Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

Distinct roles for TBP and TBP-like factor in early embryonic gene transcription in Xenopus.

The TATA-binding protein (TBP) is believed to function as a key component of the general transcription machinery. We tested the role of TBP during the onset of embryonic transcription by antisense oligonucleotide-mediated turnover of maternal TBP messenger RNA. Embryos without detectable TBP initiated gastrulation but died before completing gastrulation. The expression of many genes transcribed by RNA polymerase II and III was reduced; however, some genes were transcribed with an efficiency identical to that of TBP-containing embryos. Using a similar antisense strategy, we found that the TBP-like factor TLF/TRF2 is essential for development past the mid-blastula stage. Because TBP and a TLF factor play complementary roles in embryonic development, our results indicate that although similar mechanistic roles exist in common, TBP and TLF function differentially to control transcription of specific genes.

Gene-selective developmental roles of general transcription factors.

Innumerable transcription factors integrate cellular and intercellular signals to generate a profile of expressed genes that is characteristic of the biochemical and cellular properties of the cell. This profile of expressed genes changes dynamically along with the developmental stage and differentiation state of the cell. The biochemical machinery upon which transcription factors integrate their signals is referred to as the general transcription machinery. However, this machinery is not of universal composition, and variants of the general transcription factors play specific roles in embryonic development, reflecting the constraints and requirements of developmental gene regulation.

Translation of maternal TATA-binding protein mRNA potentiates basal but not activated transcription in Xenopus embryos at the midblastula transition.

Early embryonic development in Xenopus laevis is characterized by transcriptional repression which is relieved at the midblastula stage (MBT). Here we show that the relative abundance of TATA-binding protein (TBP) increases robustly at the MBT and that the mechanism underlying this increase is translation of maternally stored TBP RNA. We show that TBP is rate-limiting in egg extract under conditions that titrate nucleosome assembly. Precocious translation of TBP mRNA in Xenopus embryos facilitates transcription before the MBT, without requiring TBP to be prebound to the promoter before injection. This effect is transient in the absence of chromatin titration and is sustained when chromatin is titrated. These data show that translational regulation of TBP RNA contributes to limitations on the transcriptional capacity before the MBT. Second, we examined the ability of trans-acting factors to contribute to promoter activity before the MBT. Deletion of cis-acting elements does not affect histone H2B transcription in egg extract, a finding indicative of limited trans-activation. Moreover, in the context of the intact promoter, neither the transcriptional activator Oct-1, nor TBP, nor TFIID enable transcriptional activation in vitro. HeLa cell extract, however, reconstitutes activated transcription in mixed extracts. These data suggest a deficiency in egg extract cofactors required for activated transcription. We show that the capacity for activated H2B transcription is gradually acquired at the early gastrula transition. This transition occurs well after the blastula stage when the basal transcription machinery can first be complemented with TBP.

Constitutive genomic methylation during embryonic development of Xenopus.

Methylation of CpG dinucleotides is a predominant modification of genomic DNA in many species, especially in vertebrates. This modification, generally associated with transcriptional repression, is rapidly and globally lost during mammalian pre-implantation development. This loss of methylation is gradually reversed during subsequent stages of development. Here we show that the amphibian Xenopus laevis maintains high levels of DNA methylation during early embryonic development. The methylation status of specific loci is independent of the temporal expression profile. The observations have profound implications for the regulation of early embryonic gene regulation and genome function.

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo.

Oct-1, a member of the POU family of transcription factors, is expressed at relatively high levels in ectodermal and mesodermal cell lineages during early Xenopus embryogenesis (Veenstra et al, 1995). Here we show that overexpression of Oct-1 induces programmed cell death concomitant with the loss of the posterior part of the body axis. Truncated Oct-1 variants, missing either the C-terminal or N-terminal trans-activation domain, exhibit a different capacity to cause such developmental defects. Oct-1-induced cell death is rescued in unilaterally injected embryos by non-injected cells, indicative of the non-cell autonomous character of the developmental effects of Oct-1. This was confirmed by marker gene analysis, which showed a significant decrease in brachyury expression, suggesting that Oct-1 interferes with an FGF-type signalling pathway.

Dynamic and differential Oct-1 expression during early Xenopus embryogenesis: persistence of Oct-1 protein following down-regulation of the RNA.

As a first step towards the elucidation of the role of the transcription factor Oct-1 in development, we prepared a monoclonal antibody to study the spatio-temporal distribution of Oct-1 protein in vivo. Here we report differential expression of the Oct-1 gene in the Xenopus embryo both at the RNA and the protein level. Transcripts and protein are detected in ectodermal and mesodermal cell lineages, in which the expression exhibits a pattern of progressive spatial restriction in the course of development. The Oct-1 expression as reported here is not correlated with cell density or cell proliferation in the embryo. Our results suggest a role of Oct-1 in the specification and differentiation of neuronal and neural crest cells. In many other cells, the developmental decision to down regulate Oct-1 is delayed, probably due to a high stability of the protein.

Differential expression of the human, mouse and rat IGF-II genes.

The IGF-II gene is a complex transcription unit. Multiple transcripts are synthesized as a result of alternate promoter usage and the splicing of unique 5' untranslated regions to common coding exons. In order to characterize the mechanisms of IGF-II gene regulation we performed comparative studies to define essential features of IGF-II expression in human, rat and mouse. Homologous promoter regions of the human, mouse and rat IGF-II genes were fused to the luciferase reporter gene and expression was measured in various cell lines that have an endogenously active or inactive IGF-II gene expression pattern, respectively. The transient promoter activity of the human, mouse and rat IGF-II constructs was further compared with the endogenous activity of the IGF-II gene in various tissues and cell lines of human, mouse and rat origin. The results indicate that in transient expression assays employing heterologous systems (e.g., mouse promoter in human cells), most IGF-II promoter constructs are active, albeit at low levels. Maximal promoter activity is only observed, however, in homologous systems (e.g., human promoter constructs tested in human cells). This suggests that each promoter, despite the strong sequence conservation of the homologous human, rat and mouse promoters, is adapted to the levels of the transcription factors present in its natural environment. Finally, IGF-II gene expression is not only regulated at the level of transcription but also depends on mRNA stability. We show that human, rat and also mouse IGF-II mRNAs are subjected to specific endonucleolytic cleavage, suggesting that specific cleavage of IGF-II mRNAs must be of general physiological importance.

A rapid determination of brass composition and plating weight on brass-plated steel wire and cord by X-ray fluorescence spectrometry.

A rapid and simple means for determination of the brass composition and plating weight on brass-plated steel wire and cord is described. The sample preparation procedure is very simple; wires can be mounted as such, and cords can be mounted either as such or as unstranded single wires. The copper content of the brass and the plating weight are determined by measuring the intensities of the different elements by sequential X-ray fluorescence spectrometry (XRF). There is good agreement between the results obtained by XRF and those obtained by differential pulse polarography or spectrophotometry/ complexometry; the precision is even better.

The Oct-1 POU domain directs developmentally regulated nuclear translocation in Xenopus embryos.

Early embryonic development in Xenopus is characterized by transcriptional repression which is relieved at the mid-blastula stage. Here we show that most of the maternally inherited POU domain transcription factor Oct-1 is retained in the cytoplasm during early development, and that it gradually translocates to the nucleus around the mid-blastula transition. Overexpressed epitope-tagged Oct-1 exhibits highly similar localization properties compared to endogenous protein. The amino acid sequence that directs this developmentally regulated nuclear translocation resides in the POU domain. Our findings may suggest that cytoplasmic retention of Oct-1 facilitates or contributes to the repression of Oct-1 target genes before the mid-blastula transition.

Ultrastructural organization and regulation of a biomaterial adhesin of Staphylococcus epidermidis.

Coagulase-negative staphylococci have emerged as important pathogens in infections associated with intravascular devices. Microbial adherence to biomaterial surfaces is a crucial step in the pathogenesis of these infections. Staphylococcal surface proteins (herein referred to as SSP-1 and SSP-2) are involved in the attachment of Staphylococcus epidermidis 354 to polystyrene. In the present study we show that the adhesin protrudes from the cell surface as a fimbria-like polymer. Furthermore, in vitro proteolytic cleavage of SSP-1 produces an SSP-2-like protein which coincides with a loss of adhesive function. SSP-1 expression is down-regulated in a phenotypical variant of S. epidermidis 354 whereas SSP-2 expression is not. These results could suggest that proteolytic cleavage is a key to the regulation of the adhesive state of S. epidermidis in vivo.

The promoter of the Xwnt-5C gene contains octamer and AP-2 motifs functional in Xenopus embryos.

The Xwnt-5C gene is expressed in Xenopus embryos from the early gastrula stage onwards. The transcription of Xwnt-5C is regulated differentially with respect to transcript size, timing and localization. To gain insight into the generation of the Xwnt-5C expression pattern, we started to analyze the transcriptional regulation of this gene. We isolated Xwnt-5C genomic DNA sequences. By microinjection of chimaeric reporter constructs into Xenopus embryos we demonstrate that the upstream region contains a promoter functional in vivo. Of the several putative binding sites for trans-acting factors, present in a minimal promoter fragment, some have been studied in more detail. Mutations in an octamer motif and in an AP-2 consensus sequence interfere with the activity of the Xwnt-5C minimal promoter. In vitro binding assays with extracts from gastrula stage Xenopus embryos show that the octamer motif of the Xwnt-5C promoter can bind several Octamer binding factors, one of which is Oct1.