Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites

Abstract Aging causes the global disorganization of nuclear chromatin architecture. In a normal young nucleus, silent heterochromatin is associated with the nuclear lamina layer underlying nuclear envelope, thus spatially separated from euchromatin at the nuclear center. Notably, aging causes the disruption of nuclear lamina and the decondensation of associated heterochromatin. However, it is not clearly understood how these changes of chromatin architectures contribute to age-related diseases. Through large-scale computational analyses, we present that CpG islands (CGIs) give important clues to answering this question. CGIs are DNA elements with high Cytosine-phosphate-Guanine dinucleotide frequencies. In human, about 60% of total genes contain CGIs at their promoters (CGI+ genes) and are broadly expressed throughout the body. The other 40% of genes that do not have CGIs (CGI- genes) exhibit tissue-restricted expression patterns. Our results demonstrate that, in normal young nuclei, only CGI- genes can reside within lamina-associated heterochromatin when transcriptionally inactive, while CGI+ genes associate with nuclear central euchromatin even when they are repressed. In parallel, we show that age-associated heterochromatin decondensation can specifically de-repress tissue-specific CGI- genes leading to their uncontrolled expressions. Our results further demonstrate that global misregulation of CGI- genes increases the noise in gene transcription that, in turn, causes the loss of cellular identities during aging. Taken together, our study establishes critical implication of CGI-mediated chromatin architecture in age-associated degenerative changes and loss of tissue homeostasis.

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Joint profiling of chromatin accessibility and CAR-T integration site analysis at population and single-cell levels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919259117 ◽

2020 ◽

Vol 117 (10) ◽

pp. 5442-5452 ◽

Cited By ~ 1

Author(s):

Wenliang Wang ◽

Maria Fasolino ◽

Benjamin Cattau ◽

Naomi Goldman ◽

Weimin Kong ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Integration Site ◽

Chromatin Accessibility ◽

Site Analysis ◽

Car T ◽

Integration Site Analysis ◽

Integration Sites ◽

Lentiviral Integration

Chimeric antigen receptor (CAR)-T immunotherapy has yielded impressive results in several B cell malignancies, establishing itself as a powerful means to redirect the natural properties of T lymphocytes. In this strategy, the T cell genome is modified by the integration of lentiviral vectors encoding CAR that direct tumor cell killing. However, this therapeutic approach is often limited by the extent of CAR-T cell expansion in vivo. A major outstanding question is whether or not CAR-T integration itself enhances the proliferative competence of individual T cells by rewiring their regulatory landscape. To address this question, it is critical to define the identity of an individual CAR-T cell and simultaneously chart where the CAR-T vector integrates into the genome. Here, we report the development of a method called EpiVIA (https://github.com/VahediLab/epiVIA) for the joint profiling of the chromatin accessibility and lentiviral integration site analysis at the population and single-cell levels. We validate our technique in clonal cells with previously defined integration sites and further demonstrate the ability to measure lentiviral integration sites and chromatin accessibility of host and viral genomes at the single-cell resolution in CAR-T cells. We anticipate that EpiVIA will enable the single-cell deconstruction of gene regulation during CAR-T therapy, leading to the discovery of cellular factors associated with durable treatment.

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Large-Scale Identification of Disease Genes Involved in Acute Myeloid Leukemia

Journal of Virology ◽

10.1128/jvi.78.4.1971-1980.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1971-1980 ◽

Cited By ~ 27

Author(s):

Stefan J. Erkeland ◽

Marijke Valkhof ◽

Claudia Heijmans-Antonissen ◽

Antoinette van Hoven-Beijen ◽

Ruud Delwel ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Large Scale ◽

Leukemia Virus ◽

Point Mutations ◽

Insertion Mutagenesis ◽

Disease Genes ◽

Integration Sites ◽

Virus Integration ◽

Acute Myeloid

ABSTRACT Acute myeloid leukemia (AML) is a heterogeneous group of diseases in which chromosomal aberrations, small insertions or deletions, or point mutations in certain genes have profound consequences for prognosis. However, the majority of AML patients present without currently known genetic defects. Retroviral insertion mutagenesis in mice has become a powerful tool for identifying new disease genes involved in the pathogenesis of leukemia and lymphoma. Here we have used the Graffi-1.4 strain of murine leukemia virus, which causes predominantly AML, in a screen to identify novel genes involved in the pathogenesis of this disease. We report 79 candidate disease genes in common integration sites (CISs) and 15 genes whose family members previously were found to be affected in other studies. The majority of the identified sequences (60%) were not found in lymphomas and monocytic leukemias in previous screens, suggesting a specific involvement in AML. Although most of the virus integrations occurred in or near the 5′ or 3′ ends of the genes, suggesting deregulation of gene expression as a consequence of virus integration, 18 CISs were located exclusively within the genes, conceivably causing gene disruption.

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New Bioinformatic Strategies to Rapidly Characterize Retroviral Integration Sites of Gene Therapy Vectors

Methods of Information in Medicine ◽

10.1160/me0423 ◽

2007 ◽

Vol 46 (05) ◽

pp. 542-547 ◽

Cited By ~ 10

Author(s):

F. A. Giordano ◽

A. Hotz-Wagenblatt ◽

D. Lauterborn ◽

J.-U. Appelt ◽

K. Fellenberg ◽

...

Keyword(s):

Viral Vectors ◽

Large Scale ◽

Integration Site ◽

High Throughput Analysis ◽

Sequence Detection ◽

Retroviral Integration ◽

Gene Therapy Vectors ◽

Integration Site Analysis ◽

Integration Sites ◽

Insertion Sites

Summary Objective: Increasing use of retroviral vector-mediated gene transfer created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time-consuming manual data processing, are commonly applied. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. Methods: To allow large-scale and standardized comparison of insertion sites of viral vectors we developed two programs, IntegrationSeqand IntegrationMap. IntegrationSeq can trim sequences, and valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units, and further parameters like the distance from the transcription start site to the integration. Results: We validated the method using 259 files originating from integration site analysis (LM-PCR). Sequences processed by IntegrationSeq led to an increased yield of valid integration sequence detection, which were shown to be more sensitive than conventional analysis and 15 times faster, while the specificities are equal. Output files generated by IntegrationMap were found to be 99.8% identical with results retrieved by much slower conventional mapping with the ENSEMBL alignment tool. Conclusion: Using IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

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No Evidence for Clonal Selection Due to Lentiviral Integration Sites in Human Induced Pluripotent Stem Cells

Stem Cells ◽

10.1002/stem.322 ◽

2010 ◽

Vol 28 (4) ◽

pp. 687-694 ◽

Cited By ~ 29

Author(s):

Thomas Winkler ◽

Amy Cantilena ◽

Jean-Yves Métais ◽

Xiuli Xu ◽

Anh-Dao Nguyen ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Clonal Selection ◽

Integration Sites ◽

Induced Pluripotent ◽

Lentiviral Integration

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Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis

Genome Research ◽

10.1101/gr.233866.117 ◽

2019 ◽

Vol 29 (3) ◽

pp. 494-505 ◽

Cited By ~ 34

Author(s):

Leslie O. Goodwin ◽

Erik Splinter ◽

Tiffany L. Davis ◽

Rachel Urban ◽

Hao He ◽

...

Keyword(s):

Large Scale ◽

Structural Variation ◽

Insertional Mutagenesis ◽

Integration Sites

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CPSF6-Dependent Targeting of Speckle-Associated Domains Distinguishes Primate from Nonprimate Lentiviral Integration

mBio ◽

10.1128/mbio.02254-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

Author(s):

Wen Li ◽

Parmit K. Singh ◽

Gregory A. Sowd ◽

Gregory J. Bedwell ◽

Sooin Jang ◽

...

Keyword(s):

Target Genes ◽

Binding Protein ◽

Nuclear Periphery ◽

Dependent Manner ◽

Nuclear Speckles ◽

Retroviral Integration ◽

Integration Sites ◽

Primate Lentiviruses ◽

Lentiviral Integration ◽

Hiv 1

ABSTRACT Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs. IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.

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