Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator

The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP-1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element.     

Retrovirus silencer blocking by the cHS4 insulator is CTCF independent

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRβ-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.     

Assay of insulator enhancer-blocking activity with the use of transient transfection

We used a transient transfection of cultured cells with linearized plasmids to analyze the enhancer-blocking activity of potential insulators including the standard cHS4 chicken beta-globin insulator and several DNA fragments selected from the human genome sequence. About 60–80% of the potential insulators do reveal the enhancer-blocking activity when probed by the transient transfection assay. The activity of different sequences is characterized by certain tissue specificity and by dependence on the orientation of the fragments relative to the promoter. Thus, the transfection model may be used for quantitative analysis of the enhancer-blocking activity of the potential insulators.     

The host range of gammaretroviruses and gammaretroviral vectors includes post-mitotic neural cells

Background

Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest.

Methodology/Principal Findings

Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors.

Conclusions/Significance

These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease.     

Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.     

Improved short-term engraftment of lentivirally versus gammaretrovirally transduced allogeneic canine repopulating cells

Gammaretroviral vectors require cell division for efficient transduction. Thus, extended cell culture times are necessary for efficient transduction with gammaretroviral vectors, which in turn can lead to stem cell loss and impaired engraftment. Lentiviral vectors transduce nondividing cells and are therefore able to transduce stem cells in short transduction protocols. Here, we compared the short-term engraftment of lentivirally and gammaretrovirally transduced canine allogeneic DLA-matched littermate cells. A reduced conditioning regimen of 400 cGy total body irradiation was used in preparation for clinical studies. Two dogs received a graft of gammaretrovirally transduced CD34-selected cells. CD34(+) cells were prestimulated for 30 h and then exposed twice to concentrated RD114 pseudotype vector. Three dogs received lentivirally transduced CD34-selected cells. Cells were transduced overnight with concentrated VSV-G pseudotype lentiviral vector. The animals in the lentiviral group showed a significantly faster granulocyte recovery. VNTR analysis 40-50 days after transplantation revealed higher donor chimerism for the lentiviral group compared to the retroviral group. These data suggest that short lentiviral transduction protocols may be superior to extended gammaretroviral transduction protocols with respect to engraftment potential of transduced CD34(+) hematopoietic repopulating cells.     

Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants

Background  

Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are boundary elements that can prevent enhancer-promoter interactions, if placed between these elements, and protect transgene cassettes from silencing and positional effects. It has been suggested that insulators can improve the safety and performance of lentiviral vectors. Therefore insulators have been incorporated into lentiviral vectors in order to enhance their safety profile and improve transgene expression. Commonly such insulator vectors are produced at lower titers than control vectors thus limiting their potential use.     

SUMO conjugation attenuates the activity of the gypsy chromatin insulator

Chromatin insulators have been implicated in the establishment of independent gene expression domains and in the nuclear organization of chromatin. Post-translational modification of proteins by Small Ubiquitin-like Modifier (SUMO) has been reported to regulate their activity and subnuclear localization. We present evidence suggesting that two protein components of the gypsy chromatin insulator of Dorsophila melanogaster, Mod(mdg4)2.2 and CP190, are sumoylated, and that SUMO is associated with a subset of genomic insulator sites. Disruption of the SUMO conjugation pathway improves the enhancer-blocking function of a partially active insulator, indicating that SUMO modification acts to regulate negatively the activity of the gypsy insulator. Sumoylation does not affect the ability of CP190 and Mod(mdg4)2.2 to bind chromatin, but instead appears to regulate the nuclear organization of gypsy insulator complexes. The results suggest that long-range interactions of insulator proteins are inhibited by sumoylation and that the establishment of chromatin domains can be regulated by SUMO conjugation.     

Prototypic chromatin insulator cHS4 protects retroviral transgene from silencing in Schistosoma mansoni

Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) virions can transduce schistosomes, leading to chromosomal integration of reporter transgenes. To develop VSVG-MLV for functional genomics in schistosomes, the influence of the chicken β-globin cHS4 element, a prototypic chromatin insulator, on transgene expression was examined. Plasmid pLNHX encoding the MLV 5'- and 3'-Long Terminal Repeats flanking the neomycin phosphotransferase gene (neo) was modified to include, within the U3 region of the 3'-LTR, active components of cHS4 insulator, the 250 bp core fused to the 400 bp 3'-region. Cultured larvae of Schistosoma mansoni were transduced with virions from producer cells transfected with control or cHS4-bearing plasmids. Schistosomules transduced with cHS4 virions expressed 2-20 times higher levels of neo than controls, while carrying comparable numbers of integrated proviral transgenes. The findings not only demonstrated that cHS4 was active in schistosomes but also they represent the first report of activity of cHS4 in any Lophotrochozoan species, which has significant implications for evolutionary conservation of heterochromatin regulation. The findings advance prospects for transgenesis in functional genomics of the schistosome genome to discover intervention targets because they provide the means to enhance and extend transgene activity including for vector based RNA interference.     

Simultaneous sequence transfer into two independent locations of a reporter vector using MultiSite Gateway technology

The bacteriophage lambda recombination system is increasingly used for recombinant DNA applications that involve the frequent transfer of sequences into and between shuttle and reporter vectors. This approach bypasses the need for restriction endonucleases or ligases and, as such, is easily scalable and automated. However this system has not yet been tested for the ability to support the simultaneous introduction of donor fragments into two separate target sites of a single reporter plasmid. This attribute would greatly facilitate studies of cis-regulatory elements that only function in specific combinations, such as a class of regulatory elements known as chromatin insulators. With the goal of facilitating a screen for chromatin insulators, we sought to determine whether the commercially available MultiSite Gateway Technology recombination system could be used to simultaneously insert candidate insulator elements into two separate locations of a functional reporter plasmid. We show that this application is both highly efficient and specific, generating the desired recombination products nearly three quarters of the time without disrupting the specificity of the reporter system. As such, these studies establish a novel application of the MultiSite Gateway Technology for the generation of recombinant reporter plasmids where the constituent elements function in a combinatorial fashion.     

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.