CRISPR-mediated synergistic epigenetic and transcriptional control
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE163518 | GEO | 2022/12/22
REPOSITORIES: GEO
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Project description:While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we aim to bypass the need for sequence-specific transcription factors and recruit activators directly using catalytically dead Cas9 fused to the synthetic activator VPR. We perform a CRISPRa tiling screen in mouse ESCs that express doxycycline-inducible dCas9-VPR and were engineered with an endogenous T2A-EGFP reporter at the end of the Fgf5 gene. We used a library of over 13,000 guides spanning a 500kb window centered at the Prdm8-Fgf5 TAD. We demonstrate that this approach is unable to achieve detectable dCas9-VPR binding to arbitrary genomic sites making it impossible to address whether any genomic site to which activators are recruited can function as an enhancer. To overcome this limitation, we turned to CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.
2024-12-02 | GSE278772 | GEO
Project description:Lentiviral accessory genes enhance replication through diverse mechanisms. HIV-1 accessory protein Vpr modulates the host DNA damage response (DDR) at multiple steps through DNA damage, cell cycle arrest, the degradation of host proteins, and both the activation and repression of DDR signaling. Vpr also alters host and viral transcription; however, the connection between Vpr-mediated DDR modulation and transcriptional activation remains unclear. Here, we determined the cellular consequences of Vpr-induced DNA damage using Vpr mutants that allow us to separate the ability of Vpr to induce DNA damage from cell cycle arrest and other DDR phenotypes including host protein degradation and repression of DDR. RNA-sequencing of cells expressing Vpr or Vpr mutants identified that Vpr alters cellular transcription through mechanisms both dependent and independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), Vpr-induced DNA damage activates the ATM-NEMO pathway and alters cellular transcription via NF-κB/RelA signaling. HIV-1 infection of primary MDMs validated Vpr-dependent NF-κB transcriptional activation during infection. Both virion delivered and de novo expressed Vpr induced DNA damage and activated ATM-NEMO dependent NF-κB transcription, suggesting that engagement of the DDR and transcriptional reprogramming can occur during early and late stages of viral replication. Together, our data identifies a mechanism by which Vpr activates NF-κB through DNA damage and the ATM-NEMO pathway, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral transcription and replication.
2024-08-23 | GSE253779 | GEO
Project description:Efficient targeted control of exon splicing is a major goal of functional genomic and therapeutic applications. Guide RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility and presumed specificity of these systems. However, efficient, selective, and generalizable activation of targeted endogenous exons using this approach has not been reported. Here, we identify dCasRx-RBM25 as a potent activator of exons by screening over 300 dCasRx splicing factor fusions tethered to splicing reporters. dCasRx-RBM25 also strongly activates splicing of endogenous alternative exons, when recruited to downstream intron sequences using single guide RNAs. In transcriptome wide analyses we observe a high degree of specificity of dCasRx-RBM25 for endogenous exon targeting. We further leverage the guide array-processing activity of dCasRx to simultaneously target multiple endogenous exons for activation and repression by dCasRx-RBM25. Our results pave the way for versatile exon-resolution functional assays and splicing-directed therapeutic applications.
2024-09-19 | GSE243979 | GEO
Project description:The specificity of gene activation, both the dCasMINI-VPR and dhpCasMINI-VPR significantly and obviously activated IL1RN and only a few genes showed expression change, indicating high genome-wide targeted transcriptional specificity.
2024-06-29 | GSE270586 | GEO
Project description:The events controlling transition of T cells from effector to memory remain poorly understood. Herein, we defined two types of effectors on the basis of cell division, expression of activation markers and production of effector cytokines and investigated their potential for memory generation. The results indicated that the moderately activated early effectors readily transit to memory while highly activated late effectors develop minimal memory. Boosting with antigen-free adjuvant, however, rescues the late effectors from cell death and sustains both survival and cytokine responses through toll-like receptor function. Also, the genome-wide microarray analysis among early and late effectors pointed to a set og defined as well as less known genes that may play a role in transition to memory. CFSE-labeled DO11.10 CD4 naïve T cells were transferred into Balb/c mice and the hosts were immunized with OVA peptide in CFA. Within 3 days, the lymph node cells were harvested and each effector division (from division 2 to 8) was sorted based on CFSE dilution and then they were either transferred into naive hosts to generate memory cells or their RNAs were extracted to perform genome-wide microarray analysis. To correlate gene expression with memory potential, division 4 (D4), which readily transit to memory, and divisions 3 (D3), 6 (D6) and 8 (D8), which display reduced potential for memory transition, were included. The data were derived from 3 independent experiments (3 samples per division, 12 samples total).
2011-01-01 | E-GEOD-25141 | biostudies-arrayexpress
Project description:Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders. Human fetal neurons were chosen to examine the impact of HIV-1 Vpr protein on gene expression
2012-12-07 | E-GEOD-42424 | biostudies-arrayexpress
Project description:Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders. Human neurons SH-SY5Y were chosen to examine the impact of HIV-1 Vpr protein on gene expression
2013-01-17 | E-GEOD-43535 | biostudies-arrayexpress
Project description:Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders. Using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Vpr) on the expression of miRNAs and mRNAs.
2013-02-12 | E-GEOD-44266 | biostudies-arrayexpress
Project description:The accessory viral protein R (Vpr) is encoded by all primate lentiviruses. Vpr counteracts DNA repair pathways, modulates viral immune sensing, and induces cell-cycle arrest in cell culture. However, its impact in vivo is controversial. Here, we show that deletion of vpr is associated with delayed viral replication kinetics, rapid innate immune activation, development and maintenance of strong B cell responses, and generation of higher avidity p27 Gag antibodies in rhesus macaques. All wild-type SIVmac239 infected animals maintained high viral loads and five of six developed fatal immunodeficiency during ~80 weeks of follow-up. Lack of Vpr was associated with better preservation of CD4+ T cells, lower viral loads and an attenuated clinical course of infection in most animals. Our results demonstrate that Vpr contributes to efficient viral immune evasion and the full pathogenic potential of SIVmac in vivo. Inhibition of Vpr may improve humoral immune control of viral replication.
2023-12-06 | GSE229310 | GEO