Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li-Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

Identification of plant transcriptional activation domains.

Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs1. To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs.

Dimerization of hub protein DYNLL1 and bZIP transcription factor CREB3L1 enhances transcriptional activation of CREB3L1 target genes like arginine vasopressin.

bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

Pioneer Transcription Factors: The First Domino in Zygotic Genome Activation.

Zygotic genome activation (ZGA) is a pivotal event in mammalian embryogenesis, marking the transition from maternal to zygotic control of development. During the ZGA process that is characterized by the intricate cascade of gene expression, who tipped the first domino in a meticulously arranged sequence is a subject of paramount interest. Recently, Dux, Obox and Nr5a2 were identified as pioneer transcription factors that reside at the top of transcriptional hierarchy. Through co-option of retrotransposon elements as hubs for transcriptional activation, these pioneer transcription factors rewire the gene regulatory network, thus initiating ZGA. In this review, we provide a snapshot of the mechanisms underlying the functions of these pioneer transcription factors. We propose that ZGA is the starting point where the embryo's own genome begins to influence development trajectory, therefore in-depth dissecting the functions of pioneer transcription factors during ZGA will form a cornerstone of our understanding for early embryonic development, which will pave the way for advancing our grasp of mammalian developmental biology and optimizing in vitro production (IVP) techniques.

Systematic identification of transcriptional activation domains from non-transcription factor proteins in plants and yeast.

Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, "universal" activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes.

TCF12 Transcriptionally Activates SPHK1 to Induce Osteosarcoma Angiogenesis by Promoting the S1P/S1PR4/STAT3 Axis.

Transcription factor 12 (TCF12) is a known oncogene in many cancers. However, whether TCF12 can regulate malignant phenotypes and angiogenesis in osteosarcoma is not elucidated. In this study, we demonstrated increased expression of TCF12 in osteosarcoma tissues and cell lines. High TCF12 expression was associated with metastasis and poor survival rate of osteosarcoma patients. Knockdown of TCF12 reduced the proliferation, migration, and invasion of osteosarcoma cells. TCF12 was found to bind to the promoter region of sphingosine kinase 1 (SPHK1) to induce transcriptional activation of SPHK1 expression and enhance the secretion of sphingosine-1-phosphate (S1P), which eventually resulted in the malignant phenotypes of osteosarcoma cells. In addition, S1P secreted by osteosarcoma cells promoted the angiogenesis of HUVECs by targeting S1PR4 on the cell membrane to activate the STAT3 signaling pathway. These findings suggest that TCF12 may induce transcriptional activation of SPHK1 to promote the synthesis and secretion of S1P. This process likely enhances the malignant phenotypes of osteosarcoma cells and induces angiogenesis via the S1PR4/STAT3 signaling pathway.

Targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for malate-aspartate shuttle and tumour progression.

BACKGROUND: A series of studies have demonstrated the emerging involvement of transfer RNA (tRNA) processing during the progression of tumours. Nevertheless, the roles and regulating mechanisms of tRNA processing genes in neuroblastoma (NB), the prevalent malignant tumour outside the brain in children, are yet unknown. METHODS: Analysis of multi-omics results was conducted to identify crucial regulators of downstream tRNA processing genes. Co-immunoprecipitation and mass spectrometry methods were utilised to measure interaction between proteins. The impact of transcriptional regulators on expression of downstream genes was measured by dual-luciferase reporter, chromatin immunoprecipitation, western blotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods. Studies have been conducted to reveal impact and mechanisms of transcriptional regulators on biological processes of NB. Survival differences were analysed using the log-rank test. RESULTS: c-Myc was identified as a transcription factor driving tRNA processing gene expression and subsequent malate-aspartate shuttle (MAS) in NB cells. Mechanistically, c-Myc directly promoted the expression of glutamyl-prolyl-tRNA synthetase (EPRS) and leucyl-tRNA synthetase (LARS), resulting in translational up-regulation of glutamic-oxaloacetic transaminase 1 (GOT1) as well as malate dehydrogenase 1 (MDH1) via inhibiting general control nonrepressed 2 or activating mechanistic target of rapamycin signalling. Meanwhile, lamin A (LMNA) inhibited c-Myc transactivation via physical interaction, leading to suppression of MAS, aerobic glycolysis, tumourigenesis and aggressiveness. Pre-clinically, lobeline was discovered as a LMNA-binding compound to facilitate its interaction with c-Myc, which inhibited aminoacyl-tRNA synthetase expression, MAS and tumour progression of NB, as well as growth of organoid derived from c-Myc knock-in mice. Low levels of LMNA or elevated expression of c-Myc, EPRS, LARS, GOT1 or MDH1 were linked to a worse outcome and a shorter survival time of clinical NB patients. CONCLUSIONS: These results suggest that targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for MAS and tumour progression.

ACRE, a class of AP2/ERF transcription factors, activates the expression of sweet potato ß-amylase and sporamin genes through the sugar-responsible element CMSRE-1.

Sugars, synthesized by photosynthesis in source organs, are loaded and utilized as an energy source and carbon skeleton in sink organs, and also known to be important signal molecules regulating gene expression in higher plants. The expression of genes coding for sporamin and ß-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately induced by sugars. We previously reported on the identification of the carbohydrate metabolic signal-responsible element-1 (CMSRE-1) essential for the sugar-responsible expression of two genes. However, transcription factors that bind to this sequence have not been identified. In this study, we performed yeast one-hybrid screening using the sugar-responsible minimal promoter region of the ß-amylase gene as bait and a library composed only transcription factor cDNAs of Arabidopsis. Two clones, named Activator protein binding to CMSRE-1 (ACRE), encoding AP2/ERF transcription factors were isolated. ACRE showed transactivation activity of the sugar-responsible minimal promoter in a CMSRE-1-dependent manner in Arabidopsis protoplasts. Electric mobility shift assay (EMSA) using recombinant proteins and transient co-expression assay in Arabidopsis protoplasts revealed that ACRE could actually act to the CMSRE-1. Among the DEHYDRATION -RESPONSIVE ELEMENT BINDING FACTOR (DREB) subfamily, almost all homologs including ACRE, could act on the DRE, while only three ACREs could act to the CMSRE-1. Moreover, ACRE-homologs of Japanese morning glory also have the same property of DNA-binding preference and transactivation activity through the CMSRE-1. These findings suggested that ACRE plays an important role in the mechanism regulating the sugar-responsible gene expression through the CMSRE-1 conserved across plant species.

Upregulation of circ_0076684 in osteosarcoma facilitates malignant processes by mediating miRNAs/CUX1.

Circular RNAs (circRNAs) are a newly appreciated type of endogenous noncoding RNAs that play vital roles in the development of various human cancers, including osteosarcoma (OS). In this study, we investigated three circRNAs (circ_0076684, circ_0003563, circ_0076691) from the RUNX Family Transcription Factor 2 (RUNX2) gene locus in OS. We found that the expression of circ_0076684, circ_0003563, circ_0076691, and RUNX2 mRNA is upregulated in OS, which is a consequence of CBX4-mediated transcriptional activation. Among these three RUNX2-circRNAs, only circ_0076684 is significantly associated with the clinical features and prognosis of OS patients. Functional experiments indicate that circ_0076684 promotes OS progression in vitro and in vivo. Circ_0076684 acts as a sponge for miR-370-3p, miR-140-3p, and miR-193a-5p, raising Cut Like Homeobox 1 (CUX1) expression by sponging these three miRNAs. Furthermore, we presented that circ_0076684 facilitates OS progression via CUX1. In conclusion, this study found that the expression of three circRNAs and RUNX2 mRNA from the RUNX2 gene locus is significantly upregulated in OS, as a result of CBX4-mediated transcriptional activation. Circ_0076684 raises CUX1 expression by sponging miR-370-3p, miR-140-3p, and miR-193a-5p, and facilitates OS progression via CUX1.

MicroRNA164e suppresses NAC100 transcription factor-mediated synthesis of seed storage proteins in chickpea.

Development of protein-enriched chickpea varieties necessitates an understanding of specific genes and key regulatory circuits that govern the synthesis of seed storage proteins (SSPs). Here, we demonstrated the novel involvement of Ca-miR164e-CaNAC100 in regulating SSP synthesis in chickpea. Ca-miRNA164e was significantly decreased during seed maturation, especially in high-protein accessions. The miRNA was found to directly target the transactivation conferring C-terminal region of a nuclear-localized transcription factor, CaNAC100 as revealed using RNA ligase-mediated-rapid amplification of cDNA ends and target mimic assays. The functional role of CaNAC100 was demonstrated through seed-specific overexpression (NACOE) resulting in significantly augmented seed protein content (SPC) consequential to increased SSP transcription. Further, NACOE lines displayed conspicuously enhanced seed weight but reduced numbers and yield. Conversely, a downregulation of CaNAC100 and SSP transcripts was evident in seed-specific overexpression lines of Ca-miR164e that culminated in significantly lowered SPC. CaNAC100 was additionally demonstrated to transactivate the SSP-encoding genes by directly binding to their promoters as demonstrated using electrophoretic mobility shift and dual-luciferase reporter assays. Taken together, our study for the first time established a distinct role of CaNAC100 in positively influencing SSP synthesis and its critical regulation by CamiR164e, thereby serving as an understanding that can be utilized for developing SPC-rich chickpea varieties.

Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and ß-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.

ZNF263 cooperates with ZNF31 to promote the drug resistance and EMT of pancreatic cancer through transactivating RNF126.

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attribute to the aggressive local invasion, distant metastasis and drug resistance of PDAC patients, which was strongly accelerated by epithelial-mesenchymal transition (EMT). In current study, we systematically investigate the role of ZNF263/RNF126 axis in the initiation of EMT in PDAC in vitro and vivo. ZNF263 is firstly identified as a novel transactivation factor of RNF126. Both ZNF263 and RNF126 were overexpressed in PDAC tissues, which were associated with multiple advanced clinical stages and poor prognosis of PDAC patients. ZNF263 overexpression promoted cell proliferation, drug resistance and EMT in vitro via activating RNF126 following by the upregulation of Cyclin D1, N-cad, and MMP9, and the downregulation of E-cad, p21, and p27. ZNF263 silencing contributed to the opposite phenotype. Mechanistically, ZNF263 transactivated RNF126 via binding to its promoter. Further investigations revealed that ZNF263 interacted with ZNF31 to coregulate the transcription of RNF126, which in turn promoted ubiquitination-mediated degradation of PTEN. The downregulation of PTEN activated AKT/Cyclin D1 and AKT/GSK-3ß/ß-catenin signaling, thereby promoting the malignant phenotype of PDAC. Finally, the coordination of ZNF263 and RNF126 promotes subcutaneous tumor size and distant liver metastasis in vivo. ZNF263, as an oncogene, promotes proliferation, drug resistance and EMT of PDAC through transactivating RNF126.

Increased enhancer-promoter interactions during developmental enhancer activation in mammals.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals.

Enhancer selectivity in space and time: from enhancer-promoter interactions to promoter activation.

The primary regulators of metazoan gene expression are enhancers, originally functionally defined as DNA sequences that can activate transcription at promoters in an orientation-independent and distance-independent manner. Despite being crucial for gene regulation in animals, what mechanisms underlie enhancer selectivity for promoters, and more fundamentally, how enhancers interact with promoters and activate transcription, remain poorly understood. In this Review, we first discuss current models of enhancer-promoter interactions in space and time and how enhancers affect transcription activation. Next, we discuss different mechanisms that mediate enhancer selectivity, including repression, biochemical compatibility and regulation of 3D genome structure. Through 3D polymer simulations, we illustrate how the ability of 3D genome folding mechanisms to mediate enhancer selectivity strongly varies for different enhancer-promoter interaction mechanisms. Finally, we discuss how recent technical advances may provide new insights into mechanisms of enhancer-promoter interactions and how technical biases in methods such as Hi-C and Micro-C and imaging techniques may affect their interpretation.

HMGB1-mediated transcriptional activation of circadian gene TIMELESS contributes to endometrial cancer progression through Wnt-ß-catenin pathway.

TIMELESS (TIM) is a circadian gene which is implicated in the regulation of daily rhythm, DNA replication and repair, and cancer initiation and progression. Nevertheless, the role of TIM in endometrial cancer (EC) development is largely unknown. Bioinformatics analysis showed that TIM was aberrantly up-regulated in EC tissues and positively correlated with clinical or histological grade of EC. Functional studies showed that TIM knockdown reduced EC cell viability and restrained EC cell migration in vitro, as well as blocked xenograft tumor growth in vivo. Mechanistically, HMGB1 transcriptionally up-regulated TIM expression in EC cells. In addition, TIM could activate the transcription of the canonical Wnt ligand WNT8B, and TIM depletion could reduce the malignant potential of EC cells largely by targeting and down-regulating WNT8B. As a conclusion, HMGB1/TIM/WNT8B signal cascade was identified in this study for the first time. HMGB1 exerted its oncogenic role by activating the transcription of TIM, leading to the activation of Wnt signaling and EC progression.

Histone deacetylase OsHDA716 represses rice chilling tolerance by deacetylating OsbZIP46 to reduce its transactivation function and protein stability.

Low temperature is a major environmental factor limiting plant growth and crop production. Epigenetic regulation of gene expression is important for plant adaptation to environmental changes, whereas the epigenetic mechanism of cold signaling in rice (Oryza sativa) remains largely elusive. Here, we report that the histone deacetylase (HDAC) OsHDA716 represses rice cold tolerance by interacting with and deacetylating the transcription factor OsbZIP46. The loss-of-function mutants of OsHDA716 exhibit enhanced chilling tolerance, compared with the wild-type plants, while OsHDA716 overexpression plants show chilling hypersensitivity. On the contrary, OsbZIP46 confers chilling tolerance in rice through transcriptionally activating OsDREB1A and COLD1 to regulate cold-induced calcium influx and cytoplasmic calcium elevation. Mechanistic investigation showed that OsHDA716-mediated OsbZIP46 deacetylation in the DNA-binding domain reduces the DNA-binding ability and transcriptional activity as well as decreasing OsbZIP46 protein stability. Genetic evidence indicated that OsbZIP46 deacetylation mediated by OsHDA716 reduces rice chilling tolerance. Collectively, these findings reveal that the functional interplay between the chromatin regulator and transcription factor fine-tunes the cold response in plant and uncover a mechanism by which HDACs repress gene transcription through deacetylating nonhistone proteins and regulating their biochemical functions.

FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain via m6A-dependent stabilization of 5-HT3A mRNA in sensory neurons.

The N6-methyladenosine (m6A) modification of RNA is an emerging epigenetic regulatory mechanism that has been shown to participate in various pathophysiological processes. However, its involvement in modulating neuropathic pain is still poorly understood. In this study, we elucidate a functional role of the m6A demethylase alkylation repair homolog 5 (ALKBH5) in modulating trigeminal-mediated neuropathic pain. Peripheral nerve injury selectively upregulated the expression level of ALKBH5 in the injured trigeminal ganglion (TG) of rats. Blocking this upregulation in injured TGs alleviated trigeminal neuropathic pain, while mimicking the upregulation of ALKBH5 in intact TG neurons sufficiently induced pain-related behaviors. Mechanistically, histone deacetylase 11 downregulation induced by nerve injury increases histone H3 lysine 27 acetylation (H3K27ac), facilitating the binding of the transcription factor forkhead box protein D3 (FOXD3) to the Alkbh5 promoter and promoting Alkbh5 transcription. The increased ALKBH5 erases m6A sites in Htr3a messenger RNA (mRNA), resulting in an inability of YT521-B homology domain 2 (YTHDF2) to bind to Htr3a mRNA, thus causing an increase in 5-HT3A protein expression and 5-HT3 channel currents. Conversely, blocking the increased expression of ALKBH5 in the injured TG destabilizes nerve injury-induced 5-HT3A upregulation and reverses mechanical allodynia, and the effect can be blocked by 5-HT3A knockdown. Together, FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain through m6A-dependent stabilization of Htr3a mRNA in TG neurons. This mechanistic understanding may advance the discovery of new therapeutic targets for neuropathic pain management.

CRISPR activation as a platform to identify interferon stimulated genes with anti-viral function.

Interferon Stimulated Gene (ISG) expression plays a key role in the control of viral replication and development of a robust adaptive response. Understanding this dynamic relationship between the pathogen and host is critical to our understanding of viral life-cycles and development of potential novel anti-viral strategies. Traditionally, plasmid based exogenous prompter driven expression of ISGs has been used to investigate anti-viral ISG function, however there are deficiencies in this approach. To overcome this, we investigated the utility of CRISPR activation (CRISPRa), which allows for targeted transcriptional activation of a gene from its endogenous promoter. Using the CRISPRa-SAM system to induce targeted expression of a panel of anti-viral ISGs we showed robust induction of mRNA and protein expression. We then employed our CRISPRa-SAM ISG panel in several antiviral screen formats to test for the ability of ISGs to prevent viral induced cytopathic cell death (CPE) and replication of Dengue Virus (DENV), Zika Virus (ZIKV), West Nile Virus Kunjin (WNVKUN), Hepatitis A Virus (HAV) and Human Coronavirus 229E (HCoV-229E). Our CRISPRa approach confirmed the anti-viral activity of ISGs like IFI6, IFNß and IFNλ2 that prevented viral induced CPE, which was supported by high-content immunofluorescence imaging analysis. This work highlights CRISPRa as a rapid, agile, and powerful methodology to identify and characterise ISGs and viral restriction factors.

Transcriptional activation of the Myc gene by glucose in ß-cells requires a ChREBP-dependent 3-D chromatin interaction between the Myc and Pvt1 genes.

OBJECTIVE: All forms of diabetes result from insufficient functional ß-cell mass. Thus, achieving the therapeutic goal of expanding ß-cell mass requires a better mechanistic understanding of how ß-cells proliferate. Glucose is a natural ß-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. METHODS: Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. RESULTS: We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated ß-cell proliferation. CONCLUSIONS: The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.