Endogenous Transcription Factors Research Articles

Position-dependent function of human sequence-specific transcription factors.

Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF)binding sites1-3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF's distinct functional profiles-many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TFbinding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

Nuclear Factor Erythroid 2-Related Factor 2 as a Potential Therapeutic Target in Neonatal Hypoxic-Ischemic Encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is a prominent cause of neonatal mortality and neurodevelopmental disorders; however, effective therapeutic interventions remain limited. During neonatal hypoxic-ischemic injury events, increased reactive oxygen species (ROS) production and decreased antioxidant levels lead to the induction of oxidative stress, which plays a pivotal role in the pathological process of neonatal HIE. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key endogenous antioxidant transcription factor that protects against oxidative stress by promoting the transcription of various antioxidant genes. It has been demonstrated that Nrf2 signaling pathway activation by different compounds may protect against neonatal HIE. This review outlines the role of oxidative stress in neonatal HIE and summarizes the impact of antioxidants on neonatal HIE via activation of the Nrf2 signaling pathway. In conclusion, Nrf2 signaling pathway potentially exerts antioxidant, anti-inflammatory, antiapoptotic and antiferroptotic effects, thereby emerging as a focal point for future neonatal HIE treatment strategies.

Male breast cancer differs from female breast cancer in molecular features that affect prognoses and drug responses

BackgroundMale breast cancer (MBC) is a rare malignancy with a worse prognosis than female breast cancer (FBC). Current MBC treatment strategies are based on those for FBC. However, molecular differences between MBC and FBC with respect to prognosis and drug responses remain unclear. MethodsAfter controlling for confounding factors with propensity score matching (PSM), differences between MBC and FBC were comprehensively analyzed using many types of data: survival, immune microenvironments, sex hormone responses, drug sensitivity, transcriptomes, genomes, epigenomes, and proteomes. ResultsOverall survival (OS) and cancer-specific survival (CSS) were both worse for MBC than for FBC. Differentially expressed mRNAs were enriched in numerous cancer-related functions and pathways, with SPAG16 and STOX1 being as the most important prognosis-related mRNAs for MBC. Competing endogenous RNA (ceRNA) and transcription factor (TF)-mRNA regulatory networks contain potential prognostic genes. Nine genes had higher mutation frequencies in MBC than in FBC. MBC shows a comparatively poor response to immunotherapy, with five proteins that promote breast cancer progression being highly expressed in MBC. MBC may be more responsive than FBC to estrogen. We detected six United States Food and Drug Administration (FDA)-approved therapeutic target genes as being differentially expressed between MBC and FBC. ConclusionThe poor prognosis of MBC compared to FBC is due to numerous molecular differences and resulting drug responses.

Cell-free genomics: transcription factor interactions in reconstituted naïve embryonic chromatin.

Extracts from Drosophila preblastoderm embryos (DREX) form the basis of a powerful in vitro chromatin reconstitution system that assembles entire genomes into complex chromatin with physiological nucleosome spacing and polymer condensation. As the zygotic genome has not yet been activated in preblastoderm embryos, the reconstitution extract lacks endogenous transcription factors (TFs) and the RNA polymerase machinery. At the same time, it contains high levels of ATP-dependent nucleosome sliding enzymes that render the reconstituted chromatin dynamic. The naïve chromatin can be used to determine the intrinsic DNA binding properties of exogenous, usually recombinant TFs (or DNA binding proteins in general) in a complex chromatin context. Recent applications of the system include the description of cooperation and competition of Drosophila pioneer TFs for composite binding sites, and the characterization of nucleosome interactions of mammalian pioneer TFs in the heterologous system.

ChEC-seq2: an improved chromatin endogenous cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein-DNA interactions.

