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Transcriptional Modulation of Human Endogenous Retroviruses in Primary CD4+ T Cells Following Vorinostat Treatment

ABSTRACT: Purpose: In an HIV cure setting, Vorinostat may provide the “shock” capable of flushing HIV out of the persistent reservoir, while antiretroviral therapy is used to prevent new infections. However, this drug may modulate the expression of human endogenous retroviruses (HERVs). This study demonstrates significant modulation of HERVs and suggests that they should be considered as off-target effects of this drug treatment. Methods: Peripheral blood mononuclear cells were collected from 4 healthy donors. Naive CD4 T cells were isolated and utilized to assess HERV dysregulation after treatment with Vorinostat. Cells were collected for study and exposed to a dose of Vorinostat (10uM dose over a 24 hour period). After this, RNA was extracted from cells and their untreated paired counterparts for deep sequencing by Expression Analysis. Sequence reads that passed quality filters were mapped to the HERVd reference database using Bowtie and counted using HTSeq. Any HERV which did not achieve at least 1 count per million mapped and counted reads in at least 4 samples was discarded. Differential expression was performed upon the filtered dataset with edgeR and using TMM normalization. Results: Using an custom built data analysis pipeline, ~100 million reads per sample were mapped to the HERVd reference database and identified 10784 distinct dysregulated elements Bowtie/HTSeq workflow. Differential expression analysis was performed between vorinostat treated and untreated conditions which demonstrated 2102 dysregulated HERV elements with 1007 downregulated and 1095 upregulated (FDR < 0.05) with EdgeR. Results were further subset with a log2FC ± 3 which resulted in 451 upregulated and 363 downregulated HERV elements from 81 and 82 distinct families respectively. HERV elements from the LTR12 family were by far the most dramatically upregulated in family frequency and fold change magnitude. Further confirmation with droplet digital PCR confirmed upregulation of LTR12 elements and demonstrated an exponential dose response curve with HERV expression found at even moderate doses (334nM) of vorinostat treatment. Conclusions: This study represents the first detailed analysis of HERV dysregulation following vorinostat treatment using the untargeted approach of total RNA-Seq. These results demonstrate that vorinostat dysregulates multiple HERV families with a propensity to upregulate members of the LTR12 HERV family. This study also provides a methodology to analyze HERV dysregulation in future treatments with vorinostat or other drugs by providing a mechanism to choose primer and probe sets which will properly represent HERV dysregulation as expression across an HERV is not uniform or continuous. Finally, this work suggests that HERV dysregulation by vorinostat treatment should be considered an off-target effect of this drug and HERV elements, such as LTR12, should be monitored as biomarkers during shock and kill clinical trials with HDAC inhibitors and that trial subjects should be screened to explore further HIV:HERV interactions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE102187 | GEO | 2018/05/01

REPOSITORIES: GEO

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Project description:To check the acetylation levels at the positions harbouring HERV-loci and assess their variation after the treatment with HDACis (Vorinostat, romidepsin), the univocal genomic coordinates of about 3280 HERV-loci have been compared with the position of the H3AcK9 peaks identified by ChIP-seq analysis. The first analysis confirmed that HDACis are able to modulate the acetylation of repetitive elements, including HERV sequences. Subsequently, we considered only the differential peaks (diffpeaks), i.e. the peaks showing significant variation after the treatment with vorinostat and/or romidepsin. Interestingly, even if more than half of HERV-loci co-localised with acetylation peaks after HDACi treatment, only a minority of them (0.9 to 1.9%) co-localised with diffpeaks, showing mostly a reduction in their acetylation levels. Diffpeaks co-localised HERV-loci could be classified into 24 HERV groups and included the ERV-V2 locus. We focused then on the 11 HERV-loci which co-localised with diffpeaks showing an increase in acetylation levels, hence indicating an increased transcriptional activation after HDACi treatment. Remarkably, diffpeaks corresponding to the ERV-V2 locus were identified in all samples and were the most significantly upregulated in both vorinostat and romidepsin treatment. This was in part confirmed by ChIP-qPCR in different OC cells. Beside ERV-V2, only the HML8 sequence at locus 2q11,2 showed increased acetylation levels in all treated samples, independent from the HDACis used, but having lower statistical significance. The analysis revealed that the treatment with HDACis did not only account for the significant increase of ERV-V2 acetylation, but also led to the shift and concentration of acetylation peaks at the 5’ region of the HERV provirus. This observation allows us to hypothesize that HDACi treatment could not only increase ERV-V2 transcriptional activation, but also modify its pattern of expression, and eventually leading to alternative splicing.

