1564 S. D´ıaz de Leo´n-Guerrero et al. Fig. 1. Neuronal differentiation results from speciﬁc gene expression regulation. (A) In neural stem cells (NSCs), sex determining region Y-box 2 (Sox2) is highly expressed and represses the transcription of neuronal genes like Neurogenic differentiation 1 (NeuroD1). As cells differentiate from NSCs to neural progenitor cells, Sox2 expression is lost and NeuroD1 is activated by wingless-type MMTV integration site family member (Wnt) signaling-dependent binding of T-cell factor/ lymphoid enhancer factor (TCF ⁄ LEF) to its promoter (Kuwabara et al., 2009). (B) In proliferating neural cells, Sox2 and Oct4 (also known as POU5F1 for POU class 5 homeobox 1) are highly expressed and together regulate a wide variety of genes, including Sox2 itself. Sox2 and Oct4 bind to an enhancer region (named SRR2) located downstream of Sox2 gene, which is found in an unmethylated state. Sox2 and Oct4 occupancy also correlate with open chromatin marks (H3Ac) in the enhancer region and throughout the gene. As cells differentiate into neurons, Sox2 and Oct4 occupancy on the SRR2 enhancer is lost, the CpGs in the enhancer become methylated and the open chromatin marks (H3Ac) are lost (Chew et al., 2005). Methylation and gene expression Developmental stages in mammals are established by genetic and epigenetic programs. Epigenetic information is normally deﬁned as those processes that are inherited through mitosis or meiosis inﬂuencing gene expression independently of the DNA sequence. They constitute signals that are interpreted to regulate gene expression and cellular differentiation, among other cellular processes. The chromatin structure provides an additional level of regulation over gene expression as chromatin can switch between a closed or transcriptionally repressive state, to an open or transcriptionally permissive state (Berger, 2007). These changes in chromatin archi- tecture are regulated by different processes including chromatin remodelers (Clapier & Cairns, 2009), histone variants (Kamakaka & Biggins, 2005), histone post-translational modiﬁcations (Kouzarides, 2007) and DNA methylation (Miranda & Jones, 2007). DNA methylation is a covalent modiﬁcation that, in the case of vertebrates, consists of the addition of a methyl group to the 5¢ carbon of cytosine residues (Turek-Plewa & Jagodzinski, 2005). Cytosine methylation normally occurs in the context of the palindromic 5¢-CG-3¢ (CpG) dinucleotide (Holliday & Pugh, 1975; Bird, 1978). In mammals, between 60 and 90% of all CpGs are methylated (Bird, 1986). In vertebrates, there are CpG-rich regions referred to as CpG islands, which are predominantly located in regulatory regions. These islands can be found in approximately 60% of all mammalian genes (Antequera & Bird, 1993), and are mostly found in an unmethylated state (Bird, 1986). DNA methylation patterns vary signiﬁcantly throughout development. Cytosine methylation is established and maintained by DNA methyltransferases (Dnmts), whose expression is tightly regulated during development (reviewed in Goll & Bestor, 2005). In mammals, de-novo methylation is carried by two enzyme families: Dnmt3a and Dnmt3b (Okano et al., 1999). Both proteins are essential for normal development as Dnmt3a knock-out mice become runted after birth and die at around 4 weeks of age; Dnmt3b knock-out shows embryonic defects and causes death before embryonic day 15.5. Homozygous animals, mutant for both Dnmt3a and Dnmt3b, exhibit a more severe phenotype than individual mutants; they die before embryonic day 11.5, suggesting an overlapping function for these proteins in embryogenesis (Okano et al., 1999). Following replication, when the newly synthesized DNA strand is unmethylated, an enzyme binds to hemimethylated sites and catalyzes the transfer of a methyl group on the daughter strand to restore the palindromic methyl CpG conﬁguration (Leonhardt et al., 1992). This maintenance methylation activity is catalyzed by Dnmt1 and ensures that the established methylation patterns are preserved over many cell generations, and provides a means for heritable transcriptional control during development. Dnmt1 is expressed throughout embryonic development; in the adult stage Dnmt1 exists as two isoforms, Dnmt1 and Dnmt1b (Bonﬁls et al., 2000). DNA methylation has been shown to play an important role in several processes, including the suppression of tissue-speciﬁc genes and imprinted genes, X chromosome inactivation, and stability of transposable elements (Jaenisch et al., 1985; Keshet et al., 1986; Becker et al., 1987; Li et al., 1993; Panning & Jaenisch, 1996; Walsh et al., 1998). It has also been shown that methylation confers additional stability to the genome by providing a more tightly packed chromatin, in particular along repetitive sequences and constitutive heterochromatic regions to avoid unspeciﬁc recombination events (Colot et al., 1996; Chen et al., 1998; Hashimshony et al., 2003). The most common effect of DNA methylation is gene silencing, which can be achieved in two ways: (i) a transcription factor binding site can be altered by the presence of a methylated cytosine, preventing its recognition and binding by the transcription factor; and (ii) DNA methylation can recruit methyl-CpG binding proteins that, in turn, are capable of recruiting chromatin-remodeling factors (reviewed in Klose & Bird, 2006). ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 33, 1563–1574