Defining the in vivo DNA binding specificity of transcription factors (TFs) has relied nearly exclusively on chromatin immunoprecipitation (ChIP). While ChIP reveals TF binding patterns, its resolution is low. Higher resolution methods employing nucleases such as ChIP-exo, chromatin endogenous cleavage (ChEC-seq) and CUT&RUN resolve both TF occupancy and binding site protection. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC has been suggested to be lower than the specificity of the peaks identified by ChIP or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. We have simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA. ChEC-seq2 cleavage patterns were highly reproducible between replicates and with published ChEC-seq data. Combined with DoubleChEC, a new bioinformatic pipeline that removes non-specific cleavage sites, ChEC-seq2 identified high-confidence cleavage sites for three different yeast TFs that are strongly enriched for their known binding sites and adjacent to known target genes.

Identification of pivotal genes and regulatory networks associated with atherosclerotic carotid artery stenosis based on comprehensive bioinformatics analysis and machine learning.

Bioinformatics methods were applied to investigate the pivotal genes and regulatory networks associated with atherosclerotic carotid artery stenosis (ACAS) and provide new insights for the treatment of this disease. The study utilized five ACAS datasets (GSE100927, GSE11782, GESE28829, GSE41571, and GSE43292) downloaded from the NCBI GEO database. The first four datasets were combined as the training set (n = 99), while GSE43292 (n = 64) was used as the validation set. Difference analysis and functional enrichment analysis were then performed on the training set. The pathogenic targets of ACAS were screened by protein-protein interaction networks and MCODE analyses, combined with three machine learning algorithms. The results were next verified by analysis of inter-group differences and ROC curve analysis. Next, immune-related function and immune cell correlation analyses were performed, and plaques of human ACAS were applied to verify the results via immunohistochemistry (IH) and immunofluorescence (IF). Finally, the competing endogenous RNAs (ceRNA) and transcription factors (TFs) regulatory networks of the characterized genes were constructed. A total of 177 differentially expressed genes were identified, including 67 genes downregulated and 110 genes upregulated. Gene set enrichment analysis revealed that five pathways were active in the experimental group, including xenograft rejection, autoimmune thyroid disease, graft-versus-host disease, leishmaniasis infection, and lysosomes. Four key genes were identified, with C3AR1 being upregulated and FBLN5, PPP1R12A, and TPM1 being downregulated. The analysis of inter-group differences demonstrated that the four characterized genes were differentially expressed in both the control and experimental groups. The ROC analysis showed that they had high AUC values in both the training and validation sets. Therefore, a predictive ACAS patient nomogram model based on the screened genes was established. Correlation analysis revealed a positive correlation between C3AR1 expression and neutrophils, which was further validated in IH and IF. One or multiple lncRNAs may compete with the characterized genes for binding miRNAs. Additionally, each characterized gene interacts with multiple TFs. Four pivotal genes were screened, and relevant ceRNA and TFs were predicted. These molecules may exert a crucial role in ACAS and serve as potential biomarkers and therapeutic targets.

Transcriptome Analysis Reveals Candidate Genes Involved in Gibberellin-Induced Fruit Development in Rosa roxburghii.

Gibberellins (GAs) play indispensable roles in the fruit development of horticultural plants. Unfortunately, the molecular basis behind GAs regulating fruit development in R. roxburghii remains obscure. Here, GA3 spraying to R. roxburghii 'Guinong 5' at full-bloom promoted fruit size and weight, prickle development, seed abortion, ascorbic acid accumulation, and reduction in total soluble sugar. RNA-Seq analysis was conducted to generate 45.75 Gb clean reads from GA3- and non-treated fruits at 120 days after pollination. We obtained 4275 unigenes belonging to differently expressed genes (DEGs). Gene ontology and the Kyoto Encyclopedia of Genes and Genomes displayed that carbon metabolism and oxidative phosphorylation were highly enriched. The increased critical genes of DEGs related to pentose phosphate, glycolysis/gluconeogenesis, and citrate cycle pathways might be essential for soluble sugar degradation. Analysis of DEGs implicated in ascorbate revealed the myoinositol pathway required to accumulate ascorbic acid. Finally, DEGs involved in endogenous phytohormones and transcription factors, including R2R3 MYB, bHLH, and WRKY, were determined. These findings indicated that GA3-trigged morphological alterations might be related to the primary metabolites, hormone signaling, and transcription factors, providing potential candidate genes that could be guided to enhance the fruit development of R. roxburghii in practical approaches.

