Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects

The prototypic chromatin insulator cHS4 has proven effective at reducing repressive chromosomal position effects on retroviral vector expression. We report here studies designed to identify the minimal chicken hypersensitive site-4 (cHS4) sequences necessary for this activity. Using a gammaretroviral reporter vector and expression analysis in cell lines and primary mouse hematopoietic progenitor colonies, we found that a 250-bp core fragment reported to contain most of the cHS4 insulating activity failed to prevent silencing when used alone, although some barrier activity was observed when this fragment was combined with a 790-bp, but not 596-bp, spacer. Similar studies showed that four copies of a 90-bp fragment containing the cHS4 enhancer-blocking activity actually repressed vector green fluorescent protein (GFP) expression. In contrast, a 400-bp fragment containing the 250-bp core plus 3' flanking sequences protected vector expression to the same degree as the full-length 1.2-kb fragment. The 400-bp fragment activity was confirmed in a lentiviral vector expressing human beta-globin in murine erythroid leukemia (MEL) cells. Taken together, these studies indicate that the insulating activity of the 250-bp cHS4 core can be influenced by distance, and identify an extended core element that confers full barrier activity in the setting of two different classes of retroviral vectors.     

Genomic and Functional Assays Demonstrate Reduced Gammaretroviral Vector Genotoxicity Associated With Use of the cHS4 Chromatin Insulator

Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by evidence of vector-mediated genotoxicity involving the activation of cellular oncogenes flanking sites of vector integration. We report here that the rate of gammaretroviral vector genotoxicity can be significantly reduced by addition of the cHS4 chromatin insulator, based on two complementary approaches for assessing vector-mediated genotoxicity. One approach involves the direct, genomewide assessment of cellular gene dysregulation using panels of transduced cell clones and genomic microarrays, whereas the other involves the functional assessment of malignant transformation using a factor-dependent cell line. Both assays are robust and quantitative, and indicate the cHS4 chromatin insulator can reduce vector-mediated genotoxicity approximately sixfold (ranged three to eight fold). These approaches also provide a means for assessing various aspects of vector-mediated genotoxicity, including the overall rate of cellular gene dysregulation, the potential influence of vector provirus over large genomic distances, and the involvement of oncogenic pathways in vector-mediated malignant transformation.     

Improved human beta-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element.

Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hbeta/hgamma-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin insulator element and compared them with their "un-insulated" counterparts. We observed a consistent twofold-higher hbeta expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.     

Kinetics and Epigenetics of Retroviral Silencing in Mouse Embryonic Stem Cells Defined by Deletion of the D4Z4 Element

Retroviral vectors are silenced in embryonic stem (ES) cells by epigenetic mechanisms whose kinetics are poorly understood. We show here that a 3′D4Z4 insulator directs retroviral expression with persistent but variable expression for up to 5 months. Combining an internal 3′D4Z4 with HS4 insulators in the long terminal repeats (LTRs) shows that these elements cooperate, and defines the first retroviral vector that fully escapes long-term silencing. Using FLP recombinase to induce deletion of 3′D4Z4 from the provirus in ES cell clones, we established retroviral silencing at many but not all integration sites. This finding shows that 3′D4Z4 does not target retrovirus integration into favorable epigenomic domains but rather protects the transgene from silencing. Chromatin analyses demonstrate that 3′D4Z4 blocks the spread of heterochromatin marks including DNA methylation and repressive histone modifications such as H3K9 methylation. In addition, our deletion system reveals three distinct kinetic classes of silencing (rapid, gradual or not silenced), in which multiple epigenetic pathways participate in silencing at different integration sites. We conclude that vectors with both 3′D4Z4 and HS4 insulator elements fully block silencing, and may have unprecedented utility for gene transfer applications that require long-term gene expression in pluripotent stem (PS) cells.     

Transient activation of transgene expression by hydrodynamics-based injection may cause rapid decrease in plasmid DNA expression

The intranuclear disposition of exogenous DNA is quite important for the therapeutic effects of the administered DNA. The expression efficiency from one copy of exogenous DNA delivered by hydrodynamics-based injection dramatically decreases over time, and this 'silencing' occurs without CpG methylation. In this study, naked luciferase-plasmid DNA was delivered into mouse liver by hydrodynamics-based injection, and modifications of the histones bound to the plasmid DNA were analyzed by a chromatin immunoprecipitation (ChIP) analysis. In addition, the effects of a second hydrodynamics-based injection on the expression from the plasmid DNA were examined. The ChIP analysis revealed that the modification status of histone H3 remained constant from 4 h to 4 weeks. Surprisingly, the injection of saline without DNA enhanced the luciferase expression from the preexisting DNA administered 4 and 14 days previously. Our results suggest that histone modification plays no role in the silencing. Instead, our data suggest that the transgene expression is activated by the hydrodynamics-based injection manipulation, and that the return from the activated status causes the silencing.     

Histone tail modifications and noncanonical functions of histones: perspectives in cancer epigenetics

Over the past few years, the histone deacetylase (HDAC) inhibitors have occupied an important place in the effort to develop novel, but less toxic, anticancer therapy. HDAC inhibitors block HDACs, which are the enzymes responsible for histone deacetylation, and therefore they modulate gene expression. The cellular effects of HDAC inhibitors include growth arrest and the induction of differentiation. Early successes in cancer therapeutics obtained using these drugs alone or in combination with other anticancer drugs emphasize the important place of posttranslational modifications of histones in cancer therapy. Histone tail modifications along with DNA methylation are the most studied epigenetic events related to cancer progression. Moreover, extranuclear functions of histones have also been described. Because HDAC inhibitors block HDACs and thereby increase histone acetylation, we propose a model wherein exogenous acetylated histones or other related acetylated proteins that are introduced into the nucleus become HDAC substrates and thereby compete with endogenous histones for HDACs. This competition may lead to the increased acetylation of the endogenous histones, as in the case of HDAC inhibitor therapy. Moreover, other mechanisms of action, such as binding to chromatin and modulating gene expression, are also possible for exogenously introduced histones.     

急性白血病组蛋白甲基化、乙酰化修饰异常的研究

目的 研究急性白血病患者组蛋白H3K4和H3K9甲基化及H3、H4乙酰化的修饰异常.方法 用Western blot方法检测19例急性白血病患者组蛋白H3K4、H3K9甲基化水平及15例急性白血病患者H3、H4乙酰化水平,同时选取9例非肿瘤患者及4名健康志愿者作为对照.结果 19例急性白血病患者组蛋白H3K4甲基化水平(0.220±0.096)低于对照组(0.447±0.186)(P＜0.01);H3K9甲基化水平(0.409±0.106)高于对照组(0.168±0.015)(P＜0.01).15例急性白血病患者组蛋白H3乙酰化水平(0.128±0.013)低于对照组(0.386±0.104)(P＜0.01);H4乙酰化水平(0.096±0.008)低于对照组(0.341±0.096)(P＜0.01).结论 急性白血病细胞的组蛋白甲基化、乙酰化修饰存在异常,表现为组蛋白H3K4甲基化水平减低,H3K9甲基化水平增高,组蛋白乙酰化水平呈明显的减低,这些表达的异常均与染色质结构致密、基因转录抑制有关.组蛋白甲基化、乙酰化调控修饰异常与急性白血病的发病可能有关,有可能作为急性白血病治疗的靶点.     

Retrovirus silencing, variegation, extinction, and memory are controlled by a dynamic interplay of multiple epigenetic modifications.

Retrovirus silencing in stem cells produces silent or variegated provirus. Additional memory and extinction mechanisms act during differentiation. Here we show that retrovirus is silent or variegated in mouse embryonic stem (ES) cells that are de novo methyltransferase (dnmt3a and dnmt3b) null. Memory is maintained during differentiation, and extinction occurs on variegated retrovirus, indicating that DNA methylation is dispensable for all forms of retrovirus silencing. Silent and variegated provirus are marked by hypoacetylated histone H3 and bound H1. In wild-type ES cells, silent and variegated proviruses are methylated and bound by hypoacetylated H3, MeCP2, and less H1. Silencing, variegation, and extinction are partially reactivated by 5-AzaC in this context. Lentivirus vectors are also silent or variegated, marked by silent chromatin, and exhibit memory and extinction. We conclude that the universal epigenetic mark of retrovirus silencing is silent chromatin established via the dynamic interplay of multiple epigenetic modifications that include but do not require DNA methylation. A molecular mechanism of competitive H1 and MeCP2 binding may account for this epigenetic interplay, and a model for variegation is discussed.     

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

Suppressed expression of transgenes in vivo is the major obstacle in the gene therapy. For the long-term expression, we utilized a chromatin insulator from sea urchin arylsulfatase (Ars) gene locus (Ars insulator, ArsI), which has been shown to epigenetically regulate gene expression across species. ArsI was able to prevent silencing of the transgene in a myeloid cell line, HL-60, and a murine embryonic stem cell line, CCE, in an orientation-dependent manner, but not in Huh-7, K562 and MCF-7 cells, indicating that the effect of ArsI on gene silencing was cell type dependent. Although anti-silencing effect of ArsI was almost equivalent to that of chicken beta-globin insulator, incorporation of ArsI into lentiviral vector had little effect on the virus titer compared with chicken beta-globin insulator. Clonal analysis of transduced HL-60 cells revealed that ArsI protects the lentiviral vector from position effects regardless of its orientation. Furthermore, chromatin immunoprecipitation assays revealed that a high acetylation level was observed in the promoter of the insulated vector, whereas that of ArsI was independent of its anti-silencing capacity. In addition to it having little deteriorative effect on the virus titer, the identified anti-silencing effect of ArsI suggested its possibility for application in gene therapy.     

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy

Xenograft models are suitable for in vivo study of leukemia’s pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.     

H3K9甲基化在白血病中的表观遗传调控

近年来表观遗传学修饰在恶性肿瘤发生发展中的作用日益引起人们重视。白血病患者中存在着组蛋白修饰的异常,H3K9甲基化失平衡与白血病的发生发展相关。SUV39H1是第一个被发现的特异性H3K9甲基转移酶,催化H3K9甲基化从而参与异染色质形成及基因转录调控。通过纠正H3K9的甲基化异常,使高甲基化失活的抑癌基因再度表达,将有望为白血病的治疗提供新的靶点。本文就组蛋白H3K9甲基化如何影响基因转录调控、沉默基因表达及H3K9甲基化与白血病的关系进行了综述。     

Genetic Variation Stimulated by Epigenetic Modification

Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination. We show that modifications of donor chromatin structure can promote homology-directed repair. These experiments demonstrate that either the activator VP16 or the histone chaperone, HIRA, accelerated gene conversion approximately 10-fold when tethered within the donor array for Ig gene conversion in the chicken B cell line DT40. VP16 greatly increased levels of acetylated histones H3 and H4, while tethered HIRA did not affect histone acetylation, but caused an increase in local nucleosome density and levels of histone H3.3. Thus, epigenetic modification can stimulate genetic variation. The evidence that distinct activating modifications can promote similar functional outcomes suggests that a variety of chromatin changes may regulate homologous recombination, and that disregulation of epigenetic marks may have deleterious genetic consequences.     

Application of DNA methylation biomarkers for endometrial cancer management

It has become clear that aberrant gene expression, via alterations in promoter methylation or histone acetylation, is a contributing factor for carcinogenesis, perhaps as important as genetic mutation. This is particularly evident in endometrial cancer, in which multiple genes are silenced through hypermethylation. In this review, we discuss the field of epigenetics and relevant techniques to characterize methylation and acetylation alterations. The CpG island methylator phenotype, epimutations and the effects of aging on methylation are also discussed. In endometrial cancer there is evidence that hypermethylation of relevant genes can be reversed using epigenetic inhibitors, resulting in re-expression of silenced genes. Preliminary data also suggest that a panel of methylation biomarkers could be useful for diagnosis and even screening in selected populations at high risk. This disease is particularly well suited for such a strategy given that the endometrium is readily accessible for testing and endometrial cancer precursors are well defined.