Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication.

International audienceIn the mouse, transcriptional permissiveness is established in the fertilized egg prior to the activation of zygotic genes at the 2-cell stage. Therefore, gene inactivity initiated at the end of gametogenesis results from a complex process, involving more than an inhibition of the basal transcriptional apparatus. We have examined the ability of the first intron (I1) of the human hypoxanthine phosphoribosyl transferase gene, which functions as an enhancer in embryonic stem cells, to activate a reporter gene when placed proximally to or at a distance from the HSV-tk promoter, or when integrated into the mouse genome as part of a stable transgene. In microinjected embryos, I1 functions as an enhancer sequence; however, its competence for long-range activation appears only after the late 1-cell stage and depends on the first DNA replication. Moreover, activation of microinjected transgenes from proximal enhancers occurs in the late 2-cell embryo and in the male pronucleus of 1-cell embryos blocked for DNA replication; whereas, for integrated transgenes, proximal enhancer activity is subject to position effects in the 2-cell embryo and first occurs at the 2- or 4-cell stage, but only after completion of DNA replication. Therefore, the absence of long-range activation and a non-permissive genomic state (the relief of which both depend on DNA replication), together with an inactive transcriptional apparatus, appear to converge to prevent any gene activity in the 1-cell embryo. We propose that the embryo exploits the process of DNA replication to relieve the transcriptionally repressive state that was initially established to fulfil two purposes: (1) to arrest maternal gene expression in the maturing oocyte and (2) to protect the unicellular egg and 1-cell embryo from premature differentiation. Reactivation of gene expression by DNA replication would therefore serve to coordinate cell proliferation and differentiation in the preimplantation embryo

Establishment and relief of CpG-dependent transgene repression during germ line passage and mouse development

Methylation of genomic DNA at CpG sequences has a repressive effect on gene expression in vertebrates. The level of methylation of the genome varies widely during development in mammals. The DNA of early germ cells and blastocysts is largely hypomethylated but that of implantation embryos is hypermethylated. To test whether these variations might affect gene expression, we have studied the pattern of expression of a CpG-rich and of a CpG-poor LacZ transgene driven by a strong promotor of an ubiquitous gene. We find that the changes of the expression of the CpG-rich LacZ gene directly correlate with variations of methylation and that lowering the level of CpG in the reporter gene decreases dramatically its susceptibility to these variations. Therefore, in association with fluctuations in genome methylation a CpG-dependent system of repression must control positively and negatively gene expression during development and gametogenesis. We also find that the repression of the CpG-rich LacZ reporter can be abolished in 2-cell embryo by inhibitors of histone deacetylases suggesting that, at least at this stage, this CpG-dependent repression acts through histone deacetylation. In addition, we show that the β-globin locus control region and other genomic elements completely reverse the repression established at implantation in embryonic cells. The complex patterns of expression of the CpG-rich and poor LacZ reporter genes during gametogenesis and development-including a sex-dependent expression in the zygotic nucleus before the morula stage-are described. The possible implication of the constraints imposed on gene expression by DNA methylation revealed by this study are discussed

CpG content affects gene silencing in mice: evidence from novel transgenes

International audienceBackgroundTransgenes are often engineered using regulatory elements from distantly related genomes. Although correct expression patterns are frequently achieved even in transgenic mice, inappropriate expression, especially with promoters of widely expressed genes, has been reported. DNA methylation has been implicated in the aberrant expression, but the mechanism by which the methylation of a CpG-rich sequence can perturb the functioning of a promoter is unknown.ResultsWe describe a novel method for analyzing epigenetic controls that allows direct testing of CpGs involvement by using LacZ reporter genes with a CpG content varying from high to zero that are combined with a CpG island-containing promoter of a widely expressed gene - the α-subunit of the translation elongation factor 1. Our data revealed that a LacZ transgene with null CpG content abolished the strong transgene repression observed in the somatic tissues of transgenic lines with higher CpG content. Investigation of transgene expression and methylation patterns suggests that during de novo methylation of the genome the CpG island-containing promoter escapes methylation only when combined with the CpG-null transgene. In the other transgenic lines, methylation of the promoter may have led to transcriptional silencing.ConclusionsWe demonstrate that the density of CpG sequences in the transcribed regions of transgenes can have a causal role in repression of transcription. These results show that the mechanism by which CpG islands escape de novo methylation is sensitive to CpG density of adjacent sequences. These findings are of importance for the design of transgenes for controlled expression