Comprehensive analysis on subchondral bone marrow lesions of human osteoarthritis by integrating bulk and single-cell transcriptomes

ObjectiveThis study aims to demonstrate the cellular composition and underlying mechanisms in subchondral bone marrow lesions (BMLs) of knee osteoarthritis (OA).MethodsBMLs were assessed by MRI Osteoarthritis Knee Score (MOAKS)≥2. Bulk RNA-sequencing (bulk-seq) and BML-specific differentially expressed genes (DEGs) analysis were performed among subchondral bone samples (including OA-BML=3, paired OA-NBML=3; non-OA=3). The hub genes of BMLs were identified by verifying in independent datasets and multiple bioinformatic analyses. To further estimate cell-type composition of subchondral bone, we utilized two newly developed deconvolution algorithms (MuSiC, MCP-counter) in transcriptomic datasets, based on signatures from open-accessed single-cell RNA sequencing (scRNA-seq). Finally, competing endogenous RNA (ceRNA) and transcription factor (TF) networks were constructed through multiple predictive databases, and validated by public non-coding RNA profiles.ResultsA total of 86 BML-specific DEGs (up 79, down 7) were identified. IL11 and VCAN were identified as core hub genes. The “has-miR-424-5p/lncRNA PVT1” was determined as crucial network, targeting IL11 and VCAN, respectively. More importantly, two deconvolution algorithms produced approximate estimations of cell-type composition, and the cluster of heterotopic-chondrocyte was discovered abundant in BMLs, and positively correlated with the expression of hub genes.ConclusionIL11 and VCAN were identified as the core hub genes of BMLs, and their molecular networks were determined as well. We profiled the characteristics of subchondral bone at single-cell level and determined that the heterotopic-chondrocyte was abundant in BMLs and was closely linked to IL11 and VCAN. Our study may provide new insights into the microenvironment and pathological molecular mechanism of BMLs, and could lead to novel therapeutic strategies.

Small Molecules for Chemically Induced Pluripotency Inception

Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception refers to using specific small molecules to activate endogenous transcription factors to reprogram adult cells into induced pluripotent stem cells (iPSCs). This approach aims to bypass the use of viral vectors or genetic manipulation to introduce exogenous factors, making it a potentially safer and more efficient method for generating iPSCs. Jin and colleagues show that small molecule CD3254 activates endogenous existing transcription factor RXRα to significantly promote mouse chemical reprogramming. Mechanistically, the CD3254-RXRα axis can directly activate all the 11 RNA exosome component genes (Exosc1-10 and Dis3) at the transcriptional level. Unexpectedly, rather than degrading mRNAs as its substrates, RNA exosome mainly modulates the degradation of transposable element (TE)-associated RNAs, particularly MMVL30, which is identified as a new barrier for cell-fate determination. In turn, MMVL30-mediated inflammation (IFN-γ and TNF-α pathways) is reduced, promoting successful reprogramming. Collectively, their study provides conceptual advances for translating environmental cues into pluripotency inception, particularly identifies that the CD3254-RXRα-RNA exosome axis can promote chemical reprogramming and suggests modulation of TE-mediated inflammation via CD3254-inducible RNA exosome as important opportunities for controlling cell fates and regenerative medicine. Proc Natl Acad Sci USA. 2023 May 23;120(21):e2215155120. doi: 10.1073/pnas.2215155120.

Engineering an Efficient Expression Using Heterologous GAL Promoters and Transcriptional Activators in Saccharomyces cerevisiae.

Galactose-inducible (GAL) promoters have been widely used in metabolic engineering in Saccharomyces cerevisiae for production of valuable products. Endogenous GAL promoters and GAL transcription factors have often been engineered to improve GAL promoter activities. Heterologous GAL promoters and GAL activator (Gal4p-like transcriptional activators), although existing in other yeasts or fungi, have not been well explored. In this study, we comprehensively characterized the activation effects of Gal4p activators from different yeasts or fungi on a variant of GAL promoters. Overexpressing endogenous Gal4p driven by PHHF1 increased the activities of native PGAL1 and heterologous PSkGAL2 by 131.20% and 72.45%, respectively. Furthermore, eight transcriptional activators from different organisms were characterized and most of them exhibited functions that were consistent with ScGal4p. Expression of KlLac9p from Kluyveromyces lactis further increased the activity of PScGAL1 and PSkGAL2 by 41.56% and 100.63%, respectively, compared to ScGal4p expression, and was able to evade Gal80p inhibition. This optimized GAL expression system can be used to increase the production of β-carotene by 9.02-fold in S. cerevisiae. Our study demonstrated that a combination of heterologous transcriptional activators and GAL promoters provided novel insights into the optimization of the GAL expression system.

Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception

Chemistry-alone approach has recently been applied for incepting pluripotency in somatic cells, representing a breakthrough in biology. However, chemical reprogramming is hampered by low efficiency, and the underlying molecular mechanisms remain unclear. Particularly, chemical compounds do not have specific DNA-recognition domains or transcription regulatory domains, and then how do small molecules work as a driving force for reinstating pluripotency in somatic cells? Furthermore, how to efficiently clear materials and structures of an old cell to prepare the rebuilding of a new one? Here, we show that small molecule CD3254 activates endogenous existing transcription factor RXRα to significantly promote mouse chemical reprogramming. Mechanistically, CD3254-RXRα axis can directly activate all the 11 RNA exosome component genes (Exosc1-10 and Dis3) at transcriptional level. Unexpectedly, rather than degrading mRNAs as its substrates, RNA exosome mainly modulates the degradation of transposable element (TE)-associated RNAs, particularly MMVL30, which is identified as a new barrier for cell-fate determination. In turn, MMVL30-mediated inflammation (IFN-γ and TNF-α pathways) is reduced, contributing to the promotion of successful reprogramming. Collectively, our study provides conceptual advances for translating environmental cues into pluripotency inception, particularly, identifies that CD3254-RXRα-RNA exosome axis can promote chemical reprogramming, and suggests modulation of TE-mediated inflammation via CD3254-inducible RNA exosome as important opportunities for controlling cell fates and regenerative medicine.

Species origin of exogenous transcription factors affects the activation of endogenous pluripotency markers and signaling pathways of porcine induced pluripotent stem cells.

The incomplete silencing of exogenous transcription factors (TFs) and the lack of endogenous counterpart activation hampers the application of porcine induced pluripotent stem cells (piPSCs). We used porcine, bovine and murine TFs to reprogram porcine fetal fibroblasts. Porcine TFs-derived piPSCs (ppiPSCs) showed the highest levels of endogenous pluripotency markers activation, were able to differentiate into three germ layers and primordial germ cell-like cells (PGCLCs) and integrated into neural ectoderm of E7.5 mouse embryos in vitro. The bovine TFs derived piPSCs (bpiPSCs) expressed endogenous pluripotency markers higher than murine TFs derived piPSCs (mpiPSCs), but both had limited differentiation ability in vitro and depended on continuous expression of exogenous TFs for the maintenance. RNA sequencing confirmed ppiPSCs had distinct global transcriptional profiling, upregulated Hippo, PI3K-Akt, MAPK and relevant pluripotency signaling pathways as porcine blastocyst inner cell mass and expressed PGC early related genes. In addition, a positive and a negative correlation between exogenous and endogenous TFs' expression level were observed in ppiPSCs and bpiPSCs lines, respectively. The TFs' protein structures in pig were more similar to cattle than to mouse. In conclusion, the species affinity of the exogenous TFs is a key element, and the own species origin of TFs is optimal for iPSCs generation and application.

