The Functional Role of S/MARs in Episomal Vectors as Defined by the Stress-Induced Destabilization Profile of the Vector Sequences

The scaffold/matrix attachment regions (S/MARs) are chromosomal elements that participate in the formation of chromatin domains and have origin of replication support functions. Because of all these functions, in recent years, they have been used as part of episomal vectors for gene transfer. The S/MAR of the human β-interferon gene has been shown to support efficient episome retention and transgene expression in various mammalian cells. In Jurkat and other cells, DNA plasmid vectors containing Epstein-Barr virus origin of replication (EBV OriP) and the EBV nuclear antigen-1 gene mediate prolonged episome retention in the host cell nucleus, which, however, diminishes over time. In order to enhance retention, we combined this system with an S/MAR element. Unexpectedly, this completely eliminated the capacity of episomes to replicate. Calculation of the stress-induced DNA duplex destabilization profile of the vectors suggested that the S/MAR element had created an increase in molecular stability at the OriP site that may have disturbed replicative potential. In contrast, introduction of an alternative initiation of replication region from the β-globin gene locus, instead of EBV OriP and the EBV nuclear antigen-1 gene, restored replicative capacity and enhanced episome retention mediated by the S/MAR. These effects were associated with a destabilization profile at the initiation of replication region. These data demonstrate a correlation between S/MAR-mediated vector retention and the presence of an unstable duplex at a replication origin, in this particular setting. We consider that the calculation of stress-induced duplex destabilization may be an informative first step in the design of units that replicate extrachromosomally, particularly as the latter present a safer and, therefore, attractive alternative to integrating viral vectors for gene therapy applications.     

Initiation of DNA replication at the human beta-globin 3' enhancer

The origin of DNA replication in the human β-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the β-globin 3′ enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3′ enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3′ enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3′ enhancer also cooperates with elements in an expressing HS3β/γ-globin construct to initiate replication. These data indicate that the β-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.     

Organization of specific DNA sequence elements in the region of the replication origin and matrix attachment site in the chicken alpha-globin gene domain.

Summary The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site (MAR) of the -globin gene domain was investigated. The fragment was shown to contain a CR1-type repetitive element and two stably bent DNA sequences. One of them colocalizes with the previously described MAR element and with the recognition site for a proliferating-cell-specific, DNA-binding protein. The melting pattern of a set of subfragments of the region proved to be non random. No correlation between the distribution of readily melting sequences and bent DNA was found. The possible importance of curved, low-melting and repetitive DNA sequences for the organization of the upstream boundary of the -globin gene domain and the function of the replication origin is discussed.     

The tkNeo Gene, but Not the pgkPuro Gene, Can Influence the Ability of the β-Globin LCR to Enhance and Confer Position-Independent Expression onto the β-Globin Gene

Whether drug-selectable genes can influence expression of the β-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the β-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the β-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3′ end of the β-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3′ end of the β-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the β-globin gene linked to its LCR. The findings could partly explain why data on β-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.     

Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes. J. Cell. Biochem. 67:439–450, 1997. © 1997 Wiley-Liss, Inc.     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

Distinctive Patterns of Evolution of the δ-Globin Gene (HBD) in Primates

In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a constrained evolution for HBD in Anthropoid lineages, which is unlikely to be related to protein function. Collectively, these findings suggest that sequence change at the δ-globin gene has been under strong selective constraints over 65 Myr of primate evolution, likely due to a regulatory role in ontogenic switches of gene expression.     

Characterization of the 5′-to-5′linked adult α- and β-globin genes from three sciaenid fish species (Pseudosciaena crocea, Sciaenops ocellatus, Nibea miichthioides)

Recently, we cloned the adult α-globin genes from large yellow croaker Pseudosciaena crocea, cuneate drum Nibea miichthioides and red drum Sciaenops ocellatus. All these α-globins have a unique Gly insertion at the 47th residue. In this paper, the three sciaenid globin complexes were identified and compared in detail. Linkage analysis indicated that the sciaenid α- and β-globin genes were oriented head-to-head relative to each other. The sciaenid intergenic regions between the linked α- and β-globin genes were the smallest in reported fish globin gene complexes to date. Classical promoter elements were condensed and the CCAAT box unstable duplication was found in these regions. The promoter function of the intergenic region from large yellow croaker was tested by transient expression of EGFP in Vero cells. We also described a method for studying luciferase reporter gene transient expression in primary fish erythrocytes. We used the method to assess the promoter strength of the three intergenic regions between the sciaenid α- and β-globin genes.     

CTCF-dependent enhancer blockers at the upstream region of the chicken alpha-globin gene domain

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5′ flanking region of the chicken α-globin domain, 11–16 kb upstream of the embryonic α-type π gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two α-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific α-globin gene domain located in a permanently open chromatin area.     

Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human ^Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin ^Aγ-globin (βsp/^Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/^Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the ^Aγ-globin gene promoter showed developmental switching and expressed ^Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.     

Budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses

Background

Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells.

Methods

To test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 μM), formoterol (0.1 μM), both, or neither, and then inoculated with RV-16 (5×10⁶ plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFα, IFN-β, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL28A&B) were analyzed.

Results

RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma.

Conclusions

Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.