Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.

Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomicscale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cisregulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.

The steroid/hormone nuclear receptor superfamily comprises several subfamilies of receptors that interact with overlapping DNA sequences and/or related ligands. The thyroid/retinoid hormone receptor subfamily has recently attracted much interest because of the complex network of its receptor interactions. The retinoid X receptors (RXRs), for instance, play a very central role in this subfamily, forming heterodimers with several receptors. Here we describe a novel member of this subfamily that interacts with RXR. Using a v-erbA probe, we obtained a cDNA which encodes a novel 445-amino-acid protein, RLD-1, that contains the characteristic domains of nuclear receptors. Northern (RNA) blot analysis showed that in mature rats, the receptor is highly expressed in spleen, pituitary, lung, liver, and fat. In addition, weaker expression is observed in several other tissues. Amino acid sequence alignment and DNA-binding data revealed that the DNA-binding domain of the new receptor is related to that of the thyroid/retinoid subgroup of nuclear receptors. RLD-1 preferentially binds as a heterodimer with RXR to a direct repeat of the half-site sequence 5'-G/AGGTCA-3', separated by four nucleotides . Surprisingly, this binding is dependent to a high degree on the nature of the spacing nucleotides. None of the known nuclear receptor ligands activated RLD-1. In contrast, a DR-4-dependent constitutive transcriptional activation of a chloramphenicol acetyltransferase reporter gene by the RLD-1/ RXRa heterodimer was observed. Our data suggest a highly specific role for this novel receptor within the network of gene regulation by the thyroid/retinoid receptor subfamily.The steroid/thyroid/vitamin receptors, or nuclear hormone receptors, are a superfamily of ligand-modulated transcription factors which link extracellular signals directly to transcriptional responses. By binding small molecules like vitamin D, steroids, retinoids, and thyroid hormones, this class of receptors can regulate such diverse processes as homeostasis, reproduction, development, differentiation, and oncogenesis (45). In fact, nuclear receptors represent one of the largest transcription factor families known (3,12,18). The receptors share a highly conserved cysteine-rich region, the DNA-binding domain (DBD), that forms two zinc finger structures that allow protein-DNA as well as protein-protein interaction (41). A second but less well conserved region is found in all receptors in the hydrophobic carboxy-terminal part and is usually referred to as ligand-binding domain (LBD). In addition to ligand recognition, this domain encodes either receptor dimerization and transactivation or repressor functions (11,15,60,64,65). The receptors mediate their action by binding to specific DNA sequences (response elements) of target genes as homo-or heterodimers (for reviews, see references 3, 49, and 63 and references therein) or by interacting with other factors, notably the transcription factor AP-1 (reviewed in reference 48).

The vitamin hormone retinoic acid (RA) regulates many complex biological programs. The hormonal signals are mediated at the level of transcription by multiple nuclear receptors. These receptors belong to the steroid/thyroid hormone receptor superfamily that also includes a large number COUPa and -1 bind strongly to these response elements, including a palindromic thyroid hormone response element and a direct repeat RA response element as well as an RXR-specific response element. In addition, we found that the previously identified COUP-TF binding site in the ovalbumin gene functions in vitro as an RA response element that is repressed in the presence of COUP. Our data suggest that the COUP receptors are a novel class of RAR and RXR regulators that can restrict RA signaling to certain elements. The COUP orphan receptors may thus play an important role in cell-or tissue-specific repression of subsets of RA-sensitive programs during development and in the adult.The vitamin A derivative retinoic acid (RA) and other retinoids regulate a very large spectrum of biological processes, including development, growth, differentiation, metabolism, homeostasis, and morphogenesis. How most of these individual programs are restricted to certain developmental stages, cell types, or tissues is not known. Like the steroid and thyroid hormones, RA and retinoid signals are mediated by nuclear receptors (1,2,7,8,20,27,33) that belong to one of the largest transcription factor families known today. The nuclear receptors are characterized by a highly conserved DNA binding domain and a moderately conserved ligand binding domain (for a review, see references 4 and 11). The DNA binding domain encodes two zinc finger structures; the first determines DNA binding specificity, while the second zinc finger appears to be involved in receptor dimerization (26). The ligand binding domain comprises the large carboxy-terminal region that, besides ligand binding, has a number of additional functions, including dimerization (6, 51, 53, 54) (hetero and homo) and transcriptional activation (50,51,54). Besides the ligand-activated receptors, the receptor superfamily also comprises many orphan receptors for which specific ligands are not known and of which biological roles have not yet been defined (for a review, see references 4 and 11). One possible role of the orphan receptors may be to regulate the activity of ligandactivated receptors through mechanisms that could include heterodimer formation or competition for specific response elements.The complexity of the retinoid response pathways is * Corresponding author.displayed at the receptor level by six receptor genes, encoding the three RA receptors (RARs) (1,2,7,8,20,33) and three retinoid X receptors (RXRs) (12,23,27,48). In addition, multiple isoforms of several of these receptors are generated by a combination of alternative splicing and multiple promoter usage (17,22,24,49

Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.