Identification of immune-related molecular markers in intracranial aneurysm (IA) based on machine learning and cytoscape-cytohubba plug-in

BackgroundIntracranial aneurysm (IA) is a common cerebrovascular disease. The immune mechanism of IA is more complicated, and it is unclear so far. Therefore, it is necessary to continue to explore the immune related molecular mechanism of IA.MethodsAll data were downloaded from the public database. Limma package and ssGSEA algorithm was used to identify differentially expressed mRNAs (DEmRNAs) and analyze immune cell infiltration, respectively. Machine learning and cytoscape-cytohubba plug-in was used to identify key immune types and multicentric DEmRNAs of IA, respectively. Multicentric DEmRNAs related to key immune cells were screened out as key DEmRNAs by Spearman correlation analysis. Diagnostic models, competing endogenous RNA (ceRNA) regulatory network and transcription factor regulatory network were constructed based on key DEmRNAs. Meanwhile, drugs related to key DEmRNAs were screened out based on DGIdb database. The expression of key DEmRNAs was also verified by real time-PCR.ResultsIn this study, 7 key DEmRNAs (NRXN1, GRIA2, SLC1A2, SLC17A7, IL6, VEGFA and SYP) associated with key differential immune cell infiltration (CD56bright natural killer cell, Immature B cell and Type 1 T helper cell) were identified. Functional enrichment analysis showed that VEGFA and IL6 may be involved in the regulation of the PI3K-Akt signaling pathway. Moreover, IL6 was also found to be enriched in cytokine-cytokine receptor interaction signaling pathway. In the ceRNA regulatory network, a large number of miRNAs and lncRNAs were found. In the transcription factor regulatory network, the transcription factor SP1 was correlated with VEGFA, SYP and IL6. It is also predicted that drugs related to key DEmRNAs such as CARBOPLATIN, FENTANYL and CILOSTAZOL may contribute to the treatment of IA. In addition, it was also found that SVM and RF models based on key DEmRNAs may be potential markers for diagnosing IA and unruptured intracranial aneurysm (UIA), respectively. The expression trend of key DEmRNAs verified by real-time PCR was consistent with the bioinformatics analysis results.ConclusionThe identification of molecules and pathways in this study provides a theoretical basis for understanding the immune related molecular mechanism of IA. Meanwhile, the drug prediction and diagnosis model construction may also be helpful for clinical diagnosis and management.

HIF-1α-activated TMEM237 promotes hepatocellular carcinoma progression via the NPHP1/Pyk2/ERK pathway.

Hypoxia-inducible factors (HIFs) are the most essential endogenous transcription factors in the hypoxic microenvironment and regulate multiple genes involved in the proliferation, migration, invasion, and EMT of hepatocellular carcinoma (HCC) cells. However, the regulatory mechanism of HIFs in driving HCC progression remains poorly understood. Gain- and loss-of-function experiments were carried out to investigate the role of TMEM237 in vitro and in vivo. The molecular mechanisms involved in HIF-1α-induced TMEM237 expression and TMEM237-mediated enhancement of HCC progression were confirmed by luciferase reporter, ChIP, IP-MS and Co-IP assays. TMEM237 was identified as a novel hypoxia-responsive gene in HCC. HIF-1α directly bound to the promoter of TMEM237 to transactivate its expression. The overexpression of TMEM237 was frequently detected in HCC and associated with poor clinical outcomes in patients. TMEM237 facilitated the proliferation, migration, invasion, and EMT of HCC cells and promoted tumor growth and metastasis in mice. TMEM237 interacted with NPHP1 and strengthened the interaction between NPHP1 and Pyk2 to trigger the phosphorylation of Pyk2 and ERK1/2, thereby contributing to HCC progression. The TMEM237/NPHP1 axis mediates hypoxia-induced activation of the Pyk2/ERK1/2 pathway in HCC cells. Our study demonstrated that HIF-1α-activated TMEM237 interacted with NPHP1 to activate the Pyk2/ERK pathway, thereby promoting HCC progression.

An Optogenetic-Controlled Cell Reprogramming System for Driving Cell Fate and Light-Responsive Chimeric Mice.

Pluripotent stem cells (PSCs) hold great promise for cell-based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light-inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease-deficient CRISPR-associated protein 9for induced PSCs reprogramming. This system enables remote, non-invasive optogenetical regulation of endogenous Sox2and Oct4loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSCLIRE ) under light-emitting diode-based illumination. iPSCLIRE cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSCLIRE cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non-invasive control of user-defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non-invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications.

Identification of GLS as a cuproptosis-related diagnosis gene in acute myocardial infarction.

Acute myocardial infarction (AMI) has the characteristics of sudden onset, rapid progression, poor prognosis, and so on. Therefore, it is urgent to identify diagnostic and prognostic biomarkers for it. Cuproptosis is a new form of mitochondrial respiratory-dependent cell death. However, studies are limited on the clinical significance of cuproptosis-related genes (CRGs) in AMI. In this study, we systematically assessed the genetic alterations of CRGs in AMI by bioinformatics approach. The results showed that six CRGs (LIAS, LIPT1, DLAT, PDHB, MTF1, and GLS) were markedly differentially expressed between stable coronary heart disease (stable_CAD) and AMI. Correlation analysis indicated that CRGs were closely correlated with N6-methyladenosine (m6A)-related genes through R language "corrplot" package, especially GLS was positively correlated with FMR1 and MTF1 was negatively correlated with HNRNPA2B1. Immune landscape analysis results revealed that CRGs were closely related to various immune cells, especially GLS was positively correlated with T cells CD4 memory resting and negatively correlated with monocytes. Kaplan-Meier analysis demonstrated that the group with high DLAT expression had a better prognosis. The area under curve (AUC) certified that GLS had good diagnostic value, in the training set (AUC = 0.87) and verification set (ACU = 0.99). Gene set enrichment analysis (GSEA) suggested that GLS was associated with immune- and hypoxia-related pathways. In addition, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, competing endogenous RNA (ceRNA) analysis, transcription factor (TF), and compound prediction were performed to reveal the regulatory mechanism of CRGs in AMI. Overall, our study can provide additional information for understanding the role of CRGs in AMI, which may provide new insights into the identification of therapeutic targets for AMI.

Potato virus X-delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.

SUMMARYProgrammable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens‐mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus‐derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X‐derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus‐induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non‐invasive spraying method, we show that gRNA‐mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X‐based virus‐induced gene reprogramming strategy results in program‐specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid‐enriched metabolite profiles.

Kynurenine monooxygenase inhibition and associated reduced quinolinic acid reverses depression-like behaviour by upregulating Nrf2/ARE pathway in mouse model of depression: In-vivo and In-silico studies

Kynurenine pathway, a neuroimmunological pathway plays a substantial role in depression. Consistently, increased levels of neurotoxic metabolite of kynurenine pathway; quinolinic acid (QA) found in the suicidal patients and remitted major depressive patients. QA, an endogenous modulator of N-methyl-d-aspartate receptor is produced by microglial cells, may serve as a potential candidate for a link between antioxidant defence system and immune changes in depression. Further, nuclear factor (erythroid-derived 2) like 2 (Nrf2), an endogenous antioxidant transcription factor plays a significant role in maintaining antioxidant homeostasis during basal and stress conditions. The present study was designed to explore the effects of KMO-inhibition (Kynurenine monooxygenase) and association of reduced QA on Keap1/Nrf2/ARE pathway activity in olfactory bulbectomized mice (OBX-mice). KMO catalysis the neurotoxic branch of kynurenine pathway directing the synthesis of QA. KMO inhibitionshowed significant reversal of depressive-like behaviour, restored Keap-1 and Nrf2 mRNA expression, and associated antioxidant levels in cortex and hippocampus of OBX-mice. KMO inhibition also increased PI3K/AKT mRNA expression in OBX-mice. KMO inhibition and associated reduced QA significantly decreased inflammatory markers, kynurenine and increased the 5-HT, 5-HIAA and tryptophan levels in OBX-mice. Furthermore, molecular docking studies has shown good binding affinity of QA towards ubiquitin proteasome complex and PI3K protein involved in Keap-1 dependent and independent proteasome degradation of Nrf2 respectively supporting our in-vivo findings. Hence, QA might act as pro-oxidant through downregulating Nrf2/ARE pathway along with modulating other pathways and KMO inhibition could be a potential therapeutic target for depression treatment.

