retroviral-scanning-mapping-mlv-integration-sites-to-define-cell

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Généthon

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted lentiviral vectors.

Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.

The GATA1-HS2 enhancer allows persistent and position-independent expression of a β-globin transgene.

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a "bivalent" histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate.

Genome-Wide Definition of Promoter and Enhancer Usage during Neural Induction of Human Embryonic Stem Cells.

Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

Valentina Poletti

Généthon

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions