Duri Rungger

An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA

In vertebrates, transcriptionally active promoters are undermethylated. Since the transcription factor Sp1, and more recently NF‐κB, have been implicated in the demethylation process, we examined the effect of transcription factors on demethylation by injecting in vitro methylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter region when fused to a DNA binding domain which recognizes the promoter. Furthermore, demethylation occurs only after the midblastula transition, when the general transcription machinery of the host embryo becomes available. Nevertheless, transcription factor binding need not be followed by actual transcription, since demethylation is not blocked by α‐amanitin treatment. Finally, replication of the target DNA is a prerequisite for efficient demethylation since only plasmids that carry the bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated. No demethylation is detectable in the oocyte system where DNA is not replicated. These results suggest that, in the Xenopus embryo, promoters for which transcription factors are available are demethylated by a replication‐dependent, possibly passive mechanism.

Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.

The Xenopus oocyte system was used to test functionally, putative trans‐active elements involved in the transcriptional control of the mouse interleukin‐2 (IL‐2) gene in resting and mitogen‐induced primary T‐lymphocytes. The IL‐2 gene injected into the oocyte is active over a wide range of DNA concentrations. This basal activity is silenced by the addition of protein extracts from G0‐arrested spleen cells. Extracts from 8 h‐stimulated spleen cells do not silence but moderately increase transcription over basal level. When IL‐2 transcription is silenced first by an injection of extract from resting spleen cells, the addition of proteins from stimulated cells results in a strong increase in transcription (derepression). Use of proteins from purified splenic T‐lymphocytes shows that both silencer(s) and activator(s) are contributed by these cells. Extracts from control tissues have neither a silencing nor stimulatory effect. None of the proteins tested affects the activities of co‐injected control genes. Injections with IL‐2 promoter mutants indicate that the main target sequence of the silencing and activating factors is a purine region (Pu‐box) lying between positions −261 and −292 upstream of the IL‐2 gene. Bandshift assays show differential binding of the Pu‐box with proteins from resting or activated T‐cells.

Structure and transcription termination of a lysine tRNA gene from Xenopus laevis

Termination of RNA polymerase III transcripts commonly occurs at clusters of T residues. A T4 tract located 72 base-pairs beyond a lysine tRNA gene from Xenopus laevis serves as an efficient termination site for the tRNA(Lys) precursors synthesized from this gene in homologous cell-free extracts. Nucleotides following this T tract influence the extent of read-through transcription in vitro, but in a way that differs from Xenopus 5 S RNA termination. Only approximately 50% of the transcripts initiated in vitro extend as far as this downstream T cluster. The remainder prematurely terminate at a second T4 tract located within the gene itself. The contrasting behaviour of these two T tracts in injected oocytes indicates that termination can be influenced by more than just RNA polymerase III alone, and that different components may contribute to, or hinder, termination at these sites. Prematurely terminated tRNA(Lys) transcripts are detectable in RNA from ovary tissue but not from a kidney cell line, suggesting that read-through transcription beyond intragenic T clusters can be modulated in vivo.

Mitochondrial DNA arranged into chromatin-like structures after injection into amphibian oocyte nuclei

Abstract Purified mitochondrial DNA (mitDNA) from ovaries of Xenopus laevis was injected into the nuclei (germinal vesicles) of large vitellogenic oocytes of the same organism and examined by electron microscopy of the spread nuclear contents. Normally located nuclei of untreated oocytes as well as peripherally translocated nuclei of centrifuged oocytes were used. In addition, oocyte nuclei isolated and incubated under liquid paraffin oil were injected with DNA. The integrity of transcriptional structures of endogenous chromosomal (lampbrush chromosomes) and extrachromosomal (nucleoli) genes of the injected nuclei was demonstrated. Microinjected mitDNA was identified as circles of chromatin exhibiting polynucleosome-like organization and a mean contour length of 2.6 μm, corresponding to a compaction ratio of the mitDNA of about 2:1. This DNA packing ratio is similar to that observed after preparation of various kinds of native chromatin in low salt buffers. The chromatin circles formed from injected mitDNA only very rarely exhibited lateral fibrils suggestive of transcriptional activity. These results suggest that purified mitDNA can be transformed to normally structured chromatin when exposed to oocyte nuclear contents but is rarely, if at all, transcribed in this form and in this environment.

