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ABSTRACT: Over three decades ago DNA methylation had been suggested to play a role in the regulation of gene expression. This chapter reviews the development of this field of research over the last three decades, from the time when this idea was proposed up until now when the molecular mechanisms involved in the effect of DNA methylation on gene expression are becoming common knowledge. The dynamic changes that the DNA methylation pattern undergoes during gametogenesis and embryo development have now been revealed. The three-way connection between DNA methylation, chromatin structure and gene expression has been recently clarified and the interrelationships between DNA methylation and histone modification are currently under investigation. DNA methylation is implicated in developmental processes such as X-chromosome inactivation, genomic imprinting and disease, including tumor development. This chapter discusses all these issues in depth.
Progress in molecular and subcellular biology 02/2005; 38:151-67.
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ABSTRACT: The Prader-Willi/Angelman imprinted domain on human chromosome 15q11-q13 is regulated by an imprinting control center (IC) composed of a sequence around the SNRPN promoter (PWS-SRO) and a sequence located 35 kb upstream (AS-SRO). We have previously hypothesized that the primary imprint is established on AS-SRO, which then confers imprinting on PWS-SRO. Here we examine this hypothesis using a transgene that includes both AS-SRO and PWS-SRO sequences and carries out the entire imprinting process. The epigenetic features of this transgene resemble those previously observed on the endogenous locus, thus allowing analyses in the gametes and early embryo. We demonstrate that the primary imprint is in fact established in the gametes, creating a differentially methylated CpG cluster (DMR) on AS-SRO, presumably by an adjacent de novo signal (DNS). The DMR and DNS bind specific proteins: an allele-discrimination protein (ADP) and a de novo methylation protein, respectively. ADP, being a maternal protein, is involved in both the establishment of DMR in the gametes and in its maintenance through implantation when methylation of PWS-SRO on the maternal allele takes place. Importantly, while the AS-SRO is required in the gametes to confer methylation on PWS-SRO, it is dispensable later in development.
Human Molecular Genetics 12/2004; 13(22):2767-79. · 7.64 Impact Factor
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ABSTRACT: A cluster of imprinted genes on human chromosome 15q11-q13 (the PWS/AS domain) and its ortholog on mouse chromosome 7c is believed to be regulated by an imprinting control center. Although minideletions in this region in Angelman syndrome (AS) and Prader-Willi syndrome (PWS) patients revealed that two elements, shortest deletion regions of overlap in AS families and PWS families (AS-SRO and PWS-SRO), respectively, constitute the IC, the molecular mechanism that governs this regional control remains obscure. To understand how this imprinting center works, a mouse model was sought. The striking similarity between the human and mouse sequences allowed the generation of a minitransgene (AS-SMP) composed of AS-SRO and the Snrpn minimal promoter (SMP) the mouse ortholog of PWS-SRO. This minitransgene carries out, in a highly reliable and reproducible manner, all steps of the imprinting process. In an attempt to decipher the molecular mechanism of the imprinting process, we generated and tested for imprinting five minitransgenes based on AS-SMP, in which various parts of the 160 bp SMP were deleted. These experiments revealed a set of five cis elements that carry out the various steps of the imprinting process. This set includes: (i). two copies of a de novo methylation signal (DNS) that establish the maternal imprint during oogenesis; (ii). an allele discrimination signal that establishes the paternal imprint; and (iii). two elements that act together to maintain the paternal imprint. Two functionally redundant sets of the five elements were found on the respective endogenous mouse sequence explaining the previously published contradictory results of targeted deletion experiments. Together with the fact that all five elements bind specific proteins that are presumably the factors acting in trans in the imprinting process, our observations set the stage for a comprehensive study of the molecular mechanism involved in the control of the imprinting process.
Human Molecular Genetics 05/2004; 13(7):751-62. · 7.64 Impact Factor
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ABSTRACT: DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.
Gene Expression Patterns 01/2004; 3(6):697-702. · 2.02 Impact Factor
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ABSTRACT: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by loss of gene function of the imprinted genes including Snrpn within a 2 Mb domain on chromosome 15q11-13. Based on microdeletions in PWS and AS patients, a 4.3 sequence around Snrpn promoter/exon 1, together with a 880 bp sequence upstream to Snrpn, are believed to encompass an imprinting control center for the entire 2 Mb domain. We have previously characterized the mouse Snrpn minimal promoter and a 7 bp element (SBE) within it, which is required for its activity. Here we describe the human Snrpn minimal promoter sequence, which is comprised of a 71 bp upstream sequence and 51 bp of exon 1. The SBE, which has been shown to be critical for mouse promoter activity, is also found in the human sequence and absolutely required for promoter activity. Methylation of this element, like in the mouse, prevents the binding of a protein factor and abolishes promoter activity. In addition, the 5' end of exon 1 must contain cis elements that support promoter activity. In contrast, the 3' end of exon 1 appears to repress promoter activity. This sequence specifically binds a protein factor which presumably exerts a repressory effect on the promoter. Methylation of this sequence prevents the binding of this protein.
Gene 02/2003; 304:201-6. · 2.34 Impact Factor
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ABSTRACT: We have previously shown that mast cells enhance eosinophil survival and activation. In this study we further characterized mast cell activity toward eosinophils. Sonicate of both rat peritoneal mast cells and the human mast cell line 1 (HMC-1) induced a concentration-dependent IL-6 and IL-8 release from human peripheral blood eosinophils (ELISA). HMC-1-induced IL-8 release was significantly reduced by the tryptase inhibitors GW-45 and GW-58 (90 and 87%, respectively, at an optimal concentration) but not by anti-stem cell factor, anti-TNF-alpha, or anti-IFN-gamma neutralizing Abs or by the antihistamine drugs pyrilamine and cimetidine. In a manner similar to HMC-1, human recombinant tryptase induced the expression of mRNA for IL-8 (RT-PCR) and caused IL-8 release from the eosinophils. Addition of cycloheximide, actinomycin D, dexamethasone, PD 98059, curcumin, or SB 202190 completely inhibited the tryptase-induced IL-6 and IL-8 release. In contrast, cyclosporin A had no effect on tryptase-induced IL-8 release. Tryptase caused phosphorylation of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases 1 and 2, and p38 (Western blot). Tryptase also induced the translocation of c-Jun from the cytosol to the nucleus (confocal microscopy) and enhanced AP-1 binding activity to the DNA (EMSA). Eosinophils were found to express proteinase-activated receptor 2 (FACS). When eosinophils were incubated with tryptase in the presence of anti-proteinase-activated receptor 2 antagonist Abs a significant decrease in the IL-6 and IL-8 release occurred. In summary, we have demonstrated that the preformed mast cell mediator tryptase induces cytokine production and release in human peripheral blood eosinophils by the mitogen-activated protein kinase/AP-1 pathway.
The Journal of Immunology 10/2002; 169(5):2662-9. · 5.79 Impact Factor
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ABSTRACT: Background Microdeletions in chromosome 15q13-15 of Prader-Willi (PWS) and Angelman Syndrome (AS) patients suggested that SNRPN promoter/exon 1, together with a short sequence located approximately 35 kb upstream, constitute an imprinting control centre that regulates the entire 2 Mb PWS/AS imprinted domain. We have recently shown that a minitransgene composed of the human upstream sequence and mouse Snrpn promoter/exon 1 harbours all the elements necessary for establishing and maintaining an imprinted state.Results Here we describe, using transfection experiments, the Snrpn minimal promoter (SMP), being composed of the entire 76 bp exon 1 and 84 bp of upstream sequence. A 7 bp element (SBE) within SMP that, in its unmethylated state binds a specific protein, is absolutely required for promoter activity. The orthologous human sequence, in spite of the fact that it possesses an identical SBE, failed to display promoter activity in transfection experiments and failed to create a methylated state of the maternal allele. Transgenic experiments reveal that a mutation in SBE of the mouse sequence did not completely abolish methylation of the maternal allele, indicating that sequences outside SBE participate in this process. Replacement of human exon 1 with the mouse orthologue replenished promoter activity, but left the maternal allele in the transgenic experiment unmethylated. The reciprocal chimera, in which mouse exon 1 was replaced by the human orthologue resulted in loss of promoter activity and did not support differential methylation.Conclusions The observations made by in vitro and in vivo experiments suggest that several cis elements which are involved in Snrpn promoter activity and the imprinting process are present in the mouse promoter and absent in the human orthologous sequence.
Genes to Cells 01/2002; 6(11):967 - 975. · 2.68 Impact Factor
