A Genome-wide CRISPR-Based Screen Identifies KAT7 as a Senescence Driver
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PROVIDER: PRJNA510979 | ENA |
REPOSITORIES: ENA
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Project description:Understanding the genetic and epigenetic bases of cellular senescence is instrumental to aging intervention. We performed genome-wide CRISPR/Cas9-based screens in two human mesenchymal precursor cell (hMPC) models of progeroid syndromes and identified hundreds of genes whose deficiency alleviated cellular senescence. Among them, KAT7, a histone acetyltransferase, ranked as a top hit in both models. Inactivation of KAT7 decreased H3 lysine 14 acetylation (H3K14ac), repressed p15INK4b transcription, and rejuvenated both physiologically and pathologically aged cells. Moreover, lentiviral vectors encoding Cas9/sg-Kat7 alleviated liver senescence and extended healthspan and lifespan of mice. Our findings demonstrate that CRISPR/Cas9-based genetic screening is a robust method for systematically uncovering unknown senescence genes, of which KAT7 may represent a new therapeutic target for aging intervention.
2020-10-09 | GSE124197 | GEO
Project description:Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, that RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1γ (HP1γ) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process.
2024-02-14 | GSE253231 | GEO
Project description:We carried out CRISPR-Cas9 functional screen focused on AP-1 bound enhancers that are activated upon oncogenic stress. The screen discovered Enh.AP1.OIS1 - an enhancer bound by AP1 that is required for activation of oncogene-induced senescence (OIS) response. We applied RNA-seq analysis to RASG12V-activated BJ cells transduced with either sgRNAs targeting AP-1 bound enhancer (sgRNA-69 or sgRNA71), or sgRNA targeting the target gene FOXF1 or non-targeting control sgRNA (sgNT)
2018-07-15 | GSE112458 | GEO
Project description:Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDR) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend lifespan in DC cells. Of the cellular clones isolated due to increased lifespan, 34% had a guide RNA (gRNA) targeting CEBPB while gRNAs targeting WSB1, MED28 and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA-Seq to compare DC and control cells. Expression of inflammatory genes were found to be significantly elevated (p<0.0001) in addition to a key subset of these inflammation-related genes (IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5) which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere-length dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB’s contribution in DC cells’ senescence is ROS-independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.
2023-08-30 | GSE224293 | GEO
Project description:West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen using CRISPR/Cas9. HEK 293FT cells were infected with lentivirus expressing sgRNAs and then transfected with a Cas9 expressing construct. WNV infection killed most cells during a 12d selection. Survivor cells were harvested, from which DNA was isolated. The sgRNAs integrated in genome of survivor cells were amplified with PCR. The PCR product was sequenced with Illumina MiSeq to profile the sgRNA population in the survivor cells. Three replicates were conducted. Similarly, a second round of screen was conducted. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J2, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Examination of sgRNA populations in survival 293FT cells
2015-07-05 | E-GEOD-69666 | biostudies-arrayexpress
Project description:During this last decade the development of pro-senescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their anti-tumour activity. Here, using a functional genome wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalized medicine in order to increase their efficiency, stratify patients and avoid drug resistance.
2022-03-31 | GSE192525 | GEO
Project description:In the conventional model of transcriptional activation, transcription factors bind to response elements and recruit co-factors, including histone acetyltransferases. Contrary to this model, we show that the histone acetyltransferase KAT7 (HBO1/MYST2) is required genome-wide for histone H3 lysine 14 acetylation (H3K14ac). Examining neural stem cells, we found that KAT7 and H3K14ac were present not only at transcribed genes, but also at inactive genes, intergenic regions and in heterochromatin. KAT7 and H3K14ac were not required for the continued transcription of genes that were actively transcribed at the time of loss of KAT7, but indispensable for the activation of repressed genes. The absence of KAT7 abrogated neural stem cell plasticity, diverse differentiation pathways and cerebral cortex development. Reexpression of KAT7 restored stem cell developmental potential. Overexpression of KAT7 enhanced neuron and oligodendrocyte differentiation. Our data suggest that KAT7 prepares chromatin for transcriptional activation and is a prerequisite for gene activation.
2022-12-09 | GSE220602 | GEO
Project description:Functional CRISPR Screen Identifies AP1-associated Enhancer regulating FOXF1 to modulate Oncogene-Induced Senescence
| PRJNA447432 | ENA