Project description:Human Endogenous Retroviruses (HERVs) are exogenous viral elements that integrated into the germline millions of years ago. They comprise 8% of the human genome and play a critical role in cellular differentiation during development. HERV-K(HML-2) is the most recently integrated and actively transcribed HERV. It is found to be highly expressed in pluripotent stem cells and strongly downregulated during cellular differentiation. Moreover, expression of HERV-K in terminally differentiated neurons results in cytotoxicity. Expression of HERVs has been implicated in carcinogenesis and is suggested to confer stem cell like features important for cell growth. Atypical Teratoid / Rhabdoid Tumor (AT/RT) is a rare pediatric brain cancer that results from incomplete neuronal differentiation during embryonic development. In this paper, we investigated the cytopathic effect of Ouabain, a cardiac glycoside that induces cytotoxicity in stem cells, on AT/RT cells. We characterized four AT/RT cell lines by immunostaining and observed variable levels of stem cell, Oct4 and Pax6, and neuronal differentiation markers, TUBB3 and MAP2, as well as HERV-K envelope (env), polymerase (pol), and gag transcripts. We stained formalin-fixed brain tissues from 20 AT/RT patients and 5 unaffected controls for HERV-K ENV protein expression. 40% of AT/RT patient tissues showed ENV expression. In contrast, no ENV immunostaining was observed in all 5 control brain tissues. We used Ouabain, known to be cytotoxic to stem cells, observed up to 50% toxicity at 24 hr and 80% toxicity at 48 hr after treatment. While AT/RT primarily exist as large cell aggregates in suspension, Ouabain treatment downregulated HERV-K ENV protein and caused disruption of cell-to-cell interactions. Similarly, when AT/RT cells were transfected with a CRISPR/Cas9 construct designed to repress HERV-K promotor activity, cell aggregates separated and cell viability decreased significantly. Using a luciferase reporter construct under the control of a HERV-K promotor, we observed a significant decrease in HERV-K promotor activity at 8 to 72 hr post treatment with 0.5 uM Ouabain. Given that downregulation of HERV-K alone was sufficient to cause cytotoxicity in the cells, HERV-K expression may be essential for AT/RT growth and survival. Ouabain and other compounds that modulate HERV expression may provide an alternative or adjunctive therapeutic option for treatment of embryonal tumors.

Project description:Background: Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. To date, no biomarker has shown sufficient evidence and ease of application in clinical routine to monitor the complexity of the sepsis-related immune alterations. Multiple levels of interaction of human endogenous retroviruses (HERVs) with the immune response led us to hypothesize that they may be relevant candidates both to contribute to the physiopathology of sepsis and to be part of a molecular signature. In this study, we used a recently described high-density microarray which allows exploring HERVs/MaLRs transcriptome in septic shock patients. Results: About 6.9% of the HERVs/MalRs repertoire is transcribed in whole blood. Concerning the functionality of LTRs, the majority of LTRs are silent (62.9%), 7.5% of LTRs present a putative constitutive promoter or polyA functions, while 20.4% of them may shift from silent to active. Evidence was given that a subset of HERVs/MaLRs and genes were modulated in septic shock patients according to the monocyte HLA-DR (mHLA-DR) expression level measured by flow cytometry, as a proxy of immunosuppression. We then identified a large signature consisting of 193 differentially expressed HERVs/MaLRs probesets further reduced to a smaller 10 HERVs/MaLRs signature. Both signatures, validated in an independent septic shock cohort, identified two groups of patients with different severity features including clinical criteria and the CD74 and CX3CR1 sepsis severity molecular markers. Conclusion: This microarray-based approach unveiled the expression of about 87,912 distinct HERV probesets and identified 764 putative promoter LTRs and 642 putative polyA LTRs in whole blood. HERV/MaLR expression was shown to be tightly modulated in septic shock patients according to mHLA-DR expression. We identified a set of potential molecular biomarkers in septic shock patients partially overlapping immunosuppression (mHLA-DR), and the risk of Health Care associated Infections (HAI) (CD74 ratio), complementary to existing molecular markers of a sepsis patients stratification. We identified a HERV/MaLR signature discriminating patients based on their immunosuppression state and severity.

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Transcriptional Modulation of Human Endogenous Retroviruses in Primary CD4+ T Cells Following Vorinostat Treatment

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