Identification of hepatic microvascular adhesion-related genes of human colon cancer cells using random homozygous gene perturbation.

Random homozygous gene perturbation (RHGP), in combination with liver sinusoidal endothelial cell (LSEC) adhesion screening of clonal colon cancer cells with perturbed genes, was used to identify genes contributing to the hepatic microvascular adhesion of colon cancer cells. Plasmid vector encoding transactivator and gene search vector were transfected into HT-29 human colorectal cancer cells to create a HT-29 RHGP cell library; the adhesion of these library cells to primary cultured mouse LSEC significantly decreased in the presence of RSL1 ligand (inducer), indicating that most of the genes contributing to HT-29 adhesion to LSEC were altered. Next, HT-29 RHGP cell library fractions with upregulated or silenced LSEC adhesion-related genes were isolated. Around 160 clones having altered expression in LSEC adhesion-related genes were obtained, and nine relevant protein-coding genes were identified. Some were proadhesive genes detected because of their overexpression in adherent HT-29 cells (DGCR8 and EFEMP1 genes) and their silenced status in nonadherent HT-29 cells (DGKE, DPY19L1, KIAA0753, PVR and USP11 genes). Others were antiadhesive genes detected because of their overexpression in nonadherent HT-29 cells (ITPKC gene) and their silenced status in adherent HT-29 cells (PPP6R2 gene). Silencing of PVR, DGCR8 and EFEMP1 genes decreased adhesion to LSEC and hepatic microvascular retention of HT-29 cells. The results conclude that RHGP was a valuable strategy for the discovery of mechanisms regulating microvascular adhesion of circulating colon cancer cells before hepatic metastasis formation. Identified genes may contribute to understand the metastatic process of colon cancer and to discovering molecular targets for hepatic metastasis therapeutics.

Distal regions of the human IFNG locus direct cell type-specific expression.

Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a bacterial artificial chromosome transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage-specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not in NK cells. This distal regulatory element is a Runx3 binding site in Th1 cells and is needed for RNA polymerase II recruitment to IFNG, but it is not absolutely required for histone acetylation of the IFNG locus. These results support a model whereby IFNG uses cis-regulatory elements with cell type-restricted function.

T-bet dependent removal of Sin3A-histone deacetylase complexes at the Ifng locus drives Th1 differentiation.

Forming and removing epigenetic histone marks at gene loci are central processes in differentiation. Here, we explored mechanisms establishing long-range H4 acetylation marks at the Ifng locus during Th1 lineage commitment. In Th0 cells, histone deacetylase (HDAC)-Sin3A complexes recruited to the Ifng locus actively prevented accumulation of H4 acetylation marks. Th1 differentiation caused loss of HDAC-Sin3A complexes by T-bet-dependent mechanisms and accumulation of H4 acetylation marks. HDAC-Sin3A complexes were absent from the locus in NOD Th0 cells, obviating the need for Th1 differentiation signals to establish histone marks and Th1 differentiation. Thus, Ifng transcription is actively prevented in Th0 cells via epigenetic mechanisms and epigenetic defects allow unregulated Ifng transcription that may contribute to autoimmunity.

Epigenetics and T helper 1 differentiation.

Naïve T helper cells differentiate into two subsets, T helper 1 and 2, which either transcribe the Ifng gene and silence the Il4 gene or transcribe the Il4 gene and silence the Ifng gene, respectively. This process is an essential feature of the adaptive immune response to a pathogen and the development of long-lasting immunity. The 'histone code' hypothesis proposes that formation of stable epigenetic histone marks at a gene locus that activate or repress transcription is essential for cell fate determinations, such as T helper 1/T helper 2 cell fate decisions. Activation and silencing of the Ifng gene are achieved through the creation of stable epigenetic histone marks spanning a region of genomic DNA over 20 times greater than the gene itself. Key transcription factors that drive the T helper 1 lineage decision, signal transducer and activator 4 (STAT4) and T-box expressed in T cells (T-bet), play direct roles in the formation of activating histone marks at the Ifng locus. Conversely, STAT6 and GATA binding protein 3, transcription factors essential for the T helper 2 cell lineage decision, establish repressive histone marks at the Ifng locus. Functional studies demonstrate that multiple genomic elements up to 50 kilobases from Ifng play critical roles in its proper transcriptional regulation. Studies of three-dimensional chromatin conformation indicate that these distal regulatory elements may loop towards Ifng to regulate its transcription. We speculate that these complex mechanisms have evolved to tightly control levels of interferon-gamma production, given that too little or too much production would be very deleterious to the host.

Identification of novel host-oriented targets for Human Immunodeficiency Virus type 1 using Random Homozygous Gene Perturbation.

BACKGROUND: Human Immunodeficiency Virus (HIV) is a global threat to public health. Current therapies that directly target the virus often are rendered ineffective due to the emergence of drug-resistant viral variants. An emerging concept to combat drug resistance is the idea of targeting host mechanisms that are essential for the propagation of the virus, but have a minimal cellular effect. RESULTS: Herein, using Random Homozygous Gene Perturbation (RHGP), we have identified cellular targets that allow human MT4 cells to survive otherwise lethal infection by a wild type HIV-1NL4-3. These gene targets were validated by the reversibility of the RHGP technology, which confirmed that the RHGP itself was responsible for the resistance to HIV-1 infection. We further confirmed by siRNA knockdowns that the RHGP-identified cellular pathways are responsible for resistance to infection by either CXCR4 or CCR5 tropic HIV-1 variants. We also demonstrated that cell clones with these gene targets disrupted by RHGP were not permissible to the replication of a drug resistant HIV-1 mutant. CONCLUSION: These studies demonstrate the power of RHGP to identify novel host targets that are essential for the viral life cycle but which can be safely perturbed without overt cytotoxicity. These findings suggest opportunities for the future development of host-oriented therapeutics with the broad spectrum potential for safe and effective inhibition of HIV infection.

Epigenetic Activation and Silencing of the Gene that Encodes IFN-γ.

Transcriptional activation and repression of genes that are developmentally regulated or exhibit cell-type specific expression patterns is largely achieved by modifying the chromatin template at a gene locus. Complex formation of stable epigenetic histone marks, loss or gain of DNA methylation, alterations in chromosome conformation, and specific utilization of both proximal and distal transcriptional enhancers and repressors all contribute to this process. In addition, long non-coding RNAs are a new species of regulatory RNAs that either positively or negatively regulate transcription of target gene loci. IFN-γ is a pro-inflammatory cytokine with critical functions in both innate and adaptive arms of the immune system. This review focuses on our current understanding of how the chromatin template is modified at the IFNG locus during developmental processes leading to its transcriptional activation and silencing.

The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza.

Conventional approaches for therapeutic targeting of viral pathogens have consistently faced obstacles arising from the development of resistant strains and a lack of broad-spectrum application. Influenza represents a particularly problematic therapeutic challenge since high viral mutation rates have often confounded many conventional antivirals. Newly emerging or engineered strains of influenza represent an even greater threat as typified by recent interest in avian subtypes of influenza. Based on the limitations associated with targeting virally-encoded molecules, we have taken an orthogonal approach of targeting host pathways in a manner that prevents viral propagation or spares the host from virus-mediated pathogenicity. To this end, we report herein the application of an improved technology for target discovery, Random Homozygous Gene Perturbation (RHGP), to identify host-oriented targets that are well-tolerated in normal cells but that prevent influenza-mediated killing of host cells. Improvements in RHGP facilitated a thorough screening of the entire genome, both for overexpression or loss of expression, to identify targets that render host cells resistant to influenza infection. We identify a set of host-oriented targets that prevent influenza killing of host cells and validate these targets using multiple approaches. These studies provide further support for a new paradigm to combat viral disease and demonstrate the power of RHGP to identify novel targets and mechanisms.

Identification of PTCH1 requirement for influenza virus using random homozygous gene perturbation.

Influenza infection remains a leading cause of infectious disease-mediated morbidity and mortality. Accumulating evidence indicates that most variants of seasonal and pandemic influenza have developed resistance to conventional therapies. Such information has spawned new interest in identifying novel approaches to target influenza. Our laboratories have been developing a new strategy of Host-Oriented Therapeutics, which seeks to target host molecules in a safe and effective manner that prevents the virus from causing disease. Using an improved discovery technology, Random Homozygous Gene Perturbation (RHGP), we identified the PTCH1 protein as an essential host target that critically controls influenza virus infection. We further demonstrated that targeted intervention against PTCH1 using antibodies or siRNA decreases influenza infection. Finally, we demonstrated the involvement of PTCH1 in influenza infection outside of the laboratory by showing that genetic variations of PTCH1 relate to decreased disease morbidity in the field. Altogether, these findings have important implications for the development of novel, host-directed therapeutics to improve influenza disease management.

