Endogenous retrovirus | EPFL Graph Search

Endogenous retroviruses (ERVs) are endogenous viral elements in the genome that closely resemble and can be derived from retroviruses. They are abundant in the genomes of jawed vertebrates, and they comprise up to 5–8% of the human genome (lower estimates of ~1%). ERVs are a vertically inherited proviral sequence and a subclass of a type of gene called a transposon, which can normally be packaged and moved within the genome to serve a vital role in gene expression and in regulation. ERVs however lack most transposon functions, are typically not infectious and are often defective genomic remnants of the retroviral replication cycle. They are distinguished as germline provirus retroelements due to their integration and reverse-transcription into the nuclear genome of the host cell. Researchers have suggested that retroviruses evolved from a type of transposon called a retrotransposon, a Class I element; these genes can mutate and instead o

Evolutionally dynamic control of LINE-1 elements in embryonic stem cells

Natali Pennese

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1, also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution, with at any given time a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage likely under pressure of host restriction factors, which act notably by silencing L1 expression during early embryogenesis. Here, we demonstrate that in human embryonic stem cells (hESC) KAP1, the master co-factor of KRAB-containing zinc finger proteins (KRAB-ZFP) previously implicated in the restriction of endogenous retroviruses, represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 and 7.6 million years ago. In the mouse, we documented a similar chronologically conditioned pattern, albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP, suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hESC induced the expression of KAP1-bound L1 elements, but their younger, human-specific counterparts (L1Hs) were unaffected. Instead, they were stimulated by depleting DNA methyltransferases, consistent with recent evidence demonstrating that the PIWI-piRNA pathway regulates L1Hs in hESC. Altogether, these data indicate that the early embryonic control of L1 is an evolutionary dynamic process, and support a model whereby newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms, before KAP1-recruiting protein repressors are selected.

EPFL2015

Identification of genetic insulator elements to increase the safety of viral gene therapy vectors

Armelle Gaussin

Haematopoeitic system diseases, both acquired and inherited, can be currently cured by allogeneic haematopoietic stem cell transplantation. This treatment provides highly successful immune function recovery for patients receiving grafts of HLA-compatible donors but has still a great risk of complications and even failure if no suitable donor is available. Among the alternative therapeutic options, ex vivo retrovirus-mediated gene transfer into haematopoietic progenitor cells has been shown to be an efficient strategy for a substantial number of severe combined immunodeficiency-suffering patients. A recent gene therapy trial has been remarkably effective for the immunological reconstitution of patients suffering from X-linked severe combined immunodeficiency. This treatment was able to provide full correction of disease phenotype and thus, clinical benefit. However, the appearance of leukemia in several patients has put in question the safety of the procedure. This severe adverse event has been attributed to the integration of the therapeutic transgene-carrying viral vector into a known T-cell oncogene, LMO2, thereby contributing to the development of T-cell leukemia by causing aberrant expression of LMO2. Further studies mentioned the possible retroviral-mediated cis-activation of the LMO2 promoter underlying the potential ability of retroviral regulatory elements to influence neighboring gene transcription. This project aimed at decreasing the risk associated with the use of viral vectors for gene therapy through the identification of genetic insulator elements capable of isolating the vector regulatory elements in order to prevent the activation of chromosomal genes by the viral enhancers. We have established a standardized screening procedure whereby the potency of insulators can be assessed quantitatively on relevant vector elements. This assay system consists of a series of plasmids containing two reporter genes: one mimicking a therapeutic gene under the control of strong viral long terminal repeat (LTR) enhancer, and the other one standing for an endogenous gene close to the chromosomal vector integration site. The assay allowed the quantification of the enhancer blocking activity of the well-characterized chicken beta-globin 5'HS4 insulator (cHS4) in cultured cells. We assessed the insulator activity of novel synthetic elements, constructed from optimized binding sites for the insulator protein CTCF. In addition, we demonstrated the enhancer blocking activity of the nuclear factor 1 protein family (CTF/NF1) and showed that it also displays barrier properties, protecting transgene expression from silencing. We showed that both CTCF and CTF/NF1 binding sites act as insulators that mediate potent enhancer-blocker activity, resulting in a commensurate reduction of genotoxicity when implemented in viral gene therapy vectors. Finally, we used the same approach to analyze the enhancer blocking activity of well-known chromatin domain delimiters, matrix attachment regions (MAR), mainly characterized by their barrier properties. We could demonstrate the enhancer blocking ability of the 1-68 MAR that is mainly harbored by an A-T rich core sub-region.

EPFL2010

Unraveling the KRAB/KAP1 control of transposable elements in pluripotent and somatic cells

Gabriela Ecco

Transposable elements (TEs) are DNA sequences able to change position in the genome, and represent more than 40% of mammalian genetic material. TEs can have positive or detrimental effects on the host, being both important motors of evolution and genomic threats, and are associated with diseases such as cancer and diabetes. Growing evidence indicates the host co-opts TEs for its benefit, with examples such as syncytins â genes important for placental formation that derive from TEs. Given their potential damaging role, the host needs to repress TEs, especially during early embryogenesis. KRAB-containing zinc finger proteins (KRAB-ZFPs) are important regulators of TEs, which they repress with their cofactor KAP1. In stem cells, the KRAB/KAP1 complex irreversibly silence TEs and is believed to be dispensable in adult cells. KRAB-ZFPs constitute one of the largest families of transcriptional regulators encoded by higher vertebrates, but functional information is missing for most of its members. To shed light on this important family of transcription factors, we first developed a large-scale functional screen matching murine TEs with their cognate KRAB-ZFP. The screen identified KRAB-ZFP809 as the ligand of its previously mapped DNA target in the murine leukemia virus genome. Our method further singled out two novel KRAB-ZFPs binding to TE sequences, as confirmed by repression and binding assays. One of them, Gm6871, was identified to regulate LINE-1 elements together with KAP1 in embryonic stem cells. KAP1 regulation followed a chronologically conditioned pattern, repressing elements that entered the mouse genome between 5.6 and 3.8 million years ago, suggestive of an evolutionary arms race between host KRAB-ZFPs and TEs. Secondly, we found the paralogs ZFP932 and Gm15446 to bind overlapping but distinguishable subsets of ERVK (endogenous retrovirus K), to repress these elements in embryonic stem cells, and to regulate secondarily the expression of neighboring genes. Furthermore, we uncovered that these KRAB-ZFPs and KAP1 control TEs in adult tissues, in cell culture and in vivo, where they partner up to modulate cellular genes. Our results establish an efficient screening method to identify KRAB-ZFPs DNA targets, opening the way to functional analyses of this major class of transcriptional repressors. Most importantly, our study strongly suggests that KRAB-ZFP genes are not only part of an evolutionary arms race with TEs, but also participate in the domestication of these elements for the benefit of the host. Given the abundance and high degree of species-specificity of TEs and KRAB-ZFPs, these results have important implications for understanding the biology of higher vertebrates, including humans.

EPFL2016