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414. PARP-1 Contributes to the Barrier Function of a Vertebrate Chromatin Insulator
GENE REGULATION regions from gene expression profiles in experimental arthritis and experimentally verified a panel of ten identified promoters. We first performed gene expression profiling of synovial knee joint tissues from mice with collagen-induced arthritis to identify the genes that show at least tenfold regulation during disease progression. We then used k-means clustering to group these genes into six distinct expression profiles. Next, we introduced a filtering based on the presence of a putative TATA-box between positions -32/-29 in the upstream promoter regions of the murine genes and their human orthologs. The over-represented cis-regulatory elements in the proximal promoters (-500/+200) that putatively govern the distinct expression profiles were identified using an algorithm that takes advantage of spatial and phylogenetic conservation of sequences. Based on this algorithm, we constructed lentiviral luciferase reporters containing proximal promoter regions that were predicted to be predominantly regulated by the transcription factors NFκB (Cxcl1, Cxcl5, Il1b), AP-1 (Mmp3, Mmp13, Timp1, Tnfaip6) or C/EBPβ (Saa3, Chi3l1, Has1). Nine out of ten promoters were responsive to a pro-inflammatory stimulus in murine fibroblasts and macrophages. The promoter regions of Saa3, Cxcl1 and Mmp3 showed the strongest, more than tenfold, increase over basal promoter activities. For in vivo validation, lentiviral reporters were injected in knee and ankle joints of C57Bl/6 mice and the kinetics of luciferase expression during acute inflammation were determined by optical imaging or ex vivo luciferase assays. The promoter activities were rapidly and strongly induced during onset of inflammation and significantly decreased when inflammation waned. Of great value for human gene therapy, the relative Saa3 promoter response was significantly increased in primary RA synovial fibroblasts derived from patients with a high synovial inflammatory gene expression profile. This study highlights the value of a bioinformatics approach in design of transcriptionally targeted gene therapy for disease. We expect the novel vectors to be widely applicable in both basic and translation research in human and experimental arthritis.
414. PARP-1 Contributes to the Barrier Function of a Vertebrate Chromatin Insulator David W. Emery, Karol Bomsztyk, Mari Aker. Divisions of Medical Genetics and UW Medicine Lake Union, Department of Medicine, University of Washington, Seattle, WA.
The prototypic chromatin insulator cHS4 has proven effective at 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 found that an extended 400 bp core region of the cHS4 element was necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors [Aker et al., Hum Gene Ther 18:333, 2007]. In more recent studies, electrophoretic gel mobility shift assays reveled specific DNA protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies lead to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1), an abundant nuclear protein that has the capacity to bind DNA through zinc finger motifs, and to catalyze the addition of poly(ADP)-ribose chains to itself and other proteins. 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. Footprinting studies using ssDNA probes suggest that PARP-1 binding to this extended cHS4 core region is specific to a unique DNA cruciform secondary structure, rather than a primary sequence. Functional studies with gammaretroviral reporter vectors in cell lines showed that cHS4 barrier activity was abrogated upon deletion or mutation of the putative PARP-1 binding site, reducing the frequency of cells S162
expressing vector GFP 2-fold to the levels seen without the insulator. Transduction studies in primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was also abrogated following treatment with a PARP inhibitor, reducing the level of vector GFP expression 2 to 7-fold to an average of 8-45 mean fluorescent units, levels seen with the uninsulated control. Finally, barrier activity of the cHS4 element was also found to be abrogated in similar progenitor transduction studies using bone marrow from Parp1-null mice. In this case, the frequency of cells expressing vector GFP from the cHS4-insulated vector decreased from an average of 51-59% in cells from normal mice to an average of 30-39% in cells from Parp1 null mice (compared to 23-30% for the uninsulated control). All of these differences were statistically significant. PARP-1 has been shown by others to play significant roles in chromatin remodeling and transcriptional control, with some evidence suggesting it may play a role in enhancer-blocking insulator activity. Taken together, our studies demonstrate that binding of PARP-1 also plays a key functional role in the barrier activity of the prototypic cHS4 chromatin insulator, and helps to explain why the extended 400 bp cHS4 core exhibits more activity than smaller versions of this element.
415. Further Improvements and Applications of OPEN (Oligomerized Pool ENgineering): A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases
Morgan L. Maeder,1 Jing-Ruey J. Yeh,2 Jonathan E. Foley,1 Jizhong Zou,3 Stacey Thibodeau-Beganny,1 Linzhao Cheng,3 Randall T. Peterson,2 J. Keith Joung.1 1 Molecular Pathology Unit, Massachusetts General Hospital/ Harvard Medical School, Charlestown, MA; 2Cardiovascular Research Center, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA; 3Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD. Engineered zinc finger nucleases (ZFNs) can induce highly efficient genome modifications in a wide variety of cell types. ZFNs consist of a customized DNA-binding zinc finger array fused to a non-specific nuclease domain. ZFN-induced double-stranded DNA breaks can be repaired by either non-homologous end-joining or homologous recombination, processes that can be used to introduce desired alterations with very high efficiencies at or near the site of the break. The development of a rapid, robust, and publicly available capability to re-engineer the DNA-binding specificities of ZFNs is a critically important requirement for the practice of this technology. We have recently described OPEN (Oligomerized Pool ENgineering), a userfriendly selection-based method for engineering ZFNs that accounts for the context-dependent behavior of individual zinc fingers (Maeder et al., 2008). ZFNs engineered using OPEN have been used to induce highly efficient modification of numerous endogenous human, plant, and zebrafish genes (Maeder et al., 2008; Townsend et al., 2009; Foley & Yeh et al., 2009). Here we describe recent alterations to the OPEN protocol which have allowed us to increase the speed of the method and to improve the scalability of selections. These modifications, which permit the use of multi-well plates/blocks and multi-channel pipets, now enable us to perform selections for 48 target sites in less than eight weeks time. We have used this higher-throughput method to engineer ZFNs for target sites in nine endogenous zebrafish genes. To date, we have shown that many of these OPEN ZFNs can efficiently modify their endogenous gene targets in somatic cells and that these alterations can be passed through the germline. In addition, we report on the engineering of new ZFNs targeted to the endogenous human PIG-A gene. Nonsense mutations in PIG-A are associated with paroxysmal nocturnal hemoglobinuria (PNH) disease. We have shown that OPEN ZFNs targeted to PIG-A can stimulate gene targeting in both human somatic and stem cells. Taken together, our results enable Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
414. PARP-1 Contributes to the Barrier Function of a Vertebrate Chromatin Insulator David W. Emery, Karol Bomsztyk, Mari Aker. Divisions of Medical Genetics and UW Medicine Lake Union, Department of Medicine, University of Washington, Seattle, WA.
The prototypic chromatin insulator cHS4 has proven effective at 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 found that an extended 400 bp core region of the cHS4 element was necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors [Aker et al., Hum Gene Ther 18:333, 2007]. In more recent studies, electrophoretic gel mobility shift assays reveled specific DNA protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies lead to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1), an abundant nuclear protein that has the capacity to bind DNA through zinc finger motifs, and to catalyze the addition of poly(ADP)-ribose chains to itself and other proteins. 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. Footprinting studies using ssDNA probes suggest that PARP-1 binding to this extended cHS4 core region is specific to a unique DNA cruciform secondary structure, rather than a primary sequence. Functional studies with gammaretroviral reporter vectors in cell lines showed that cHS4 barrier activity was abrogated upon deletion or mutation of the putative PARP-1 binding site, reducing the frequency of cells S162
expressing vector GFP 2-fold to the levels seen without the insulator. Transduction studies in primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was also abrogated following treatment with a PARP inhibitor, reducing the level of vector GFP expression 2 to 7-fold to an average of 8-45 mean fluorescent units, levels seen with the uninsulated control. Finally, barrier activity of the cHS4 element was also found to be abrogated in similar progenitor transduction studies using bone marrow from Parp1-null mice. In this case, the frequency of cells expressing vector GFP from the cHS4-insulated vector decreased from an average of 51-59% in cells from normal mice to an average of 30-39% in cells from Parp1 null mice (compared to 23-30% for the uninsulated control). All of these differences were statistically significant. PARP-1 has been shown by others to play significant roles in chromatin remodeling and transcriptional control, with some evidence suggesting it may play a role in enhancer-blocking insulator activity. Taken together, our studies demonstrate that binding of PARP-1 also plays a key functional role in the barrier activity of the prototypic cHS4 chromatin insulator, and helps to explain why the extended 400 bp cHS4 core exhibits more activity than smaller versions of this element.
415. Further Improvements and Applications of OPEN (Oligomerized Pool ENgineering): A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases
Morgan L. Maeder,1 Jing-Ruey J. Yeh,2 Jonathan E. Foley,1 Jizhong Zou,3 Stacey Thibodeau-Beganny,1 Linzhao Cheng,3 Randall T. Peterson,2 J. Keith Joung.1 1 Molecular Pathology Unit, Massachusetts General Hospital/ Harvard Medical School, Charlestown, MA; 2Cardiovascular Research Center, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA; 3Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD. Engineered zinc finger nucleases (ZFNs) can induce highly efficient genome modifications in a wide variety of cell types. ZFNs consist of a customized DNA-binding zinc finger array fused to a non-specific nuclease domain. ZFN-induced double-stranded DNA breaks can be repaired by either non-homologous end-joining or homologous recombination, processes that can be used to introduce desired alterations with very high efficiencies at or near the site of the break. The development of a rapid, robust, and publicly available capability to re-engineer the DNA-binding specificities of ZFNs is a critically important requirement for the practice of this technology. We have recently described OPEN (Oligomerized Pool ENgineering), a userfriendly selection-based method for engineering ZFNs that accounts for the context-dependent behavior of individual zinc fingers (Maeder et al., 2008). ZFNs engineered using OPEN have been used to induce highly efficient modification of numerous endogenous human, plant, and zebrafish genes (Maeder et al., 2008; Townsend et al., 2009; Foley & Yeh et al., 2009). Here we describe recent alterations to the OPEN protocol which have allowed us to increase the speed of the method and to improve the scalability of selections. These modifications, which permit the use of multi-well plates/blocks and multi-channel pipets, now enable us to perform selections for 48 target sites in less than eight weeks time. We have used this higher-throughput method to engineer ZFNs for target sites in nine endogenous zebrafish genes. To date, we have shown that many of these OPEN ZFNs can efficiently modify their endogenous gene targets in somatic cells and that these alterations can be passed through the germline. In addition, we report on the engineering of new ZFNs targeted to the endogenous human PIG-A gene. Nonsense mutations in PIG-A are associated with paroxysmal nocturnal hemoglobinuria (PNH) disease. We have shown that OPEN ZFNs targeted to PIG-A can stimulate gene targeting in both human somatic and stem cells. Taken together, our results enable Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy