The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation.

High-risk human papillomaviruses (hrHPVs) infect keratinocytes and successfully evade host immunity despite the fact that keratinocytes are well equipped to respond to innate and adaptive immune signals. Using non-infected and freshly established or persistent hrHPV-infected keratinocytes we show that hrHPV impairs the acetylation of NFκB/RelA K310 in keratinocytes. As a consequence, keratinocytes display a decreased pro-inflammatory cytokine production and immune cell attraction in response to stimuli of the innate or adaptive immune pathways. HPV accomplishes this by augmenting the expression of interferon-related developmental regulator 1 (IFRD1) in an EGFR-dependent manner. Restoration of NFκB/RelA acetylation by IFRD1 shRNA, cetuximab treatment or the HDAC1/3 inhibitor entinostat increases basal and induced cytokine expression. Similar observations are made in IFRD1-overexpressing HPV-induced cancer cells. Thus, our study reveals an EGFR-IFRD1-mediated viral immune evasion mechanism, which can also be exploited by cancer cells.

Lentiviral vectors encoding zinc-finger nucleases specific for the model target locus HPRT1.

Zinc-finger nucleases (ZFNs) are artificial proteins designed to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. These site-specific genomic lesions facilitate the study of translocations and cellular DNA repair processes and serve as powerful stimuli for the editing of complex genomes. The delivery of ZFNs into a wide range of cell types is of utmost importance for the broad evaluation and deployment of the technology. Lentiviral vectors (LVs) are versatile gene delivery vehicles that transduce alike transformed and primary cells regardless of their division rate. In this chapter, we describe the generation of conventional and integrase-defective LVs encoding ZFNs targeting the human hypoxanthine phosphoribosyltransferase 1 (HPRT1) locus. Furthermore, we introduce a general LV titration method based on a cost-effective quantitative PCR protocol and implement a rapid and simple restriction enzyme site polymorphism assay to detected DSB formation at the HPRT1 target sequence. Owing in part to the small molecule-based clone selection schemes conferred by HPRT1 allelic knockouts, this X-linked gene has become a "classical" target model locus in mammalian cells. The reagents and techniques detailed herein yield LV preparations that induce HPRT1-specific DSBs. As a result, they should constitute a valuable resource to increase the robustness and decrease the timelines of the various protocols based on HPRT1 gene disruption and targeting.

Histone deacetylase inhibition rescues gene knockout levels achieved with integrase-defective lentiviral vectors encoding zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration-approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance.

Histone deacetylase inhibition activates transgene expression from integration-defective lentiviral vectors in dividing and non-dividing cells.

Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone- and cell type-dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles.

Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms.

The transcription start sites (TSS) and promoters of many genes are located in upstream CpG islands. Methylation within such islands is known for both imprinted and oncogenes, although poorly studied for other genes, especially those with complex CpG islands containing multiple first exons and promoters. The glucocorticoid receptor (GR) CpG island contains seven alternative first exons and their promoters. Here we show for the five GR promoters activated in PBMCs that methylation patterns are highly variable between individuals. The majority of positions were methylated at levels >25% in at least one donor affecting each promoter and TSS. We also examined the evolutionarily conserved transcription factor binding sites (TFBS) using an improved in silico phylogenetic footprinting technique. The majority of these contain methylatable CpG sites, suggesting that methylation may orchestrates alternative first exon usage, silencing and controlling tissue-specific expression. The heterogeneity observed may reflect epigenetic mechanisms of GR fine tuning, programmed by early life environment and events. With 78% of evolutionarily conserved alternative first exons falling into such complex CpG islands, their internal structure and epigenetic modifications are bound to be biologically important, and may be a common transcriptional control mechanism used throughout many phyla.

Tissue specific glucocorticoid receptor expression, a role for alternative first exon usage?

The CpG island upstream of the GR is highly structured and conserved at least in all the animal species that have been investigated. Sequence alignment of these CpG islands shows inter-species homology ranging from 64 to 99%. This 3.1kb CpG rich region upstream of the GR exon 2 encodes 5' untranslated mRNA regions. These CpG rich regions are organised into multiple first exons and, as we and others have postulated, each with its own promoter region. Alternative mRNA transcript variants are obtained by the splicing of these alternative first exons to a common acceptor site in the second exon of the GR. Exon 2 contains an in-frame stop codon immediately upstream of the ATG start codon to ensure that this 5' heterogeneity remains untranslated, and that the sequence and structure of the GR is unaffected. Tissue specific differential usage of exon 1s has been observed in a range of human tissues, and to a lesser extent in the rat and mouse. The GR expression level is tightly controlled within each tissue or cell type at baseline and upon stimulation. We suggest that no single promoter region may be capable of containing all the necessary promoter elements and yet preserve the necessary proximity to the transcription initiation site to produce such a plethora of responses. Thus we further suggest that alternative first exons each under the control of specific transcription factors control both the tissue specific GR expression and are involved in the tissue specific GR transcriptional response to stimulation. Spreading the necessary promoter elements over multiple promoter regions, each with an associated alternative transcription initiation site would appear to vastly increase the capacity for transcriptional control of GR.