing transcription factors to directly activate downstream genes and indirectly regulate GA biosynthesis genes increasing GA responsiveness and feedback control by promoting GID1 transcription. GA communicates extensively with other plant hormones and uses crosstalk to regulate plant growth and development. In this review, we summarize current understanding of evolutionary DELLA-mediated gibberellin signaling and functional diversification of DELLA, focusing primarily on interactions of DELLAs with diverse phytohormones.

Kinases are pivotal regulators in tumorigenesis and metastasis by modulating the expression of oncogenes and the transcription of antioncogenes directly or indirectly. Correspondingly, multifarious 3-substituted indolin-2-one derivatives as selective kinase inhibitors for cancer therapy exhibited a low nanomolar activity with prominent efficacy, superior response rate and admirable tolerability. Particularly, certain 3-substituted indolin- 2-one derivatives have met the requirements for clinical trials or the pharmaceutical market. Herein, we focus on the traits of 3-substituted indolin-2-one derivatives as kinase inhibitors for cancer therapy, overview recent progress of 3-substituted indolin-2-one derivatives as kinase inhibitors for cancer therapy, analyze the selectivity for tyrosine kinases inhibitors and serine/threonine kinases inhibitors from the molecular aspects based on the molecular docking studies, summarize the structure-activity relationships (SARs) as selective kinase inhibitors and provide our perspectives for the development of 3- substituted indolin-2-one derivatives as kinase inhibitors for cancer therapy.

The protein interactome mediates crucial functions in transcription, chromatin remodeling, and higher-order structural organization. Elucidating the proteins that interact with chromatin-associated RNA or proteins is key to understanding fundamental epigenetic regulatory pathways. We discuss the opportunities and challenges of mass spectrometry (MS)-based proteomics for characterizing the plant chromatin interactome.

The B3 transcription factors (TFs) in plants play vital roles in numerous biological processes. Although B3 genes have been broadly identified in many plants, little is known about their potential functions in mediating seed development and material accumulation. Castor bean (Ricinus communis L.) is a non-edible oilseed crop considered an ideal model system for seed biology research. Here, we identified a total of 61 B3 genes in the castor bean genome, which can be classified into five subfamilies, including ABI3/VP1, HSI, ARF, RAV and REM. The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development, indicating that these genes may be associated with the accumulation of storage oils. Furthermore, through yeast one-hybrid and tobacco transient expression assays, we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2, which encodes an oil-body structural protein. This finding suggests that RcLEC2, as a seed-specific TF, may be involved in the regulation of storage materials accumulation. This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

SummaryGlnR activates nitrogen metabolism genes under nitrogen-limited conditions whereas MtrA represses these genes under nutrient-rich conditions in Streptomyces. In this study, we compared the transcription patterns of nitrogen metabolism genes in a double deletion mutant (ΔmtrA-glnR) lacking both mtrA and glnR and in mutants lacking either mtrA (ΔmtrA) or glnR (ΔglnR). The nitrogen metabolism genes were expressed similarly in ΔmtrA-glnR and ΔglnR under both nitrogen-limited and nutrient-rich conditions, with patterns distinctly different from that of ΔmtrA, suggesting a decisive role for GlnR in the control of nitrogen metabolism genes and further suggesting that regulation of these genes by MtrA is GlnR-dependent. MtrA and GlnR utilize the same binding sites upstream of nitrogen metabolism genes, and we showed stronger in vivo binding of MtrA to these sites under nutrient-rich conditions and of GlnR under nitrogen-limited conditions, consistent with the higher levels of MtrA or GlnR under those respective conditions. In addition, we showed that both mtrA and glnR are auto-regulatory. Our study provides new insights into the regulation of nitrogen metabolism genes in Streptomyces.

Background LINC00152 (long intergenic non-protein coding RNA 152) was identified as an oncogenic lncRNA in multiple cancers. In the current study, we aimed to explore the transcriptional profile of LINC00152 in oral squamous cell carcinoma (OSCC) and its regulations at the transcriptional level. Methods Bioinformatic analysis was performed by extracting the OSCC subset from The Cancer Genome Atlas (TCGA)-Head and Neck Squamous Cell Carcinoma (HNSC). LINC00152 subcellular localization and its interacting transcriptional factors (TFs) were explored. Dual-luciferase assay and ChIP-qPCR were applied to study transcriptional regulation. In vitro and in-vivo tumor cell growth models were used for functional assays. Results NR_024206.2 was the dominant isoform that accounts for 80% of all transcripts of LINC00152. LINC00152 upregulation was associated with unfavorable survival of patients with OSCC. LINC00152 knockdown significantly impaired OSCC cell growth in vitro and in vivo. RNA FISH assay confirmed nuclear and cytoplasmic distribution of LINC00152. It physically interacted with Upstream Transcription Factor 1 (USF1), a common transcription factor in mammalian cells. USF1 could bind to the promoter region of MRPL52 (Mitochondrial Ribosomal Protein L52) and activate its transcription. LINC00152 could enhance the binding, thereby indirectly elevating MRPL52 expression. USF1 or MRPL52 knockdown slowed the proliferation of OSCC cells and partly canceled LINC00152 mediated growth-promoting effects. Conclusion This study revealed a novel LINC00152-USF1/MRPL52 axis promoting OSCC tumor growth.

