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CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites
Published on: November 14, 2018
A CRISPR/Cas9 screen identifies the histone demethylase MINA53 as a novel HIV-1 latency-promoting gene (LPG)
Huachao Huang1, Weili Kong2, Maxime Jean3
1Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA.
Abstract:
Although combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53's function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.
Insights
Scientists identified MINA53 as a key factor promoting HIV-1 latency. Inhibiting MINA53 and its associated epigenetic modifications may help reactivate latent HIV-1 reservoirs for potential clearance.
Area of Science:
- Virology
- Epigenetics
- Molecular Biology
Background:
- Combination antiretroviral therapy effectively suppresses HIV-1 replication but cannot eliminate latent reservoirs.
- Latent HIV-1 proviruses, transcriptionally inactive, persist in infected cells, posing a major barrier to HIV-1 eradication.
- Identifying host factors that regulate HIV-1 latency is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To investigate the role of the histone demethylase MINA53 in maintaining HIV-1 latency.
- To elucidate the epigenetic mechanisms by which MINA53 influences HIV-1 latency.
- To explore therapeutic strategies targeting MINA53 for the reactivation of latent HIV-1.
Main Methods:
- CRISPR/Cas9 sgRNA screens to identify potential HIV-1 latency-promoting genes.
- RNA interference (RNAi) to deplete MINA53 and assess its effect on HIV-1 latency.
- Biochemical assays to determine MINA53's histone demethylase activity and substrate specificity (H3K36me3).
- Analysis of epigenetic cross-talk between H3K36me3 and H4K16ac, mediated by KAT8.
- Treatment with a pan-histone demethylase inhibitor (JIB-04) and latency-reversing agents (LRAs).
Main Results:
- MINA53 was identified as a novel HIV-1 latency-promoting gene.
- Depletion of MINA53 using RNAi led to increased levels of H3K36me3 at the HIV-1 LTR region.
- MINA53 preferentially demethylates H3K36me3, and its depletion impacts the H3K36me3/H4K16ac epigenetic balance.
- The histone demethylase inhibitor JIB-04 effectively inhibits MINA53 activity.
- JIB-04 synergized with LRAs to reactivate latent HIV-1.
Conclusions:
- MINA53 plays a critical role in maintaining HIV-1 latency through H3K36me3 demethylation.
- Targeting MINA53-mediated epigenetic mechanisms offers a potential strategy for purging HIV-1 latent reservoirs.
- Pharmacological inhibition of MINA53, such as with JIB-04, combined with LRAs, can reactivate latent HIV-1, paving the way for viral clearance.
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