RESUMO
Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.
Assuntos
Eczema , Transplante de Células-Tronco Hematopoéticas , Síndrome de Wiskott-Aldrich , Humanos , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Proteína da Síndrome de Wiskott-Aldrich/genética , Células-Tronco Hematopoéticas/metabolismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Terapia Genética/métodos , Eczema/etiologia , Eczema/metabolismo , Eczema/terapiaRESUMO
The integration of the HIV-1 genome into the host genome is an essential step in the life cycle of the virus and it plays a critical role in the expression, long-term persistence, and reactivation of HIV expression. To better understand the local genomic environment surrounding HIV-1 proviruses, we assessed the influence of non-canonical B-form DNA (non-B DNA) on the HIV-1 integration site selection. We showed that productively and latently infected cells exhibit different integration site biases towards non-B DNA motifs. We identified a correlation between the integration sites of the latent proviruses and non-B DNA features known to potently influence gene expression (e.g., cruciform, guanine-quadruplex (G4), triplex, and Z-DNA). The reactivation potential of latent proviruses with latency reversal agents also correlated with their proximity to specific non-B DNA motifs. The perturbation of G4 structures in vitro using G4 structure-destabilizing or -stabilizing ligands resulted in a significant reduction in integration within 100 base pairs of G4 motifs. The stabilization of G4 structures increased the integration within 300-500 base pairs from G4 motifs, increased integration near transcription start sites, and increased the proportion of latently infected cells. Moreover, we showed that host lens epithelium-derived growth factor (LEDGF)/p75 and cleavage and polyadenylation specificity factor 6 (CPSF6) influenced the distribution of integration sites near several non-B DNA motifs, especially G4 DNA. Our findings identify non-B DNA motifs as important factors that influence productive and latent HIV-1 integration and the reactivation potential of latent proviruses.
Assuntos
DNA de Forma B , Quadruplex G , Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , HIV-1/genética , Motivos de Nucleotídeos , Latência Viral , DNA , Provírus/genéticaRESUMO
Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.
RESUMO
Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.
RESUMO
BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).
Assuntos
Gammaretrovirus/genética , Terapia Genética , Vetores Genéticos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Antígenos CD34 , DNA Complementar/uso terapêutico , Expressão Gênica , Inativação Gênica , Terapia Genética/efeitos adversos , Humanos , Lactente , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Mutação , Linfócitos T/imunologia , Transdução Genética , Transgenes/fisiologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologiaRESUMO
MOTIVATION: Gene therapy with retroviral vectors can induce adverse effects when those vectors integrate in sensitive genomic regions. Retroviral vectors are preferred that target sensitive regions less frequently, motivating the search for localized clusters of integration sites and comparison of the clusters formed by integration of different vectors. Scan statistics allow the discovery of spatial differences in clustering and calculation of false discovery rates providing statistical methods for comparing retroviral vectors. RESULTS: A scan statistic for comparing two vectors using multiple window widths is proposed with software to detect clustering differentials and compute false discovery rates. Application to several sets of experimentally determined HIV integration sites demonstrates the software. Simulated datasets of various sizes and signal strengths are used to determine the power to discover clusters and evaluate a convenient lower bound. This provides a toolkit for planning evaluations of new gene therapy vectors. AVAILABILITY AND IMPLEMENTATION: The geneRxCluster R package containing a simple tutorial and usage hints is available from http://www.bioconductor.org.
Assuntos
Vetores Genéticos , HIV/genética , Integração Viral , Algoritmos , Inteligência Artificial , DNA/química , Interpretação Estatística de Dados , Genômica , Humanos , Células Jurkat , SoftwareRESUMO
BACKGROUND: HIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models. RESULTS: Here we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models. CONCLUSIONS: The silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency.
Assuntos
Linfócitos T CD4-Positivos/virologia , HIV/fisiologia , Integração Viral , Latência Viral , Células Cultivadas , HIV/genética , Humanos , Provírus/genética , Provírus/fisiologiaRESUMO
MOTIVATION: The relative abundance of retroviral insertions in a host genome is important in understanding the persistence and pathogenesis of both natural retroviral infections and retroviral gene therapy vectors. It could be estimated from a sample of cells if only the host genomic sites of retroviral insertions could be directly counted. When host genomic DNA is randomly broken via sonication and then amplified, amplicons of varying lengths are produced. The number of unique lengths of amplicons of an insertion site tends to increase according to its abundance, providing a basis for estimating relative abundance. However, as abundance increases amplicons of the same length arise by chance leading to a non-linear relation between the number of unique lengths and relative abundance. The difficulty in calibrating this relation is compounded by sample-specific variations in the relative frequencies of clones of each length. RESULTS: A likelihood function is proposed for the discrete lengths observed in each of a collection of insertion sites and is maximized with a hybrid expectation-maximization algorithm. Patient data illustrate the method and simulations show that relative abundance can be estimated with little bias, but that variation in highly abundant sites can be large. In replicated patient samples, variation exceeds what the model implies-requiring adjustment as in Efron (2004) or using jackknife standard errors. Consequently, it is advantageous to collect replicate samples to strengthen inferences about relative abundance.
Assuntos
Algoritmos , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano , Integração Viral , Infecções por HTLV-I/genética , HumanosRESUMO
Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.
Assuntos
HIV/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , beta Carioferinas/metabolismo , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , RNA Interferente Pequeno/genética , Replicação Viral , beta Carioferinas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.
Assuntos
Marcação de Genes , Terapia Genética , Análise de Sequência de DNA/métodos , Bacteriófago mu/genética , Linhagem Celular , Humanos , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: The outcomes of gene therapy to correct congenital immunodeficiencies are unknown. We reviewed long-term outcomes after gene therapy in nine patients with X-linked severe combined immunodeficiency (SCID-X1), which is characterized by the absence of the cytokine receptor common gamma chain. METHODS: The nine patients, who lacked an HLA-identical donor, underwent ex vivo retrovirus-mediated transfer of gamma chain to autologous CD34+ bone marrow cells between 1999 and 2002. We assessed clinical events and immune function on long-term follow-up. RESULTS: Eight patients were alive after a median follow-up period of 9 years (range, 8 to 11). Gene therapy was initially successful at correcting immune dysfunction in eight of the nine patients. However, acute leukemia developed in four patients, and one died. Transduced T cells were detected for up to 10.7 years after gene therapy. Seven patients, including the three survivors of leukemia, had sustained immune reconstitution; three patients required immunoglobulin-replacement therapy. Sustained thymopoiesis was established by the persistent presence of naive T cells, even after chemotherapy in three patients. The T-cell-receptor repertoire was diverse in all patients. Transduced B cells were not detected. Correction of the immunodeficiency improved the patients' health. CONCLUSIONS: After nearly 10 years of follow-up, gene therapy was shown to have corrected the immunodeficiency associated with SCID-X1. Gene therapy may be an option for patients who do not have an HLA-identical donor for hematopoietic stem-cell transplantation and for whom the risks are deemed acceptable. This treatment is associated with a risk of acute leukemia. (Funded by INSERM and others.)
Assuntos
Terapia Genética , Subunidade gama Comum de Receptores de Interleucina/genética , Imunodeficiência Combinada Severa/terapia , Antígenos CD34 , Linfócitos B/imunologia , Seguimentos , Terapia Genética/efeitos adversos , Humanos , Imunoglobulinas/sangue , Lactente , Subunidade gama Comum de Receptores de Interleucina/deficiência , Células Matadoras Naturais/fisiologia , Contagem de Linfócitos , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologiaRESUMO
X-linked severe-combined immunodeficiency (SCID-X1) has been treated by therapeutic gene transfer using gammaretroviral vectors, but insertional activation of proto-oncogenes contributed to leukemia in some patients. Here we report a longitudinal study of gene-corrected progenitor cell populations from 8 patients using 454 pyrosequencing to map vector integration sites, and extensive resampling to allow quantification of clonal abundance. The number of transduced cells infused into patients initially predicted the subsequent diversity of circulating cells. A capture-recapture analysis was used to estimate the size of the gene-corrected cell pool, revealing that less than 1/100th of the infused cells had long-term repopulating activity. Integration sites were clustered even at early time points, often near genes involved in growth control, and several patients harbored expanded cell clones with vectors integrated near the cancer-implicated genes CCND2 and HMGA2, but remain healthy. Integration site tracking also documented that chemotherapy for adverse events resulted in successful control. The longitudinal analysis emphasizes that key features of transduced cell populations--including diversity, integration site clustering, and expansion of some clones--were established early after transplantation. The approaches to sequencing and bioinformatics analysis reported here should be widely useful in assessing the outcome of gene therapy trials.
Assuntos
Terapia Genética/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Epigênese Genética , Terapia Genética/efeitos adversos , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/virologia , Humanos , Estudos Longitudinais , Mutagênese Insercional , Proto-Oncogenes , Retroviridae/genética , Transdução Genética , Resultado do Tratamento , Integração Viral/genéticaRESUMO
OBJECTIVE: The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. DESIGN: Primary resting or activated CD4 T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. METHODS: We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. RESULTS: HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. CONCLUSION: These data indicate that integration site distributions show modest but significant differences between resting and activated CD4 T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression.
Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Integração Viral/genética , Sequência de Bases , Células Cultivadas , DNA Viral/genética , Genoma , HIV-1/genética , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , Latência ViralRESUMO
At least 8% of the human genome was formed by integration of retroviral DNA sequences. Here we analyze the forces directing the accumulation of human endogenous retroviruses (HERVs) by comparing de novo HERV integration targeting with the distribution of fixed HERV elements in the human genome. All known genomic HERVs are inactive due to mutation, but we were able to study integration targeting using a reconstituted consensus HERV-K (designated HERV-K(Con)). We found that HERV-K(Con) integrated preferentially in transcription units, in gene-rich regions, and near features associated with active transcription units and associated regulatory regions. In contrast, genomic HERV-K proviruses are found preferentially outside transcription units. The minority of genomic HERVKs present inside transcription units are in opposite transcriptional orientation relative to the host gene, the orientation predicted to be minimally disruptive to host mRNA synthesis, but de novo HERV-K(Con) integration within transcription units showed no orientation bias. We also found that the youngest HERV-K elements in the human genome showed a distribution intermediate between de novo HERV-K(Con) integration sites and older fixed HERV-Ks. These findings indicate that accumulation of HERVs in the human germline is a two-step process: integration targeting biases direct initial accumulation, then purifying selection leads to loss of proviruses disrupting gene function.
Assuntos
Retrovirus Endógenos/metabolismo , Genoma Humano/genética , Integração Viral/fisiologia , Células/virologia , Cromatina/metabolismo , Mapeamento Cromossômico , Retrovirus Endógenos/genética , Histonas/metabolismo , Humanos , Metilação , Integração Viral/genéticaRESUMO
Lentiviral vector-based gene therapy has been used to target the human immunodeficiency virus (HIV) using an antisense env payload. We have analyzed lentiviral-vector integration sites from three treated individuals. We compared integration sites from the ex vivo vector-transduced CD4+ cell products to sites from cells recovered at several times after infusion. Integration sites were analyzed using 454 pyrosequencing, yielding a total of 7,782 unique integration sites from the ex vivo product and 237 unique sites from cells recovered after infusion. Integrated vector copies in both data sets were found to be strongly enriched within active genes and near epigenetic marks associated with active transcription units. Analysis of integration relative to nucleosome structure on target DNA indicated favoring of integration in outward facing DNA major grooves on the nucleosome surface. There was no indication that growth of transduced cells after infusion resulted in enrichment for integration sites near proto-oncogene 5'-ends or within tumor suppressor genes. Thus, this first look at the longitudinal evolution of cells transduced with a lentiviral vector after infusion of gene modified CD4+ cells provided no evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Vetores Genéticos/genética , Infecções por HIV/genética , Infecções por HIV/terapia , Lentivirus/genética , Integração Viral/genética , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Biologia Computacional , Humanos , Proto-Oncogene Mas , Análise de Sequência de DNARESUMO
The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host-virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences.
Assuntos
Marcação de Genes/métodos , Genoma Viral/genética , Espaço Intracelular/virologia , Integração Viral/genética , Animais , Cromossomos/genética , DNA Viral/genética , HumanosRESUMO
Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.
Assuntos
Arabidopsis/genética , Metilação de DNA , Epigênese Genética , Genoma de Planta , Ilhas de CpG , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Perfilação da Expressão Gênica , Mutação , RNA de Plantas/metabolismo , RNA não Traduzido/metabolismo , Análise de Sequência de DNA/métodosRESUMO
Integration of retroviral DNA into host cell DNA is a defining feature of retroviral replication. HIV integration is known to be favored in active transcription units, which promotes efficient transcription of the viral genes, but the molecular mechanisms responsible for targeting are not fully clarified. Here we used pyrosequencing to map 40,569 unique sites of HIV integration. Computational prediction of nucleosome positions in target DNA indicated that integration sites are periodically distributed on the nucleosome surface, consistent with favored integration into outward-facing DNA major grooves in chromatin. Analysis of integration site positions in the densely annotated ENCODE regions revealed a wealth of new associations between integration frequency and genomic features. Integration was particularly favored near transcription-associated histone modifications, including H3 acetylation, H4 acetylation, and H3 K4 methylation, but was disfavored in regions rich in transcription-inhibiting modifications, which include H3 K27 trimethylation and DNA CpG methylation. Statistical modeling indicated that effects of histone modification on HIV integration were partially independent of other genomic features influencing integration. The pyrosequencing and bioinformatic methods described here should be useful for investigating many aspects of retroviral DNA integration.
Assuntos
Mapeamento Cromossômico/métodos , Epigênese Genética , HIV/genética , Integração Viral/genética , DNA Viral/metabolismo , Difosfatos/metabolismo , Perfilação da Expressão Gênica , Genoma , Humanos , Células Jurkat , Modelos Genéticos , Dados de Sequência Molecular , Nucleossomos/metabolismo , Análise de Sequência de DNARESUMO
Sleep researchers invariably struggle with decisions regarding the optimal design for their studies. Whether such studies involve treatment for insomnia, obstructive sleep apnea, or any other sleep disorder, questions arise regarding the respective trade-offs between a parallel group and a crossover design. This study analyzed the variance structure of commonly measured polysomnographic variables in an effort to describe the statistical impact of these alternate designs. The study examined the effects of opioids on sleep and employed multiple crossovers between placebo, MS-contin, and methadone using a double-blind, randomized crossover design. Thirty-seven healthy subjects were studied. Four of the subjects were unable to complete the protocol for a variety of reasons, and polysomnogram data was unavailable for one subject. Data from 37 subjects provide the basis for this analysis. Despite dropouts, the crossover study was approximately four times as efficient as the parallel group design in terms of being able to recognize differences in deep sleep across these conditions. Other polysomnographic variables also favored the crossover design to varying extents. Despite the operational complexity of a crossover design, the statistical efficiency of this approach makes it a preferable approach for designing intervention studies in sleep research.
Assuntos
Metadona/farmacologia , Morfina/farmacologia , Entorpecentes/farmacologia , Polissonografia/efeitos dos fármacos , Sono/efeitos dos fármacos , Adulto , Gráficos por Computador , Estudos Cross-Over , Coleta de Dados/estatística & dados numéricos , Interpretação Estatística de Dados , Feminino , Humanos , Masculino , Computação Matemática , Polissonografia/estatística & dados numéricos , Projetos de PesquisaRESUMO
Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I-hypersensitive sites (i.e., +/- 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.