Background Palustrin-2CE2 and brevinin-2CE3 are antimicrobial peptides from Rana chensinensis. In R. chensinensis tadpoles, the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3 increased with the developmental stage. In addition, the expression of the two genes was dramatically upregulated with stimulation by Escherichia coli, Staphylococcus aureus, and the chemical lipopolysaccharide (LPS). The genomic organization of the two antimicrobial peptide genes was confirmed. Both prepropalustrin-2CE2 and preprobrevinin-2CE3 contain three exons separated by two large introns. Additionally, several presumed transcription factor binding sites were identified in the promoter sequence. Functional analysis of the promoter was performed using a luciferase reporter system, and further confirmed by yeast one-hybrid experiment and EMSA assay. The results indicated that the transcription factors NF-κB and RelA are involved in regulating the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3. As amphibian populations decline globally, this study provides new data demonstrating how frogs defend against pathogens from the environment by regulating AMP expression. For amphibians, antimicrobial peptides are innate immune molecules that resist adverse external environmental stimuli. However, the regulation mechanism of antimicrobial peptide gene expression in frogs is still unclear. Objective The two antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, are produced under external stimulation in Rana chensinensis. Using this model, we analyzed the gene structure and regulatory elements of the two antimicrobial peptide genes and explored the regulatory effects of related transcription factors on the two genes. Method Different stimuli such as E. coli, S. aureus, and chemical substance lipopolysaccharide (LPS) were applied to Rana chensinensis tadpoles at different developmental stages, and antimicrobial peptide expression levels were detected by RT-PCR. Bioinformatics analysis and 5'-RACE and genome walking technologies were employed to analyze the genome structure and promoter region of the antimicrobial peptide genes. With dual-luciferase reporter gene assays, yeast one-hybrid experiment and EMSA assays, we assessed the regulatory effect of the endogenous regulators of the cell on the antimicrobial peptide promoter. Results The transcription levels of prepropalustrin-2CE2 and preprobrevinin-2CE3 were significantly upregulated after different stimulations. Genomic structure analysis showed that both genes contained three exons and two introns. Promoter analysis indicated that there are binding sites for regulatory factors of the NF-κB family in the promoter region, and experiments showed that endogenous NF-κB family regulatory factors in frog cells activate the promoters of the antimicrobial peptide genes. Yeast one-hybrid experiment and EMSA assay demonstrated that RelA and NF-κB1 might interact with specific motifs in the prepropalustrin-2CE2 promoter. Conclusion In this paper, we found that the gene expression levels of the antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, in R. chensinensis will increase under environmental stimuli, and we verified that the changes in gene expression levels are affected by the transcription factors RelA and NF-κB1. The yeast one-hybrid experiment and EMSA assay confirmed that RelA and NF-κB1 could directly interact with the frog antimicrobial peptide gene promoter, providing new data for the regulatory mechanism of antimicrobial peptides in response to environmental stimuli.

Background Interferon regulatory factor 2 (IRF-2) acts as an anti-oncogene in gastric cancer (GC); however, the underlying mechanism remains unknown. Methods This study determined the expression of IRF-2 in GC tissues and adjacent non-tumor tissues using immunohistochemistry (IHC) and explored the predictive value of IRF-2 for the prognoses of GC patients. Cell function and xenograft tumor growth experiments in nude mice were performed to test tumor proliferation ability, both in vitro and in vivo. Chromatin immunoprecipitation sequencing (ChIP-Seq) assay was used to verify the direct target of IRF-2. Results We found that IRF-2 expression was downregulated in GC tissues and was negatively correlated with the prognoses of GC patients. IRF-2 negatively affected GC cell proliferation both in vitro and in vivo. ChIP-Seq assay showed that IRF-2 could directly activate AMER-1 transcription and regulate the Wnt/β-catenin signaling pathway, which was validated using IHC, in both tissue microarray and xenografted tumor tissues, western blot analysis, and cell function experiments. Conclusions Increased expression of IRF-2 can inhibit tumor growth and affect the prognoses of patients by directly regulating AMER-1 transcription in GC and inhibiting the Wnt/β-catenin signaling pathway.

Introduction: The outburst of the novel coronavirus COVID-19, at the end of December 2019 has turned itself into a pandemic taking a heavy toll on human lives. The causal agent being SARS-CoV-2, a member of the long-known Coronaviridae family, is a positive sense single-stranded enveloped virus and quite closely related to SARS-CoV. It has become the need of the hour to understand the pathophysiology of this disease, so that drugs, vaccines, treatment regimens and plausible therapeutic agents can be produced. Methods: In this regard, recent studies uncovered the fact that the viral genome of SARS-CoV-2 encodes nonstructural proteins like RNA dependent RNA polymerase (RdRp) which is an important tool for its transcription and replication process. A large number of nucleic acid based anti-viral drugs are being repurposed for treating COVID-19 targeting RdRp. Few of them are in the advanced stage of clinical trials including Remdesivir. While performing close investigation of the large set of nucleic acid based drugs, we were surprised to find that the synthetic nucleic acid backbone is explored very little or rare. Results: We have designed scaffolds derived from peptide nucleic acid (PNA) and subjected them for in-silico screening systematically. These designed molecules have demonstrated excellent binding towards RdRp. Compound 12 was found to possess similar binding affinity as Remdesivir with comparable pharmacokinetics. However, the in-silico toxicity prediction indicates compound 12 may be a superior molecule which can be explored further due to its excellent safety-profile with LD50 (12,000mg/kg) as opposed to Remdesivir (LD50 =1000mg/kg). Conclusion: Compound 12 falls in the safe category of class 6. Synthetic feasibility, equipotent binding and very low toxicity of this peptide nucleic acid derived compounds can serve as a leading scaffold to design, synthesize and evaluate many of similar compounds for the treatment of COVID-19.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the catecholamine (CA) biosynthesis pathway, making TH a molecular target for controlling CA production, specifically dopamine. Dysregulation of dopamine is correlated with neurological diseases such as Parkinson's disease (PD) and post-traumatic stress disorder (PTSD), among others. Previously, we showed that a 49-nucleotide guanine (G)-rich sequence within the human TH promoter adopts two different sets of G-quadruplex (GQ) structures (5'GQ and 3'GQ), where the 5'GQ uses G-stretches I, II, IV, and VI in TH49, which enhances TH transcription, while the 3'GQ utilizes G-stretches II, IV, VI, and VII, which represses transcription. Herein, we demonstrated targeted switching of these GQs to their active state using rationally designed DNA GQ Clips (5'GQ and 3'GQ Clips) to modulate endogenous TH gene expression and dopamine production. As a translational approach, we synthesized a targeted nanoparticle delivery system to effectively deliver the 5'GQ Clip in vivo. We believe this strategy could potentially be an improved approach for controlling dopamine production in a multitude of neurological disorders, including PD.

Scaffold attachment factor A (SAF-A) or hnRNP U is a nuclear RNA-binding protein with a well-documented role in processing newly transcribed RNA. Recent studies also indicate that SAF-A can oligomerise in an ATP-dependent manner and interact with RNA to form a dynamic nuclear mesh. This mesh is thought to regulate nuclear and chromatin architecture, yet a mechanistic understanding is lacking. Here, we review developments in the field to understand how the SAF-A/RNA mesh affects chromatin organisation in interphase and mitosis. As SAF-A has an intrinsically disordered domain we discuss how the chromatin mesh is related to nuclear phase-separated condensates, which in other situations have been shown to regulate transcription and cell functions. Finally, we infer possible links between diseases emerging from SAF-A mutations and its role in chromatin organisation and regulation.

Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis, has latently infected about one-third of the world’s population and may lead to severe clinical symptoms and death. The WhiB4 protein, a transcription factor, plays a crucial role in the survival and pathology of Mtb. WhiB4 leads to the condensation of mycobacterial nucleoids and regulates the expression of genes involved in central metabolism, respiration, and maintaining redox homeostasis. Here, we report the solution structure of reduced apo-WhiB4 monomer, which consists of an unstructured N-terminal domain with four cysteine residues and a helix-turn-helix C-terminal domain that plays a major role in DNA binding. The C-terminal domain of WhiB4 binds DNA at the minor groove, with five positively charged lysine/arginine residues contacting DNA sugar-phosphate backbones through electrostatic interactions. AT-rich DNA sequences with narrower minor grooves are more preferred by WhiB4. The binding affinity of a single C-terminal domain of WhiB4 is weak. When oxidized, WhiB4 can form dimers and oligomers in different forms through disulfide bonds, which should significantly enhance its DNA binding ability through multivalent effect and change the local structure of target genes and influence their transcription. These structural features form the basis for WhiB4 to function as a redox-sensitive transcription factor in Mtb.

Invasion and metastasis of breast cancer cells is an important cause of death in breast cancer patients. In the tumor microenvironment, M2 polarization of macrophages can promote the invasion and metastasis of tumor cells. OVOL2 is an evolutionarily conserved transcription regulator, but its effect in macrophages has not been described previously. The aim of this study was to investigate the effects of OVOL2 on macrophage polarity and the role of these effects in the tumor metastasis. We found that overexpression of OVOL2 in macrophages significantly inhibited M2 polarization and thus inhibits breast cancer metastasis. We propose a novel mechanism in which OVOL2 inhibits M2 polarization of macrophages and thus reduces their ability to induce invasion and metastasis of breast cancer. By shedding new light on the regulation of metastasis in cancers, our study provides a new strategy for the targeted therapy of cancer.

Since the epidemic began in November 2019, no viable medicine against SARS-CoV-2 has been discovered. The typical medication discovery strategy requires several years of rigorous research and development as well as a significant financial commitment, which is not feasible in the face of the current epidemic. Through molecular docking and dynamic simulation studies, we used the FDA-approved drug mezonavir against the most important viral targets, including spike (S) glycoprotein, Transmembrane serine protease 2 (TMPRSS2), RNA-dependent RNA polymerase (RdRp), Main protease (Mpro), human angiotensin-converting enzyme 2 (ACE-2), and furin. These targets are critical for viral replication and infection propagation because they play a key role in replication/transcription and host cell recognition. Molecular docking revealed that the antiviral medication mozenavir showed a stronger affinity for SARS-CoV-2 target proteins than reference medicines in this investigation. We discovered that mozenavir increases the complex's stability and validates the molecular docking findings using molecular dynamics modeling. Furin, a target protein of COVID-19, has a greater binding affinity (-12.04 kcal/mol) than other COVID-19 target proteins, forming different hydrogen bonds and polar and hydrophobic interactions, suggesting that it might be used as an antiviral treatment against SARS-CoV-2. Overall, the present in silico results will be valuable in identifying crucial targets for subsequent experimental investigations that might help combat COVID-19 by blocking the protease furin's proteolytic activity.

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II–mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1–CDK9 complex elicited RNA m6A modification–dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1–CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM. Significance: Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti–PD-1 response through regulating the m6A modification program, linking epigenetic regulation to immunomodulatory function in GBM. This article is highlighted in the In This Issue feature, p. 275

Numerous studies have shown that viruses can utilize or manipulate ribosomal proteins to achieve viral protein biosynthesis and replication. In our recent studies using proteomics analysis of virus-infected cells, we found that ribosomal protein L18 (RPL18) was the highest up-regulated differentially expressed protein, along with the increasingly expressed viral proteins later in Newcastle disease virus (NDV) infection. However, the association of RPL18 with viral protein biosynthesis and NDV replication remains unclear. In this study, we found that the expression and transcription levels of RPL18 was reduced early in NDV infection but increased later in NDV infection. In addition, the presence of cytoplasmic NDV matrix (M) protein was responsible for the increased expression of RPL18 in both virus-infected cells and plasmid-transfected cells. Moreover, cytoplasmic M protein increased RPL18 expression in a dose-dependent manner, even though they did not interact with each other. Furthermore, siRNA-mediated knockdown of RPL18 or overexpression of RPL18 dramatically reduced or enhanced NDV replication by decreasing or increasing viral protein translation rather than viral RNA synthesis and transcription. Taken together, these results suggested that the increased expression of RPL18 might be associated with the physical clumping together of the M protein, which in turn promoted viral protein biosynthesis and NDV replication. RESEARCH HIGHLIGHTSThe increased expression of RPL18 is associated with the presence of cytoplasmic M protein.Cytoplasmic M protein increases RPL18 expression in a dose-dependent manner.Knockdown of RPL18 reduces NDV replication by decreasing viral protein translation.Overexpression of RPL18 enhances NDV replication by increasing viral protein translation.

Human genes are arranged on 23 pairs of chromosomes, but in cancer, tumor-promoting genes and regulatory elements can free themselves from chromosomes and relocate to circular, extrachromosomal pieces of DNA (ecDNA). ecDNA, because of its nonchromosomal inheritance, drives high-copy-number oncogene amplification and enables tumors to evolve their genomes rapidly. Furthermore, the circular ecDNA architecture fundamentally alters gene regulation and transcription, and the higher-order organization of ecDNA contributes to tumor pathogenesis. Consequently, patients whose cancers harbor ecDNA have significantly shorter survival. Although ecDNA was first observed more than 50 years ago, its critical importance has only recently come to light. In this review, we discuss the current state of understanding of how ecDNAs form and function as well as how they contribute to drug resistance and accelerated cancer evolution. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 17 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) is essential for gene expression control. How altering the phosphorylation of the CTD contributes to gene expression in mammalian systems remains poorly understood. Methods: Primary mouse embryonic fibroblasts, hepatocytes, and embryonic stem cells were isolated from conditional Ssu72f/f mice. To knockout the mouse Ssu72 gene, we infected the cells with adenoviruses of incorporated luciferase and Cre recombinase, respectively. RNA sequencing, ChIP sequencing, ChIP assay, immunoblot analyses, qRT-PCR assay, and immunostaining were performed to gain insights into the functional mechanisms of Ssu72 loss in Pol II dynamics. Results: Using primary cells isolated from Ssu72 conditional knockout and transgenic mice, we found that mammalian Ssu72-mediated transcriptional elongation rather than polyadenylation or RNA processing contributed to the transcriptional regulation of various genes. Depletion of Ssu72 resulted in aberrant Pol II pausing and elongation defects. Reduced transcriptional elongation efficiency tended to preferentially affect expression levels of actively transcribed genes in a tissue-specific manner. Furthermore, Ssu72 CTD phosphatase seemed to regulate the phosphorylation levels of CTD Ser2 and Thr4 through accurate modulation of P-TEFb activity and recruitment. Conclusions: Our findings demonstrate that mammalian Ssu72 contributes to the transcription of tissue-specific actively transcribed gene expression by regulating reciprocal phosphorylation of Pol II CTD.

Thyroid hormone (T3) affects many diverse physiological processes such as metabolism, organogenesis, and growth. The two highly related frog species, diploid Xenopus tropicalis and pseudo tetraploid Xenopus laevis, have been used as models for analyzing the effects of T3 during vertebrate development. T3 regulates T3-inducible gene transcription through T3 receptor (TR)-binding to T3-response elements (TREs). We have previously identified sperm associated antigen 7 (spag7) as a candidate T3 target gene that is potentially involved in adult stem cell development and/or proliferation during intestinal metamorphosis. To investigate whether T3 regulates spag7 directly at the transcriptional level via TR, we first conducted qRT-PCR to analyze its expression during natural and T3-induced metamorphosis and found that spag7 was up-regulated during natural metamorphosis in the intestine, tail, brain and hindlimb, peaking at the climax of metamorphosis in all those organs, and upon T3 treatment of premetamorphic tadpoles. Next, we demonstrated that an intronic TRE in spag7, first identified through bioinformatic analysis, could bind to TR in vitro and in vivo during metamorphosis. A dual luciferase assay utilizing a reconstituted frog oocyte transcription system showed that the TRE could mediate promoter activation by liganded TR. These results indicate that spag7 expression is directly regulated by T3 through the TRE in the first intron during metamorphosis, implicating a role for spag7 early during T3-regulated tissue remodeling and resorption.

