scholarly journals Foamy Virus Backbone Has Insulator Properties Which Remarkably Reduce Its Genotoxicity Potential

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1002-1002
Author(s):  
Michael Goodman ◽  
Paritha Arumugam ◽  
Devin Pillis ◽  
Danielle Lynn ◽  
Johannes C.M. Van der Loo ◽  
...  
Keyword(s):  
Hela Cells ◽  
Binding Sites ◽  
Transgene Expression ◽  
Integration Site ◽  
Foamy Virus ◽  
Fold Increase ◽  
Ctcf Binding ◽  
Vector Backbone ◽  
Median Increase ◽  
Enhancer Blocking

Abstract Strong viral enhancers in γ-retrovirus vectors (GV) have caused cellular proto-oncogene activation and leukemia in gene therapy trials, necessitating the use of cellular promoters in enhancer-less integrating vectors. However, data is now emerging that inadequate transgene expression from cellular promoters may limit successful correction. Vectors derived from foamy virus (FV), a nonpathogenic retrovirus, have a higher preference for non-genic integrations than GV/lentiviral vectors (LV), and may be less genotoxic. We constructed GV, LV and FV driven either by the spleen focus forming virus (SFFV) or the murine stem cell virus (MSCV) enhancer/promoters, all driving eGFP expression, and compared their relative genotoxicity using an in vitro immortalization assay on primary hematopoietic stem/progenitor cells (HSPC). In this assay, integration near a protooncogene/gene promoting cell proliferation results in quantifiable HSPC immortalization. Strong viral enhancer/promoters from SFFV or MSCV in FV caused a remarkably low immortalization of HSPC compared to analogous LV or GV: compared to the immortalization frequency of HSPC with the SFFV-GV in this assay, SFFV-LV and MSCV-LV had 12- and 14-fold lower immortalization frequency, while the SFFV-FV and MSCV-FV showed a 155- and 414-fold lower immortalization frequency, respectively. Immortalized clones had multiple (3-10) integrated copies. Integration site analysis of FV immortalized clones revealed a majority of integrants in non-gene regions; those in genic regions targeted cell proliferation or proto-oncogenes, as previously reported. FV has been previously reported to have 2-fold higher insertions in non-genic regions and higher, but nearly half the propensity to target promoters compared to GV. However, this remarkably reduced genotoxicity with FV was not explained by the integration pattern. We therefore hypothesized that FV backbone may contain sequences that have an enhancer blocking/insulator effect. Studies on chromatin insulators have shown that enhancer-blocking property of insulators is mediated via binding of CTCF to its consensus sequences within the insulator. Indeed, an in silico analysis for CCCTC-binding factor (CTCF) binding sites in the vector backbone sequences showed more predicted CTCF binding sites in the FV than in GV or LV (26, 8, and 6, respectively). To functionally validate the enhancer-blocking effect of the FV backbone and ensure that only effects of the vector backbone would be measured, without the confounding influence of integration site or the enhancer/promoter/transgene, we inserted SFFV-GV, SFFV-LV and SFFV-FV into a clinically relevant proto-oncogene, LMO2, using CRISPR/Cas9, and assessed LMO2 expression. LMO2 upregulation has previously resulted in leukemias in the X-linked severe combined immune-deficiency and Wiscott-Aldrich syndrome (WAS) GV-mediated gene transfer trials; notably SFFV-GV was used in the WAS trial and caused leukemias in 8 of 10 patients from insertional oncogenesis. HeLa cells were transfected with the proviral donor plasmids and the guide-RNA/spCas9 plasmids and GFP+ cells sorted and cloned. Nearly all clones derived had one intact LMO2 allele, while the other alleles had GV/LV/FV proviral insertions. We next assessed LMO2 mRNA and protein expression in these clones. We found a minimal increase in LMO2 mRNA expression with SFFV-FV, in sharp contrast to significantly increased LMO2 expression with SFFV-GV and SFFV-LV by qRT-PCR (Figure 1A). Overall, the SFFV enhancer in GV demonstrated the greatest fold-increase in LMO2 expression (median increase of 280+/-23-fold over unmodified HeLa cells), followed by the SFFV enhancer in LV (median 200+/-27-fold increase). However, the same SFFV enhancer in FV only showed a 45+/-7-fold median increase in expression. Western blot analysis for LMO2 protein expression in three clones for each vector showed no detectable LMO2 expression in SFFV-FV clones, which was similar to baseline in mock (non-targeted) HeLa cells (Figure 1B). However, significantly higher LMO2 protein was detectable in GV and LV clones. Hence, the remarkably low genotoxic potential of FV, even those carrying strong viral enhancers appears to be, in large part, from an insulator property of FV sequences, making FV ideal for situations where high transgene expression, necessitating strong enhancers is required for a therapeutic effect. Disclosures No relevant conflicts of interest to declare.

scholarly journals Allele-Specific Binding of CTCF to the Multipartite Imprinting Control Region KvDMR1

2007 ◽  
Vol 27 (7) ◽  
pp. 2636-2647 ◽  
Author(s):  
Galina V. Fitzpatrick ◽  
Elena M. Pugacheva ◽  
Jong-Yeon Shin ◽  
Ziedulla Abdullaev ◽  
Youwen Yang ◽  
...  
Keyword(s):  
Control Region ◽  
Binding Sites ◽  
Noncoding Rna ◽  
Specific Binding ◽  
Ctcf Binding ◽  
Luciferase Reporter ◽  
Imprinting Control Region ◽  
Enhancer Blocking ◽  
Functional Components

ABSTRACT Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.

CTCF function is modulated by neighboring DNA binding factors

2011 ◽  
Vol 89 (5) ◽  
pp. 459-468 ◽  
Author(s):  
Oliver Weth ◽  
Rainer Renkawitz
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Zinc Finger ◽  
Binding Sites ◽  
Zinc Finger Protein ◽  
Gene Activation ◽  
Ctcf Binding ◽  
Paternal Allele ◽  
Ctcf Binding Site ◽  
Enhancer Blocking

The zinc-finger protein CTCF was originally identified in the context of gene silencing and gene repression (Baniahmad et al. 1990; Lobanenkov et al. 1990). CTCF was later shown to be involved in several transcriptional mechanisms such as gene activation (Vostrov et al. 2002) and enhancer blocking (Filippova et al. 2001; Hark et al. 2000; Kanduri et al. 2000; Lutz et al. 2003; Szabó et al. 2000; Tanimoto et al. 2003; Phillips and Corces 2009; Bell et al. 1999; Zlatanova and Caiafa 2009a, 2009b). Insulators block the action of enhancers when positioned between enhancer and promoter. CTCF was found to be required in almost all cases of enhancer blocking tested in vertebrates. This CTCF-mediated enhancer blocking is in many instances conferred by constitutive CTCF action. For some examples however, a modulation of the enhancer blocking activity was documented (Lutz et al. 2003; Weth et al. 2010). One mechanism is achieved by regulation of binding to DNA. It was shown that CTCF is not able to bind to those binding-sites containing methylated CpG sequences. At the imprinting control region (ICR) of the Igf2/H19 locus the binding-site for CTCF on the paternal allele is methylated. This prevents DNA-binding of CTCF, resulting in the loss of enhancer blocking (Bell and Felsenfeld 2000; Chao et al. 2002; Filippova et al. 2001; Hark et al. 2000; Kanduri et al. 2000, 2002; Szabó et al. 2000; Takai et al. 2001). Not only can DNA methylation interfere with CTCF binding to DNA, it was also shown in one report that RNA transcription through the CTCF binding site results in CTCF eviction (Lefevre et al. 2008). In contrast to these cases most of the DNA sites are not differentially bound by CTCF. Even CTCF interaction with its cofactor cohesin does not seem to differ in different cell types (Schmidt et al. 2010). These results indicate that regulation of CTCF activity might be achieved by neighboring factors bound to DNA. In fact, whole genome analyses of CTCF binding sites identified several classes of neighboring sequences (Dickson et al. 2010; Boyle et al. 2010; Essien et al. 2009). Therefore, in this review we will summarize those results for which a combined action of CTCF with factors bound adjacently was found. These neighboring factors include the RNA polymerases I, II and III, another zinc finger factor VEZF1 and the factors YY1, SMAD, TR and Oct4. Each of these seems to influence, modulate or determine the function of CTCF. Thereby, at least some of the pleiotropic effects of CTCF can be explained.

scholarly journals Insertion of CTCF-binding sites into a first-generation adenovirus vector reduces the innate inflammatory response and prolongs transgene expression

Virology ◽  
2011 ◽  
Vol 412 (1) ◽  
pp. 136-145 ◽  
Author(s):  
Jerome Schaack ◽  
Liping Qiao ◽  
Marcin P. Walkiewicz ◽  
Martin Stonehouse ◽  
Daniel A. Engel ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Binding Sites ◽  
Transgene Expression ◽  
First Generation ◽  
Adenovirus Vector ◽  
Ctcf Binding

scholarly journals GCMa Regulates the Syncytin-mediated Trophoblastic Fusion

2002 ◽  
Vol 277 (51) ◽  
pp. 50062-50068 ◽  
Author(s):  
Chenchou Yu ◽  
Kuofeng Shen ◽  
Meiyao Lin ◽  
Porchun Chen ◽  
Chenchen Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Family Member ◽  
Cell Fusion ◽  
Hela Cells ◽  
Binding Sites ◽  
Integration Site ◽  
Human Endogenous Retrovirus ◽  
Placental Development ◽  
Fusion Event ◽  
Syncytiotrophoblast Layer

The human placental trophoblast cell can be classified as either a cytotrophoblast or a syncytiotrophoblast. Cytotrophoblasts can function as stem cells for the development of the syncytiotrophoblast layer via cell fusion. Anenvelopegene of the human endogenous retrovirus family W (HERV-W) calledsyncytinis specifically expressed in the syncytiotrophoblast layer. Syncytin is a fusogenic membrane protein; therefore, it can mediate the fusion of cytotrophoblasts into the syncytiotrophoblast layer, which is essential for pregnancy maintenance. GCMa is a placenta-specific transcription factor and is required for placental development. To study the placenta-specific fusion mediated by syncytin, we tested whether GCMa is involved in this process by regulatingsyncytingene expression. In this report, we demonstrate that GCMa was able to regulatesyncytingene expression via two GCMa-binding sites upstream of the 5′-long terminal repeat of thesyncytin-harboring HERV-W family member in BeWo and JEG3 cells but not in HeLa cells. Furthermore, adenovirus-directed expression of GCMa enhancedsyncytingene expression and syncytin-mediated cell fusion in BeWo and JEG3 cells but not in HeLa cells. Therefore, the integration site of thesyncytin-harboring HERV-W family member in the human genome is close to the functional GCMa-binding sites by which GCMa can specifically transactivatesyncytingene expression in trophoblast cells. Our results may help to explain the mechanism underlying the cell fusion event specific for syncytiotrophoblast formation.

scholarly journals Rasgrf1 Imprinting Is Regulated by a CTCF-Dependent Methylation-Sensitive Enhancer Blocker

2005 ◽  
Vol 25 (24) ◽  
pp. 11184-11190 ◽  
Author(s):  
Bongjune Yoon ◽  
Herry Herman ◽  
Benjamin Hu ◽  
Yoon Jung Park ◽  
Anders Lindroth ◽  
...  
Keyword(s):  
Binding Sites ◽  
Repeated Sequence ◽  
Ctcf Binding ◽  
Paternal Allele ◽  
Maternal Allele ◽  
Binary Switch ◽  
Blocking Activity ◽  
Enhancer Blocking

ABSTRACT Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5′ of the promoter. A repeated sequence 3′ of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.

scholarly journals Secondary Structural Model of MALAT1 Becomes Unstructured in Chronic Myeloid Leukemia and Undergoes Structural Rearrangement in Cervical Cancer

2021 ◽  
Vol 7 (1) ◽  
pp. 6
Author(s):  
Matthew C. Wang ◽  
Phillip J. McCown ◽  
Grace E. Schiefelbein ◽  
Jessica A. Brown
Keyword(s):  
Cervical Cancer ◽  
Chronic Myeloid Leukemia ◽  
Hela Cells ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Structural Changes ◽  
Structural Model ◽  
Dimethyl Sulfate ◽  
K562 Cells ◽  
Cancer Types

Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

scholarly journals Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Michael Aaron Goodman ◽  
Paritha Arumugam ◽  
Devin Marie Pillis ◽  
Anastacia Loberg ◽  
Mohammed Nasimuzzaman ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Long Terminal Repeat ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Foamy Virus ◽  
Terminal Repeat ◽  
Genotoxic Potential ◽  
Binding Factor ◽  
Activation Assay ◽  
Lentivirus Vectors

ABSTRACTStrong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into theLMO2gene and then measuringLMO2expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay,LMO2expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCEUnderstanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

scholarly journals Evolution of mouse circadian enhancers from transposable elements

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Julius Judd ◽  
Hayley Sanderson ◽  
Cédric Feschotte
Keyword(s):  
Gene Expression ◽  
Transposable Elements ◽  
Binding Sites ◽  
Mouse Liver ◽  
Integration Site ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Circadian Gene ◽  
Binding Motifs ◽  
Reporter Assays

Abstract Background Transposable elements are increasingly recognized as a source of cis-regulatory variation. Previous studies have revealed that transposons are often bound by transcription factors and some have been co-opted into functional enhancers regulating host gene expression. However, the process by which transposons mature into complex regulatory elements, like enhancers, remains poorly understood. To investigate this process, we examined the contribution of transposons to the cis-regulatory network controlling circadian gene expression in the mouse liver, a well-characterized network serving an important physiological function. Results ChIP-seq analyses reveal that transposons and other repeats contribute ~ 14% of the binding sites for core circadian regulators (CRs) including BMAL1, CLOCK, PER1/2, and CRY1/2, in the mouse liver. RSINE1, an abundant murine-specific SINE, is the only transposon family enriched for CR binding sites across all datasets. Sequence analyses and reporter assays reveal that the circadian regulatory activity of RSINE1 stems from the presence of imperfect CR binding motifs in the ancestral RSINE1 sequence. These motifs matured into canonical motifs through point mutations after transposition. Furthermore, maturation occurred preferentially within elements inserted in the proximity of ancestral CR binding sites. RSINE1 also acquired motifs that recruit nuclear receptors known to cooperate with CRs to regulate circadian gene expression specifically in the liver. Conclusions Our results suggest that the birth of enhancers from transposons is predicated both by the sequence of the transposon and by the cis-regulatory landscape surrounding their genomic integration site.

Intact Ca(2+)-binding sites are required for targeting of annexin 1 to endosomal membranes in living HeLa cells

2000 ◽  
Vol 113 (22) ◽  
pp. 3931-3938 ◽  
Author(s):  
U. Rescher ◽  
N. Zobiack ◽  
V. Gerke
Keyword(s):  
Hela Cells ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Membrane Binding ◽  
Growth Factor Receptor ◽  
Intracellular Distribution ◽  
Live Cells ◽  
Membrane Structures ◽  
Annexin 1

Annexin 1 is a Ca(2+)-regulated membrane binding protein and a major substrate of the epidermal growth factor receptor kinase. Because of its properties and intracellular distribution, the protein has been implicated in endocytic trafficking of the receptor, in particular in receptor sorting occurring in multivesicular endosomes. Up to now, however, the localization of annexin 1 to cellular membranes has been limited to subcellular fractionation and immunocytochemical analyses of fixed cells. To establish its localization in live cells, we followed the intracellular fate of annexin 1 molecules fused to the Green Fluorescent Protein (GFP). We show that annexin 1-GFP associates with distinct, transferrin receptor-positive membrane structures in living HeLa cells. A GFP chimera containing the Ca(2+)/phospholipid-binding protein core of annexin 1 also shows a punctate intracellular distribution, although the structures labeled here do not resemble early but, at least in part, late endosomes. In contrast, the cores of annexins 2 and 4 fused to GFP exhibit a cytoplasmic or a different punctate distribution, respectively, indicating that the highly homologous annexin core domains carry distinct membrane specificities within live cells. By inactivating the three high-affinity Ca(2+) binding sites in annexin 1 we also show that endosomal membrane binding of the protein in live HeLa cells depends on the integrity of these Ca(2+) binding sites. More detailed analysis identifies a single Ca(2+) site in the second annexin repeat that is crucially involved in establishing the membrane association. These results reveal for the first time that intracellular membrane binding of an annexin in living cells requires Ca(2+) and is mediated in part through an annexin core domain that is capable of establishing specific interactions.

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