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Patient derived γ-retroviral integration sites reveal active gene-regulatory regions in human repopulating long-term hematopoietic stem cells

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Hematopoietic stem cell (HSC) research largely relies on cell culture models or mouse transplantation studies. Moreover, HSCs are rare and immunophenotypic definitions are incomplete, rendering the characterization of HSCs difficult. In this study, we circumvented these restrictions using >180,000 γ-retroviral (γRV) integration sites (ISs) from a gene therapy trial on 10 Wiskott-Aldrich-Syndrome patients. γRV ISs leave a unique tag to hematopoietic stem and progenitor cells (HSPCs) that engraft in patients, which are passed on to all progeny, making them suitable to track clonal reconstitution dynamics. Moreover, γRV ISs can be used to map active promoters and enhancers, due to their predilection to integrate at such sites. ISs recovered during stable long-term hematopoiesis would therefore point towards active promoters and genes that originate from true repopulating long-term HSCs. However, due to the genotoxic potential of γRVs, ISs are often regarded as molecular tags that point towards proto-oncogenes. To examine this in more detail, we first cloned 20 protein-coding genes that showed a large number of ISs in their vicinity and established a pooled lentiviral overexpression library to study their influence on proliferation, self-renewal and differentiation of HSPCs. Although the characterization of individual candidate genes was limited by transduction efficiencies and library representation, we observed that not a single candidate gene led to clonal expansion or measurable increase in self-renewal during both in vitro and in vivo experiments, suggesting that γRV genotoxicity is less universal than expected. Based on this, we assessed the cumulative number of ISs per gene over time and statistically compared γRV IS pattern before and after transplantation, demonstrating that the clonal skewing of IS pattern is indeed restricted to only few known leukemogenic loci. We next modeled the hematopoietic reconstitution after transplantation in humans and used these insights to define long-term HSC specific ISs,which confirmatively showed the highest ATAC-seq signal intensity at HSC specific peaks, efficiently enriched for HSC specific gene sets and strongly correlated with hematopoietic risk variants. Finally, through integration of publicly available ATAC-seq, ChIP-seq, capture Hi-C as well as GWAS SNP data, we were able to create the first genome wide map for active gene-regulatory regions in functionally defined human repopulating long-term HSCs.

Item Type: Dissertation
Supervisor: Boutros, Prof. Dr. Michael
Date of thesis defense: 28 September 2018
Date Deposited: 10 Oct 2018 09:00
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
Controlled Keywords: hematopoietic stem cell, enhancer, regulatory regions
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