Arabidopsis methylation elevated mutant 1 (mem1) mutants have elevated levels of global DNA methylation. In this study, such mutant alleles showed increased sensitivity to methyl methanesulfonate (MMS). In mem1 mutants, an assortment of genes engaged in DNA damage response (DDR), especially DNA-repair-associated genes, were largely upregulated without MMS treatment, suggestive of activation of DDR pathway in them. Following MMS treatment, expression levels of multiple DNA-repair-associated genes in mem1 mutants were generally lower than in Col-0 plants, which accounted for the MMS-sensitive phenotype of the mem1 mutants. A group of DNA methylation pathway genes were upregulated in mem1 mutants under non-MMS-treated conditions, causing elevated global DNA methylation, especially in RNA-directed DNA methylation (RdDM)-targeted regions. Moreover, MEM1 seemed to help ATAXIA-TELANGIECTASIA MUTATED (ATM) and/or SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) to fully activate/suppress transcription of a subset of genes regulated simultaneously by MEM1 and ATM and/or SOG1, because expression of such genes decreased/increased consistently in mem1 and atm and/or sog1 mutants, but the decreases/increases in the mem1 mutants were not as dramatic as in the atm and/or sog1 mutants. Thus, our studies reveals roles of MEM1 in safeguarding genome, and interrelationships amongst DNA damage, activation of DDR, DNA methylation/demethylation, and DNA repair. This article is protected by copyright. All rights reserved.

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

Background: Sanghuangporus baumii is a traditional Chinese medicine with anti- cancer, anti-tumor, and anti-inflammatory effects. Triterpenoids are one of the main medicinal ingredients found in S. baumii. However, the dynamic changes of triterpenoids content and its molecular regulation mechanism are still unclear. Objective: Some studies have shown that Lanosterol synthase (LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing S. baumii triterpenoids. Therefore, the study of LS gene and expression characteristics can provide clues for the further study of triterpenoids synthesis. Methods: The PCR, RACE PCR, RT-PCR, homologous recombination and prokaryotic expression technology were used to research the gene characteristic and dynamic changes of LS transcription level. Results: The S. baumii LS sequence included a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. Results: The S. baumii LS sequence included a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. Conclusion: The S. baumii LS was cloned and characterized to help to understand the mechanism of triterpenoids synthesis. In addition, we studied the relationship between LS transcription level and triterpenoid dynamic accumulation, and we found that they had a certain correlation.

In eukaryotes, three RNA polymerases (RNAPs) play essential roles in the synthesis of various types of RNA: namely, RNAPI for rRNA; RNAPII for mRNA and most snRNAs; and RNAPIII for tRNA and other small RNAs. All three RNAPs possess a short flexible tail derived from their common subunit RPB6. However, the function of this shared N-terminal tail (NTT) is not clear. Here we show that NTT interacts with the PH domain (PH-D) of the p62 subunit of the general transcription/repair factor TFIIH, and present the structures of RPB6 unbound and bound to PH-D by nuclear magnetic resonance (NMR). Using available cryo-EM structures, we modelled the activated elongation complex of RNAPII bound to TFIIH. We also provide evidence that the recruitment of TFIIH to transcription sites through the p62–RPB6 interaction is a common mechanism for transcription-coupled nucleotide excision repair (TC-NER) of RNAPI- and RNAPII-transcribed genes. Moreover, point mutations in the RPB6 NTT cause a significant reduction in transcription of RNAPI-, RNAPII- and RNAPIII-transcribed genes. These and other results show that the p62–RPB6 interaction plays multiple roles in transcription, TC-NER, and cell proliferation, suggesting that TFIIH is engaged in all RNAP systems.

We identified cytosine-rich regions adjacent to guanine-rich regions in protease genes. A typical GC-rich sequence derived from the TMPRSS2 gene showed structural competition between a G-quadruplex and a hairpin loop, and this competition significantly affected transcription efficiency. These results suggest an impact of neighboring sequences on the gene expression of guanine-rich sequences.

Bi-oriented attachment of microtubules to the centromere is a pre-requisite for faithful chromosome segregation during mitosis. Budding yeast have point centromeres containing the cis-element proteins CDE-I, -II, and -III, which interact with trans-acting factors such as Cbf1, Cse4, and Ndc10. Our previous genetic screens, using a comprehensive library of histone point mutants, revealed that the TBS-I, -II, and -III regions of nucleosomes are required for faithful chromosome segregation. In TBS-III deficient cells, peri-centromeric nucleosomes containing the H2A.Z homolog Htz1 are lacking, however, it is unclear why chromosome segregation is defective in these cells. Here, we show that, in cells lacking TBS-III, both chromatin binding at the centromere and the total amount of some of the centromere proteins are reduced, and transcription through the centromere is up-regulated during M-phase. Moreover, the chromatin binding of Cse4, Mif2, Cbf1, Ndc10, and Scm3 was reduced upon ectopic transcription through the centromere in wild-type cells. These results suggest that transcription through the centromere displaces key centromere proteins and, consequently, destabilizes the interaction between centromeres and microtubules, leading to defective chromosome segregation. The identification of new roles for histone binding residues in TBS-III will shed new light on nucleosome function during chromosome segregation.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), which required developing novel therapies targeting the inhibition of HBV transcription and replication due to current limited treatment options. We explored novel target for the development of novel therapies targeting the inhibition of HBV replication and transcription. The expression of Id1 and E2F4 in HCC cells and tissues was detected by qRT-PCR and western blot. We investigated the Id1 and E2F4-mediated transcription of HBV infection by using HepG2.2.15, HepAD38, HepG2-NTCP cell lines and AAV/HBV-infected mice. Interactions between the two host proteins and viral covalently closed circular DNA (cccDNA) were assessed using subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays. Ectopic Id1 significantly reduced HBV transcription and replication in both HBV-expressing cells and AAV/HBV-infected mice. Id1 and E2F4 could form a heterodimer to prevent E2F4 from promoting HBV transcription and replication. E2F4 could directly bind to cccDNA and activate the HBV core promoter in cell lines. Furthermore, in vitro binding experiments confirmed that the sequence 1758'-TTAAAGGTC-1766', which is highly conserved among HBV genotypes, is the target site of the E2F4 homodimer. The findings suggest that E2F4 function as novel cccDNA-binding protein to directly activate HBV transcription by binding to Cp promoter region. Our results highlight the ability that E2F4 represent a pan-potential therapeutic target against HBV transcription and provide more clues to better understand the life cycle of HBV.

Cotton that produces natural fiber materials for the textile industry is one of the most important crops in the world. Class II KNOX proteins are often considered as transcription factors in regulating plant secondary cell wall (SCW) formation. However, the molecular mechanism of the KNOX transcription factors-regulated SCW synthesis in plants (especially in cotton) remains unclear in details so far. In this study, we show a cotton class II KNOX protein (GhKNL1) as a transcription repressor functioning in fiber development. The GhKNL1-silenced transgenic cotton produced longer fibers with thicker SCWs, whereas GhKNL1 dominant repression transgenic lines displayed the opposite fiber phenotype, compared with controls. Further experiments revealed that GhKNL1 could directly bind to promoters of GhCesA4-2/4-4/8-2 and GhMYB46 for modulating cellulose synthesis during fiber SCW development of cotton. On the other hand, GhKNL1 could also suppress expressions of GhEXPA2D/4A-1/4D-1/13A through binding to their promoters for regulating fiber elongation of cotton. Taken together, these data revealed GhKNL1 functions in fiber elongation and SCW formation by directly repressing expressions of its target genes related to cell elongation and cellulose synthesis. Thus, our data provide an effective clue for potentially improving fiber quality by genetic manipulation of GhKNL1 in cotton breeding. This article is protected by copyright. All rights reserved.

Background Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Results Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. Conclusions The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition, and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened within minutes after transcription. Our data show that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs escape rapid nuclear deadenylation. Modelling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggest that nuclear deadenylation is a key mechanism for regulating mRNA stability, abundance, and subcellular localization.

Abnormal cell proliferation caused by abnormal transcription regulation mechanism seems to be one of the reasons for the progression of breast cancer and also the pathological basis. MicroRNA-142-5p (miR-142-5p) is a low-expressed miRNA in breast cancer. The role of MKL-1s regulation of DNMT1 in breast cancer cell proliferation and migration is still unclear. MKL-1 (myocardin related transcription factor A) can bind to the conserved cis-regulatory element CC (A/T) 6GG (called CarG box) in the promoter to regulate the transcription of miR-142-5p. The expressions of miR-142-5p and MKL-1 are positively correlated. In addition, it has been proved that DNMT1 is the target of miR-142-5p, which inhibits the expression of DNMT1 by targeting the 3-UTR of DNMT1, thereby forming a feedback loop and inhibiting the migration and proliferation of breast cancer. Our data provide important and novel insights into the MKL-1/miR-142-5p/DNMT1/maspin signaling pathway and may become a new idea for breast cancer diagnosis, treatment, and prognosis.

DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells.

Key Points Typical features of GATA2 deficiency were associated with a de novo tandem duplication of GATA2 and increased expression of GATA2-AS1.The duplication contained a maternally inherited deletion copy number polymorphism esv2725896/nsv513733., A 3-year-old girl of nonconsanguineous healthy parents presented with cervical and mediastinal lymphadenopathy due to Mycobacterium fortuitum infection. Routine blood analysis showed normal hemoglobin, neutrophils, and platelets but profound mononuclear cell deficiency (monocytes < 0.1 × 109/L; B cells 78/μL; NK cells 48/μL). A 548 902-bp region containing GATA2 was sequenced by targeted capture and deep sequencing. This revealed a de novo 187-kb duplication of the entire GATA2 locus, containing a maternally inherited copy number variation deletion of 25 kb (GRCh37: esv2725896 and nsv513733). Many GATA2-associated phenotypes have been attributed to amino acid substitution, frameshift/deletion, loss of intronic enhancer function, or aberrant splicing. Gene deletion has been described, but other structural variation has not been reported in the germline configuration. In this case, duplication of the GATA2 locus was paradoxically associated with skewed diminished expression of GATA2 messenger RNA and loss of GATA2 protein. Chimeric RNA fusion transcripts were not detected. A possible mechanism involves increased transcription of the anti-sense long noncoding RNA GATA2-AS1 (RP11-472.220), which was increased several fold. This case further highlights that evaluation of the allele count is essential in any case of suspected GATA2-related syndrome.

Zinc ribbons, one of the largest fold groups among zinc fingers, often include proteins involved in the transcription machinery. Here, we identify and characterize one such zinc ribbon-bearing protein in the apicomplexan parasite Toxoplasma gondii, annotated as putative transcription elongation factor 1 (ELF1), with predicted functions in transcription and chromatin maintenance. We show that this ELF1 homolog, referred to as Toxoplasma gondii ELF1-like divergent (TgELD), is expressed in both tachyzoite and bradyzoite developmental stages. TgELD associates with the cytoskeleton in the tachyzoites while it transiently becomes a part of the cyst wall in the early bradyzoites, followed by a cytosolic and peripheral localization in late bradyzoites. TgELD is phosphorylated by a casein kinase 2-like protein, which has potential implications for its localization and function in the parasite.

Telomere length regulation is essential for cell viability in eukaryotes. While many pathways that affect telomere length are known, we do not yet have a complete understanding of the mechanism of length regulation. To identify new pathways that might regulate telomere length, we carried out a genetic screen in yeast and identified the cyclin-dependent kinase complex Bur1/2 as a regulator of telomere length. Mutations in either BUR1 cyclin-dependent kinase or the associated BUR2 cyclin resulted in short telomeres. This regulation did not function through the known role of BUR1 in regulating histone modification as bur1∆ set2∆ and bur2∆ set2∆ double mutants rescued cell growth but did not rescue the telomere shortening effects. We found that both bur1∆ and bur2∆ set2∆ were also defective in de novo telomere addition, and deletion of SET2 did also not rescue this elongation defect. The Bur1/2 cyclin-dependent kinase regulates transcription of many genes. We found that TLC1 RNA levels were reduced in bur2∆ set2∆ mutants; however, overexpression of TLC1 restored the transcript levels but did not restore de novo telomere elongation or telomere length. These data suggest that the Bur1/2 kinase plays a role in telomere elongation separate from its role in transcription of telomerase components. Dissecting the role of the Bur1/2 kinase pathway at telomeres will help complete our understanding of the complex network of telomere length regulation.

Soil salinity and drought limit sweet potato yield. Scavenging of reactive oxygen species (ROS) by peroxidases (PRXs) is essential during plant stress responses, but how PRX expression is regulated under abiotic stress is not well understood. Here, we report that the B-box (BBX) family transcription factor IbBBX24 activates the expression of the class III peroxidase gene IbPRX17 by binding to its promoter. Overexpression of IbBBX24 and IbPRX17 significantly improved the tolerance of sweet potato to salt and drought stresses, whereas reducing IbBBX24 expression increased their susceptibility. Under abiotic stress, IbBBX24- and IbPRX17-overexpression lines showed higher peroxidase activity and lower H2 O2 accumulation compared with the wild-type. RNA sequencing analysis revealed that IbBBX24 modulates the expression of genes encoding ROS scavenging enzymes, including PRXs. Moreover, interaction between IbBBX24 and the APETALA2 (AP2) protein IbTOE3 enhances the ability of IbBBX24 to activate IbPRX17 transcription. Overexpression of IbTOE3 improved the tolerance of tobacco plants to salt and drought stresses by scavenging ROS. Together, our findings elucidate the mechanism underlying the IbBBX24-IbTOE3-IbPRX17 module in response to abiotic stress in sweet potato and identify candidate genes for developing elite crop varieties with enhanced abiotic stress tolerance.

Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92- are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green fluorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modification to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.

Quantitative real-time RT-PCR is considered to valuable method for the analysis of gene expression. But for the data accuracy of gene expression, the normalization of data using reference genes is essential. In this study, we focus on the analysis of the relative expression of nine commonly used candidate reference genes in the different developmental stages and female and male pupae/adults of Chilo suppressalis, and expression stabilities were analyzed by the ΔCt method, geNorm, NormFinder, and RefFinder. The results showed that H3 was the most stable reference genes in the different developmental stages while EF1 was the optimal genes in pupae/adult of different sexes of C. suppressalis. To validate, the expression profile of the gene encoding heat shock protein 60 (Hsp60) was investigated. The results showed that the transcription of target gene Hsp60 varied depending on the reference gene selected for normalization in different developmental stages, but the trend of expression of Hsp60 between the most stable reference gene combination and the least stable reference gene in pupae/adults of different sexes was the same. Therefore, it is imperative to select the reference genes to estimate target gene expression accurately in the specific experiment. This study provides scientific basis on functional analyses of the important genes contributed to the development and sex differentiation of C. suppressalis.

Stringent starvation protein A (SspA) involved in nucleotide metabolism, acid tolerance and virulence of bacteria has been demonstrated to function as a transcription factor to regulate σ70-dependent gene transcription through interacting with σ70 region 4 and the zinc binding domain (ZBD) of E. coli RNA polymerase (EcoRNAP) β′ subunit simultaneously. Despite extensive biochemical and structural analyses were reported recently, the interactions of SspA with RNAP are not comprehensively understood. Here, we reprocessed our previous cryo-EM dataset of EcoRNAP-promoter open complex with SspA (SspA-RPo) and obtained a significantly improved density map. Unexpectedly, the new map showed that SspA interacts with both N-terminal helix of β′ subunit (β′ΝΤΗ) and ω subunit, which contributes to stabilize the SspA-EcoRNAP σ70 holoenzyme complex. Sequence alignments and phylogenetic tree analyses of N-terminal sequences of β′ subunit from different classes of bacteria revealed that β′ΝΤΗ is highly conserved and exclusively found in low-GC-content Gram-negative bacteria that harbor SspA, implying a co-evolution of β′ΝΤΗ and SspA. The transcription assays of wild-type SspA and its mutants demonstrated the interaction between SspA and β′ΝΤΗ facilitates the transcription regulation of SspA. Together, our results provide a more comprehensive insight into the interactions between SspA and RNAP and their roles in bacterial transcription regulation.

Circular RNAs with covalently linked ends are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery backsplices. These mature transcripts are resistant to digestion by exonucleases and typically have much longer half-lives than their associated linear mRNAs. Circular RNAs thus have great promise as sensitive biomarkers, including for detection of transcriptional activity. Here, we show that circular RNAs can serve as markers of readthrough transcription events in Drosophila and human cells, thereby revealing mechanistic insights into RNA polymerase II transcription termination as well as pre-mRNA 3' end processing. We describe methods that take advantage of plasmids that generate a circular RNA when an upstream polyadenylation signal fails to be used and/or RNA polymerase II fails to terminate. As a proof-of-principle, we show that RNAi-mediated depletion of well-established transcription termination factors, including the RNA endonuclease Cpsf73, results in increased circular RNA output from these plasmids in Drosophila and human cells. This method is generalizable as a circular RNA can be easily encoded downstream of any genomic region of interest. Circular RNA biomarkers, therefore, have great promise for identifying novel cellular factors and conditions that impact transcription termination processes.

DNA methylation is a class of epigenetic modification manner, which is responsible for the inactivation of various tumor suppressors. Recently, long non-coding RNAs (lncRNAs) were revealed to be implicated in a variety of malignancies, including non-small cell lung cancer (NSCLC). However, the contributions of lncRNAs to DNA-methylation-induced oncogenic effects in NSCLC remain largely unknown. In this study, we identified a DNA-methylation-repressed lncRNA DIO3 opposite strand upstream RNA (DIO3OS) in NSCLC. DIO3OS is downregulated in NSCLC, and its low expression is related to poor prognosis. Ectopic expression of DIO3OS repressed NSCLC cell growth and motility and promoted NSCLC cell apoptosis in vitro. DIO3OS also repressed NSCLC tumorigenesis and metastasis in vivo. DIO3OS knockdown exhibited opposite biological effects. DIO3OS competitively bound heterogeneous nuclear ribonucleoprotein K (hnRNPK), repressed the binding of hnRNPK to MYC DNA and MYC mRNA, reduced the promoting roles of hnRNPK on MYC transcription and translation, led to the repression of MYC transcription and translation, and therefore remarkably decreased the expression of MYC and CDC25A, a downstream target of MYC. Additionally, depletion of hnRNPK blocked the tumor-suppressive roles of DIO3OS in NSCLC. In conclusion, these findings identified DIO3OS as an important protective factor against NSCLC via modulating hnRNPK-MYC-CDC25A axis., Graphical abstract, DIO3OS is downregulated by DNA methylation in NSCLC. DIO3OS repressed NSCLC progression via competitively binding hnRNPK, repressing the binding of hnRNPK to MYC DNA and MYC mRNA, reducing the promoting roles of hnRNPK on MYC transcription and translation, and therefore decreasing the expression of MYC.

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IFN-γ–inducible protein 16 (IFI16) recognizes viral DNAs from both nucleus-replicating viruses and cytoplasm-replicating viruses. Isoform 2 of IFI16 (IFI16-iso2) with nuclear localization sequence (NLS) has been studied extensively as a well-known DNA sensor. However, the characteristics and functions of other IFI16 isoforms are almost unknown. Here, we find that IFI16-iso1, with exactly the same length as IFI16-iso2, lacks the NLS and locates in the cytoplasm. To distinguish the functions of IFI16-iso1 and IFI16-iso2, we have developed novel nuclear viral DNA mimics that can be recognized by the nuclear DNA sensors, including IFI16-iso2 and hnRNPA2B1. The hexanucleotide motif 5′-AGTGTT-3′ DNA form of the nuclear localization sequence (DNLS) effectively drives cytoplasmic viral DNA nuclear translocation. These nuclear viral DNA mimics potently induce IFN-β and antiviral IFN-stimulated genes in human A549 cells, HEK293T cells, and mouse macrophages. The subcellular location difference of IFI16 isoforms determines their differential functions in recognizing viral DNA and activating type I IFN–dependent antiviral immunity. IFI16-iso1 preferentially colocalizes with cytoplasmic HSV60mer and cytoplasm-replicating vaccinia virus (VACV), whereas IFI16-iso2 mainly colocalizes with nuclear HSV60-DNLS and nucleus-replicating HSV-1. Compared with IFI16-iso2, IFI16-iso1 induces more transcription of IFN-β and IFN-stimulated genes, as well as stronger antiviral immunity upon HSV60mer transfection or VACV infection. IFI16-iso2, with the ability of nuclear-cytoplasmic shuttling, clears both invaded HSV type 1 and VACV significantly. However, IFI16-iso2 induces more type I IFN–dependent antiviral immunity than IFI16-iso1 upon HSV60-DNLS transfection or HSV type 1 infection. Our study has developed potent agonists for nuclear DNA sensors and also has demonstrated that IFI16 isoforms with cytoplasmic and nuclear locations play differential roles in innate immunity against DNA viruses.

Fatty acid binding protein 4 (FABP4) was crucial to fatty acid uptake and intracellular transport. However, the mechanisms regulating yak (Bos grunniens) FABP4 transcription were not determined. In the current study, predominant expression levels of yak FABP4 were identified in subcutaneous fat and longissimus dorsi muscles by quantitative real-time polymerase chain reactions (qPCR). The CCAAT/enhancer binding protein alpha (CEBPα) and myocyte enhancer factor 2A (MEF2A), as transcriptional activator or repressor in the promoter region of FABP4, were confirmed by both site-directed mutagenesis experiment and chromatin immunoprecipitation assay. Additionally, molecular mechanisms of CEBPɑ regulation were analyzed to explore the transcriptional regulatory property of FABP4, which indicated that transcriptional activity of CEBPɑ depended on CCAAT/ enhancer binding protein beta (CEBPβ) transcription factor. Our results demonstrated that CEBPβ binding directly to the promoter region drove CEBPɑ transcription, improving yak FABP4 transcriptional activity in adipocytes. This mechanism expanded the information on the transcriptional regulatory network of adipogenesis.