Abstract 2362: Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

Abstract Background: Gene expression networks are perturbed in every human malignancy. One common way that this occurs is through chromosomal translocations such as the the t(2;13)(q35;q14), which creates the oncogenic transcription factor, PAX3-FOXO1, in alveolar rhabdomyosarcoma (aRMS). Identification of the direct transcriptional targets and mechanisms of control by translocated transcription factors is critical to our understanding of disease etiology and the development of targeted therapeutic approaches. However, defining the genes directly controlled by DNA binding proteins and the mechanisms of regulation at those direct target genes is challenging, because traditional genetic approaches to inactivate transcription factors cannot distinguish direct from indirect changes in gene expression. Methods: A CRISPR-based approach was used to engineer the endogenous PAX3-FOXO1 locus to incorporate the FKBP12F36V degron tag. This allowed degradation of PAX3-FOXO1 within 2 hr of treatment with the proteolysis targeting chimera, dTAG-47. We performed short time courses to analyze nascent transcription (precision nuclear run-on sequencing; PRO-seq), chromatin accessibility (ATAC-seq), and genome-wide mapping of transcriptional complexes (CUT&RUN). This identified a small cohort of high-confidence PAX3-FOXO1 gene targets and associated PAX3-FOXO1-regulated enhancer elements. In addition, we used CRISPR editing to add APEX2 and 3XFLAG tags to PAX3-FOXO1 to enable deep proteomics to identify PAX3-FOXO1-associated transcriptional complexes in aRMS cell lines. Results: Degradation of PAX3-FOXO1-FKBP12F36V led to growth inhibition, hallmarks of myogenic differentiation, and reduced growth in soft agar. PAX3-FOXO1 binding was detected at over 44,000 sites throughout the genome using CUT&RUN, and loss of chromatin bound PAX3-FOXO1 was observed within 2-4hr of PAX3-FOXO1 degradation. PRO-seq analysis defined a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Unexpectedly, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. Deep proteomic analysis showed that PAX3-FOXO1 was in close proximity to many transcriptional regulatory complexes, but appeared to coordinate the activity of these complexes at only a few hundred sites. Moreover, the continued presence of PAX3-FOXO1 at these elements was required to maintain chromatin accessibility. Conclusion: This work demonstrates the utility of using rapid degradation of endogenous transcription factors to define their direct transcription targets and the mechanisms by which these factors control gene expression. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network, and emphasizes the utility of PAX3-FOXO1 as a therapeutic target in aRMS. Citation Format: Susu Zhang, Jing Wang, Qi Liu, Hayes McDonald, Monica Bomber, Hillary Layden, Jacob Ellis, Scott Borinstein, Scott Hiebert, Kristy Stengel. Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2362.

CRISPR signal conductor 2.0 for redirecting cellular information flow

A key challenge in designing intelligent artificial gene circuits is generating flexible connections between arbitrary components and directly coupling them with endogenous signaling pathways. The CRISPR signal conductor based on conditionally inducible artificial transcriptional regulators can link classic cellular protein signals with targeted gene expression, but there are still problems with multiple signal processing and gene delivery. With the discovery and characterization of new Cas systems and long noncoding RNA (lncRNA) functional motifs, and because of the compatibility of guide RNA with noncoding RNA elements at multiple sites, it is increasingly possible to solve these problems. In this study, we developed CRISPR signal conductor version 2.0 by integrating various lncRNA functional motifs into different parts of the crRNA in the CRISPR-dCasΦ system. This system can directly regulate the expression of target genes by recruiting cellular endogenous transcription factors and efficiently sense a variety of protein signals that are not detected by a classical synthetic system. The new system solved the problems of background leakage and insensitive signaling responses and enabled the construction of logic gates with as many as six input signals, which can be used to specifically target cancer cells. By rewiring endogenous signaling networks, we further demonstrated the effectiveness and biosafety of this system for in vivo cancer gene therapy.