Development of a broad-spectrum antiviral with activity against Ebola virus.

We report herein the identification of a small molecule therapeutic, FGI-106, which displays potent and broad-spectrum inhibition of lethal viral hemorrhagic fevers pathogens, including Ebola, Rift Valley and Dengue Fever viruses, in cell-based assays. Using mouse models of Ebola virus, we further demonstrate that FGI-106 can protect animals from an otherwise lethal infection when used either in a prophylactic or therapeutic setting. A single treatment, administered 1 day after infection, is sufficient to protect animals from lethal Ebola virus challenge. Cell-based assays also identified inhibitory activity against divergent virus families, which supports a hypothesis that FGI-106 interferes with a common pathway utilized by different viruses. These findings suggest FGI-106 may provide an opportunity for targeting viral diseases.

Dynamic changes in histone-methylation 'marks' across the locus encoding interferon-gamma during the differentiation of T helper type 2 cells.

The 'histone-code' hypothesis proposes that cell fate 'decisions' are achieved through the creation of stable epigenetic histone 'marks' at gene loci. Here we explored the formation of marks of repressive methylation of histone 3 at lysine 9 (H3-K9) and of H3-K27 at the locus encoding interferon-gamma (Ifng locus) during the commitment of naive T cells to the T helper type 1 (TH1) and TH2 lineages. Methylation of H3-K9 across the Ifng locus was rapidly induced in differentiating TH1 and TH2 cells and was sustained in TH1 cells. In contrast, TH2 differentiation caused loss of methylation of H3-K9 and gain of methylation of H3-K27 by mechanisms dependent on the transcription factors GATA-3 and STAT6. Thus, histone-methylation marks at the Ifng locus are highly dynamic, which may ensure higher-order transcriptional regulation during early lineage commitment.

Histone hyperacetylated domains across the Ifng gene region in natural killer cells and T cells.

Local histone acetylation of promoters precedes transcription of many genes. Extended histone hyperacetylation at great distances from coding regions of genes also occurs during active transcription of gene families or individual genes and may reflect developmental processes that mark genes destined for cell-specific transcription, nuclear signaling processes that are required for active transcription, or both. To distinguish between these, we compared long-range histone acetylation patterns across the Ifng gene region in natural killer (NK) cells and T cells that were or were not actively transcribing the Ifng gene. In T cells, long-range histone acetylation depended on stimulation that drives both T helper (Th) 1 differentiation and active transcription, and it depended completely or partially on the presence of Stat4 or T-bet, respectively, two transcription factors that are required for Th1 lineage commitment. In contrast, in the absence of stimulation and active transcription, similar histone hyperacetylated domains were found in NK cells. Additional proximal domains were hyperacetylated after stimulation of transcription. We hypothesize that formation of extended histone hyperacetylated domains across the Ifng gene region represents a developmental mechanism that marks this gene for cell- or stimulus-specific transcription.

Long-range histone acetylation of the Ifng gene is an essential feature of T cell differentiation.

Histone acetylation of promoters precedes activation of many genes. In addition, long-range histone acetylation patterns can be established over many kilobases of the chromatin of linked families of genes that are under common transcriptional control. It is not known whether establishment of long-range histone acetylation patterns is limited to gene families or is a common feature of many genes. The Ifng gene is not known to be a member of a gene family but exhibits complex strain-, cell lineage-, and stimulus-dependent regulation. For example, stimulation of naive T cells through their antigen receptor does not initiate Ifng gene transcription. However, stimulation of naive T cells through their antigen and IL-12 receptors initiates differentiation programs that yield effector cells with 100-fold greater rates of transcription of the Ifng gene after stimulation through the antigen receptor. Here, we demonstrate that these differentiation programs establish long-range histone hyperacetylation patterns that extend at least 50 kb in both upstream and downstream directions of the Ifng gene. Establishment of these histone acetylation patterns and Ifng gene expression is relatively IL-12-independent in T cells from autoimmune-prone nonobese diabetic mice. These results indicate that gene expression programs that mediate T cell differentiation are regulated by long-range histone acetylation patterns and that defective control of these patterns may contribute to development of autoimmunity.