Ars insulator protects transgenes from long-term silencing in sea urchin larva

Reporter genes have been used as a powerful tool to analyze cis-regulatory elements responsible for temporal and spatial expression in the early development of sea urchin. However, here we show that the transgenes introduced into the sea urchin embryos undergo suppression in larval stage. The transgene silencing could be one of major obstacle in the analysis of regulatory regions in the late stages of development. We previously demonstrated that a DNA fragment (ArsI) located in the upstream region of sea urchin (Hemicentrotus pulcherrimus) arylsulfatase gene has the property of an insulator. We show that tandem ArsI prevents silencing of a transgene in sea urchin larvae when the ArsI is fused to the 5′ end of the reporter gene. Furthermore, we demonstrate that DNA of the reporter gene introduced into the sea urchin eggs is methylated during development and that the ArsI protects the transgene from the DNA methylation.     

Gene introduction into mouse blastocysts via “pricking”

It is a well-known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation ‘pricking’ technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid-gestation stage were obtained. Twenty-two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)-Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli β-galactosidase (β-gal) gene to a mouse metallothionein-I (MT-I) promoter and assessed for β-gal activity histochemically after 1 and 5 days of culture in the presence of 1 μM CdCI₂, at least 65% of the embryos exhibited β-gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique. © 1993 Wiley-Liss, Inc.     

Upstream element of the sea urchin arylsulfatase gene serves as an insulator.

Insulator DNAs functionally isolate neighboring genes by blocking interactions between distal cis-regulatory elements and promoters. Here we report that a DNA fragment located in the upstream region of sea urchin, H. pulcherrimus, arylsulfatase (HpArs) gene blocks the interaction of the Ars enhancer when positioned between the enhancer and the target promoter, in an orientation dependent manner. The Ars insulator works only 3' to 5' direction and has no significant stimulatory or inhibitory effects on its own promoter. In transgenic Drosophila, the Ars insulator blocks the interaction between even-skipped stripe enhancer and its target promoter. The insulation mechanism operates also unidirectionally in Drosophila. We also show that the efficiency of transformation of HeLa cells is enhanced when the integrated gene is flanked by the Ars insulator, suggesting the sea urchin insulator overcomes the position-dependent transgene expression in mammalian cells. These results demonstrate that the mechanism of action of the insulator has been conserved throughout evolution.     

Survivin contributes to the anti-apoptotic activities of transforming growth factor alpha in mouse blastocysts through phosphatidylinositol 3'-kinase pathway

Transforming growth factor alpha (TGFA) is produced by epithelial cells in the oviducts and uteri and has the potential to act as an anti-apoptotic factor on preimplantation embryos expressing its receptor. Previously, we demonstrated that survivin (also known as BIRC5), an anti-apoptotic gene expressed in mouse preimplantation embryos, protects embryos from apoptosis. In this study, we investigated the role of survivin on TGFA-mediated inhibition of apoptosis in mouse blastocysts. Under the suboptimal conditions produced by single embryo culture, blastocysts showed an increase of apoptosis that correlated with a decrease of survivin expression. TGFA treatment significantly decreased apoptosis and increased the levels of survivin mRNA in a dose-dependent manner in blastocyst, and conversely, these activities were neutralized by an anti-TGFA antibody. Antibody treatment alone exerted little effect on either the occurrence of apoptosis or the levels of survivin mRNA. Upregulation of survivin expression by TGFA treatment was insignificant before the blastocyst stage. Using an antisense approach, we examined whether upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts. Apoptosis was inhibited by TGFA treatment in blastocysts, but the effect was abrogated by cotreatment with antisense oligonucleotides directed against survivin. These data suggest that survivin contributes to the anti-apoptotic activities of TGFA in blastocysts. We also found that the upregulation of survivin expression was mediated by activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Thus, TGFA inhibits apoptosis in mouse blastocysts through upregulation of survivin expression via the PI3K pathway.     

Regulation of gene expression in bovine blastocysts in response to oxygen and the iron chelator desferrioxamine

Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.     

Sox regulates transcription of the sea urchin arylsulfatase gene

A 50 bp region from -194 bp to -144 bp of the arylsulfatase gene (HpArs) of the sea urchin, Hemicentrotus pulcherrimus, is related to the temporally regulated expression of this gene. This region contains a Sox (Sry-related HMG box)-binding site, and the introduction of sequence mutations to this site significantly reduced the activity of the HpArs promoter, even in the presence of the C15 enhancer, which consists of HpOtx and CAAT motifs. A protein that binds to the Sox-binding site in the 50 bp region of the HpArs gene was detected in nuclear extracts of mesenchyme blastulae and a protein synthesized in vitro using SoxB1 cDNA of another sea urchin, Strongylocentrotus purpuratus, also bound to this Sox site. These results suggest that HpSox, which is maternally expressed and remains abundant by the pluteus stage, is clearly implicated in regulation of the HpArs gene. The presence of a negatively acting cis element in this 50 bp region has also been detected.     

Functional analysis of the promoter of a sea urchin metallothionein gene.

The 5'-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides +31 to -309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injection into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5'-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.     

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Mouse telokin and SM22 promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22 promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22 transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22 transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells. visceral smooth muscle; development; myosin light chain kinase; embryos; CArG element     

Distal cis-acting elements restrict expression of the CyIIIb actin gene in the aboral ectoderm of the sea urchin embryo

The distal region of the S. purpuratus actin CyIIIb gene, between −400 and −1400 nucleotides, contains at least three distinct cis-acting elements (C1R, C1L and E1) which are necessary for correct expression of fusion reporter genes in transgenic sea urchin embryos. The contribution of these elements in the temporal and spatial regulation of the gene was analyzed by single and double site-directed mutagenesis in fusion constructs which carry the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter. Following microinjection of the transgenes in sea urchin embryos, the activity of the mutants was compared to the wild type in time and space by measuring CAT activity at the blastula and pluteus embryonic stages and by in situ hybridization to the CAT mRNA at pluteus stage. Our results indicate that E1 involved in the temporal regulation of CyIIIb and that all three elements are necessary and sufficient to confer aboral (dorsal) ectoderm specificity to the proximal promoter. This is achieved by suppressing the promoter's activity in all other tissues by the cooperative interaction of the cis-acting elements. The C1R element, binding site of the nuclear receptors SpCOUP-TF and SpSHR2, is by itself sufficient to restrict expression in the ectoderm, whereas the aboral ectoderm restricted expression requires in addition the presence of both C1L adn E1. It is therefore evident, that the actin CyIIIb gene is exclusively expressed in the aboral ectoderm by a combinatorial repression in all other cell lineages of the developing embryo.