Retinal patterning by Pax6‐dependent cell adhesion molecules

Long‐standing evidence gained from Pax6 mutant embryos pointed to an involvement of Pax6‐dependent cell adhesion molecules in patterning the central nervous system and, in particular, the retina. However, direct evidence for such pathways remained elusive. We here present direct evidence that knockdown of Pax6 expression by morpholino antisense molecules in Xenopus embryos and knockdown of maternal N‐cadherin (mNcad), N‐cadherin (Ncad) and neural cell adhesion molecule (NCAM) produce similar phenotypes. Eye formation is reduced and retinal lamination is heavily disorganized. In Pax6 knockdown embryos, the levels of mRNAs coding for these cell adhesion molecules are markedly reduced. Overexpression of Pax6 efficiently rescues the phenotype of Pax6 knockdown embryos and restores expression of these putative target genes. Rescue of Pax6‐deficiency by the putative target gene mNcad moderately rescues eye formation. The promoters of the genes coding for cell adhesion molecules contain several putative Pax6 binding sites, as determined by computer analysis. Chromatin immunoprecipitation shows that, in embryonic heads, Pax6 binds to promoter regions containing such predicted binding sites. Thus, several cell adhesion molecules are direct target genes of Pax6 and cooperate in retinal patterning.

Visualization of rDNA spacer transcription in Xenopus oocytes treated with fluorouridine.

Abstract Under the influence of 5-fluoro-uridine, the ultrastructure of the rDNA transcription units in Xenopus oocytes is altered. Whereas part of the matrix units maintains a normal aspect or shows various degrees of inhibition, in a strong proportion of the transcription units the alternating pattern of matrix units and fibril-free spacer regions is no longer recognized. Transcriptional complexes are found along the entire DNP axis, including the regions of the spacers. These observations support biochemical data on transcription in rDNA spacer regions.

Heat-regulated expression of the hepatitis B virus surface antigen in the human Wish cell line

The DNA fragment coding for the hepatitis B virus surface antigen (HBsAg) was placed under the control of a human 70 kDa heat-shock protein (hsp70) promotor sequence. This plasmid construct has been used in transfection experiments to establish a stable amnion cell line of human origin (Wish), expressing an HBsAg in a heat-regulated fashion. Post-translational modifications, such as assembly, glycosylation, secretion and production of both major and middle S proteins appear to function normally. In addition, production of HBsAg under various protocols of heat induction is described. After inoculation into nude mice, development of tumours has been observed at the site of injection. Tumour cells, dispersed by means of collagenase or trypsin treatment from excised tumours, and subsequently seeded into Petri dishes, were able to secrete the same quantities of HBsAg after heat induction as were cells of the original cell line.

Inhibition of Xhox1A gene expression in Xenopus embryos by antisense RNA produced from an expression vector read by RNA polymerase III.

Antisense inhibition of gene expression during Xenopus development was obtained by injecting, into the zygote, an expression vector carrying the adenovirus VAI gene read by RNA polymerase III. This vector yields high levels of antisense RNA in most embryonic cells between mid-blastula transition and tailbud stage. As a target we chose the Xenopus homeobox gene Xhox1A. A 26 bp long oligonucleotide, including the initiation codon of this gene, was inserted in opposite polarity into the vector. Antisense treatment reduces Xhox1A mRNA in embryos up to stage 22 and Xhox1A protein expression up to stage 30. Half of the antisense-treated embryos develop a characteristic phenotype with disorganized somites in the anterior trunk and delayed development of the intestinal tract.

Cyclosporin A and FK506 prevent the derepression of the IL-2 gene in mitogen-induced primary T lymphocytes.

In resting primary T lymphocytes the interleukin 2 (IL-2) gene is silenced by a repressor binding to the Pud element spanning positions -292 to -264 upstream of the cap site. Upon T-cell activation, this silencer is displaced by a positive transcription factor (TF) and the gene is derepressed and transcribed. Cyclosporin A (CsA) and FK506 interfere with normal derepression of the IL-2 gene. Both drugs exert no direct effect on basal transcription of the IL-2 or control viral genes. Direct addition does not abolish the active state of positive TFs present in proteins from activated T cells. However, if T cells are activated in the presence of either drug, their proteins not only fail to derepress, but efficiently and irreversibly silence IL-2 transcription. DNA-protein binding data show that proteins present in drug-treated cells form retarded complexes corresponding in size to the silencer and positive TF. Thus, in drug-treated cells a functional silencer persists, and a positive TF-like factor appears which is functionally abnormal. Moreover, drug-treated T cells appear to form a component that prevents functioning of normal positive TF.

In vivo transcription of the spacer sequences of rDNA in Xenopus

Abstract Fluoro-uracil (FUc) and fluoro-uridine (FU) inhibit the maturation of pre-rRNA to 18S and 28S rRNA in Xenopus oocytes and cell lines. As a consequence of the incorporation of the nucleotide analogue, a heterogeneous population of RNA molecules heavier than 2.6 million D is conserved. A certain proportion of these molecules has a size of about 4 million D (45S). It is shown by hybridization experiments, that part of the large transcripts induced by the drugs are ribosomal precursors which contain sequences complementary to most of the rDNA repeating unit, including the so-called non-transcribed spacer.