Similar Datasets
Project description:The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo. We also detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells.
2024-09-17 | GSE276777 | GEO
Project description:The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor are appealing towards inducing HIV reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 in both cell line models and in CD4 T cells from virally suppressed PLWH improves reactivation at the transcriptional level and is more specific than AZD5582 & I-BET151 treatment alone. INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observe more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151 indicating the combination may specifically overcome an elongation block to reactivation. We also observe viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 controls full-length HIV RNA and virus production ex vivo.
2024-09-17 | GSE277305 | GEO
Project description:The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor are appealing towards inducing HIV reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 in both cell line models and in CD4 T cells from virally suppressed PLWH improves reactivation at the transcriptional level and is more specific than AZD5582 & I-BET151 treatment alone. INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observe more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151 indicating the combination may specifically overcome an elongation block to reactivation. We also observe viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 controls full-length HIV RNA and virus production ex vivo.
2024-09-17 | GSE276747 | GEO
Project description:Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal
| PRJNA1161690 | ENA
Project description:Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal [CRISPR]
| PRJNA1158849 | ENA
Project description:Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal [AutoCUT&Tag]
| PRJNA1161694 | ENA
Project description:Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal [RNA-seq]
| PRJNA1158793 | ENA
Project description:Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal.
| S-EPMC10172343 | biostudies-literature
Project description:Genetic studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3'end processing of small nuclear RNAs.We found that, in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighbouring GSTCD expression. In contrast to the previous reports in other species, we only observed a minor alteration of snRNA processing following INTS12 depletion. RNAseq analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, ChIPseq experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, we defined the INTS12 regulome which includes genes belonging to the protein synthesis pathways.INTS12 has functions beyond the canonical snRNA processing. We show that it regulates translation by regulating the expression of genes belonging to protein synthesis pathways. This study provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the biology behind the genetic association for lung function at 4q24.
| S-EPMC5364626 | biostudies-literature
Project description:The Drosophila integrator complex consists of 14 subunits that associate with the C terminus of Rpb1 and catalyze the endonucleolytic cleavage of nascent snRNAs near their 3' ends. Although disruption of almost any integrator subunit causes snRNA misprocessing, very little is known about the role of the individual subunits or the network of structural and functional interactions that exist within the complex. Here we developed an RNAi rescue assay in Drosophila S2 cells to identify functional domains within integrator subunit 12 (IntS12) required for snRNA 3' end formation. Surprisingly, the defining feature of the Ints12 protein, a highly conserved and centrally located plant homeodomain finger domain, is not required for reporter snRNA 3' end cleavage. Rather, we find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein. This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. Moreover, mutations within the microdomain not only disrupt IntS12 function but also abolish binding to other integrator subunits. Finally, the IntS12 microdomain is sufficient to interact and stabilize the putative scaffold integrator subunit, IntS1. Collectively, these results identify an unexpected interaction between the largest and smallest integrator subunits that is essential for the 3' end formation of Drosophila snRNA.
| S-EPMC3576091 | biostudies-literature