We’ve investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. prevent their reactivation. Synopsis Regulation of gene expression during development and differentiation occurs at many levels including transcriptional gene Rabbit Polyclonal to CSGALNACT2 silencing and activation. Understanding the mechanism of gene silencing has practical importance because genes inserted in the genome for gene therapy are often silenced, decreasing the efficiency and increasing the risk associated with these procedures. In this report, the authors created a gene that cannot be methylated by mutating all of its CpG dinucleotides and examined in detail the role of DNA methylation and other epigenetic modifications in the establishment and maintenance of gene silencing. The authors found that DNA methylation is not required for the establishment or the maintenance of the silent chromatin state, but that this covalent DNA modification was unique among the epigenetic marks tested because it conferred to the chromatin structure a long-term intrinsic epigenetic memory that prevents gene reactivation. Introduction Silencing of transgenes, viruses, and transposons is usually a general phenomenon that has been documented KRN 633 distributor from yeast to mammals [1] and is believed to be a part of a genome defense mechanism against mobile genetic elements [2]. Understanding the epigenetic mechanisms underlying transgene silencing should provide insights into this important nuclear process and also have practical importance: for instance, designing safer gene therapy cassettes. Numerous studies have shown that gene expression in general, and transgene expression in particular, is usually regulated in part by DNA methylation, post-translational histone modifications, and timing of replication, three of the major epigenetic factors controlling chromatin structure. DNA methylation is usually associated with transgene silencing, X chromosome inactivation, genomic imprinting, silencing of mobile genetic elements, and the developmental regulation of some tissue-specific KRN 633 distributor genes [3]. KRN 633 distributor In mammalian cells, DNA methylation is usually maintained and KRN 633 distributor established by three DNA methyltransferases (Dnmt) [4]. Dnmt3a and Dnmt3b are particularly important for de novo methylation, while Dnmt1 is critical for the maintenance of DNA methylation after DNA replication [5]. The greater affinity of Dnmt1 for hemi-methylated DNA than for unmethylated DNA has been suggested as the mechanism for the maintenance of methylation patterns after each cell division (reviewed in [6]). Histone modifications have emerged as major mediators of the formation of repressive chromatin structures [7]. Deacetylation of lysines in the tails of histone H3 and H4 is usually associated with silent chromatin, while acetylation of the same lysine residues is usually associated with gene activation [8,9]. Methylation of lysine 4 of histone H3 (H3-K4-Me) is usually associated with active chromatin, while methylation of H3-K9 and H3-K27 has been shown to be important for the formation of constitutive and KRN 633 distributor facultative heterochromatin [10C14]. The maintenance of histone modifications after each DNA replication is not fully comprehended. Once certain silent chromatin structures are established, they can be stably transmitted through cell division in the absence of the initiating signal [15]. This epigenetic memory is best comprehended for genes regulated by the Polycomb proteins in and by the Sir proteins in yeast [15C17]. A similar phenomenon has been elegantly exhibited in mammalian cells by the Rauscher lab [10]. In that report, the memory was conferred to the transgenes by the recruitment of HP1. Models for the inheritance of histone modifications are based on the observations that this effectors of silencing that bind post-translationally altered histones can also recruit the enzymes that change the histones. After DNA replication, the newly assembled nucleosomes are believed to be made of a random mixture of new unmodified histones and of the aged altered histones. Binding of effector proteins to the aged histones would recruit histone-modifying enzymes and lead to the re-establishment of the pattern of histone modifications that existed before DNA replication [16,18]. In mammals, multiple histone-modifying enzymes and chromodomain proteins are known to be important for silencing, but the interactions between DNA methylation and histone modifications are complex. The Dnmts have been shown to interact with at least 15 proteins including HP1, histone methyltransferases, and histone deacetylases [19C21]. Whether histone modifications and DNA methylation are redundant, cooperative, or impartial pathways of.