Cell line-dependent variability in HIV activation employing DNMT inhibitors

Long-lived reservoirs of Human Immunodeficiency Virus (HIV) latently infected cells present the main barrier to a cure for HIV infection. Much interest has focused on identifying strategies to activate HIV, which would be used together with antiretrovirals to attack reservoirs. Several HIV activating agents, including Tumor Necrosis Factor alpha (TNFα) and other agents that activate via NF-kB are not fully effective in all latent infection models due to epigenetic restrictions, such as DNA methylation and the state of histone acetylation. DNA methyltransferases (DNMT) inhibitors like 5-aza-2'deoxycytidine (Aza-CdR) and histone deacetylase (HDAC) inhibitors like Trichostatin A (TSA) have been proposed as agents to enhance reactivation and have shown activity in model systems. However, it is not clear how the activities of DNMT and HDAC inhibitors range across different latently infected cell lines, potential models for the many different latently infected cells within an HIV patient. We determined HIV activation following treatment with TNFα, TSA and Aza-CdR across a range of well known latently infected cell lines. We assessed the activity of these compounds in four different Jurkat T cell-derived J-Lat cell lines (6.3, 8.4, 9.2 and 10.6), which have a latent HIV provirus in which GFP replaces Nef coding sequence, and ACH-2 and J1.1 (T cell-derived), and U1 (promonocyte-derived) cell lines with full-length provirus. We found that Aza-CdR plus TNFα activated HIV at least twice as well as TNFα alone for almost all J-Lat cells, as previously described, but not for J-Lat 10.6, in which TNFα plus Aza-CdR moderately decreased activation compared to TNFα alone. Surprisingly, a much greater reduction of TNFα-stimulated activation with Aza-CdR was detected for ACH-2, J1.1 and U1 cells. Reaching the highest reduction in U1 cells with a 75% reduction. Interestingly, Aza-CdR not only decreased TNFα induction of HIV expression in certain cell lines, but also decreased activation by TSA. Since DNMT inhibitors reduce the activity of provirus activators in some HIV latently infected cell lines the use of epigenetic modifying agents may need to be carefully optimized if they are to find clinical utility in therapies aimed at attacking latent HIV reservoirs

Repressive LTR Nucleosome Positioning by the BAF Complex Is Required for HIV Latency

The SWI/SNF BAF chromatin remodeling complex generates a repressive nucleosome structure at the HIV LTR conducive to establishment and maintenance of HIV latency, while PBAF augments HIV transcription

A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

Reprogramming of somatic cells to different extents has been reported using different methods. However, this is normally accompanied by the use of exogenous materials, and the overall reprogramming efficiency has been low. Chemicals and small molecules have been used to improve the reprogramming process during somatic cell nuclear transfer (SCNT) and induced pluripotent stem (iPS) cell generation. We report here the first application of a combined epigenetic and non-genetic approach for reprogramming somatic cells, i.e., DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and human embryonic stem cell (hESC) extracts. When somatic cells were pretreated with these inhibitors before exposure to hESC (MEL1) extracts, morphological analysis revealed a higher rate of hESC-like colony formation than without pretreatment. Quantitative PCR (qPCR) demonstrated that pluripotency genes were upregulated when compared to those of somatic cells or treated with hESC extracts alone. Overall changes in methylation and acetylation levels of pretreated somatic cells suggests that epigenetic states of the cells have an effect on reprogramming efficiency induced by hESC extracts. KnockOutserum replacement (KOSR™) medium (KO-SR) played a positive role in inducing expression of the pluripotency genes. hESC extracts could be an alternative approach to reprogram somatic cells without introducing exogenous materials. The epigenetic pre-treatment of somatic cells could be used to improve the efficiency of reprogramming process. Under differentiation conditions, the reprogrammed cells exhibited differentiation ability into neurons suggesting that, although fully reprogramming was not achieved, the cells could be transdifferentiated after reprogramming

Glucose and Fatty Acids Synergize to Promote B-Cell Apoptosis through Activation of Glycogen Synthase Kinase 3β Independent of JNK Activation

The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.Increasing glucose (5-25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target

Epigenetic Regulation of HIV-1 Latency by Cytosine Methylation

Human immunodeficiency virus type 1 (HIV-1) persists in a latent state within resting CD4+ T cells of infected persons treated with highly active antiretroviral therapy (HAART). This reservoir must be eliminated for the clearance of infection. Using a cDNA library screen, we have identified methyl-CpG binding domain protein 2 (MBD2) as a regulator of HIV-1 latency. Two CpG islands flank the HIV-1 transcription start site and are methylated in latently infected Jurkat cells and primary CD4+ T cells. MBD2 and histone deacetylase 2 (HDAC2) are found at one of these CpG islands during latency. Inhibition of cytosine methylation with 5-aza-2′deoxycytidine (aza-CdR) abrogates recruitment of MBD2 and HDAC2. Furthermore, aza-CdR potently synergizes with the NF-κB activators prostratin or TNF-α to reactivate latent HIV-1. These observations confirm that cytosine methylation and MBD2 are epigenetic regulators of HIV-1 latency. Clearance of HIV-1 from infected persons may be enhanced by inclusion of DNA methylation inhibitors, such as aza-CdR, and NF-κB activators into current antiviral therapies

HIV Promoter Integration Site Primarily Modulates Transcriptional Burst Size Rather Than Frequency

Mammalian gene expression patterns, and their variability across populations of cells, are regulated by factors specific to each gene in concert with its surrounding cellular and genomic environment. Lentiviruses such as HIV integrate their genomes into semi-random genomic locations in the cells they infect, and the resulting viral gene expression provides a natural system to dissect the contributions of genomic environment to transcriptional regulation. Previously, we showed that expression heterogeneity and its modulation by specific host factors at HIV integration sites are key determinants of infected-cell fate and a possible source of latent infections. Here, we assess the integration context dependence of expression heterogeneity from diverse single integrations of a HIV-promoter/GFP-reporter cassette in Jurkat T-cells. Systematically fitting a stochastic model of gene expression to our data reveals an underlying transcriptional dynamic, by which multiple transcripts are produced during short, infrequent bursts, that quantitatively accounts for the wide, highly skewed protein expression distributions observed in each of our clonal cell populations. Interestingly, we find that the size of transcriptional bursts is the primary systematic covariate over integration sites, varying from a few to tens of transcripts across integration sites, and correlating well with mean expression. In contrast, burst frequencies are scattered about a typical value of several per cell-division time and demonstrate little correlation with the clonal means. This pattern of modulation generates consistently noisy distributions over the sampled integration positions, with large expression variability relative to the mean maintained even for the most productive integrations, and could contribute to specifying heterogeneous, integration-site-dependent viral production patterns in HIV-infected cells. Genomic environment thus emerges as a significant control parameter for gene expression variation that may contribute to structuring mammalian genomes, as well as be exploited for survival by integrating viruses

At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1

Background: Genomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/ Angelman syndromes and cancer. Methodology/Principal Findings: To identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression. Conclusions/Significance: Ten imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/‘‘Rian’’

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation

Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-κB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway