Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development and in repression of viral genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

The ability to derive a whole-genome map of transcription-factor binding sites (TFBS) is crucial for elucidating gene regulatory networks. Herein, we describe a robust approach that couples chromatin immunoprecipitation (ChIP) with the paired-end ditag (PET) sequencing strategy for unbiased and precise global localization of TFBS. We have applied this strategy to map p53 targets in the human genome. From a saturated sampling of over half a million PET sequences, we characterized 65,572 unique p53 ChIP DNA fragments and established overlapping PET clusters as a readout to define p53 binding loci with remarkable specificity. Based on this information, we refined the consensus p53 binding motif, identified at least 542 binding loci with high confidence, discovered 98 previously unidentified p53 target genes that were implicated in novel aspects of p53 functions, and showed their clinical relevance to p53-dependent tumorigenesis in primary cancer samples.

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. Packaging of DNA into chromatin allows the cell to store its genetic information efficiently and has an important role in regulating gene expression (Workman and Kingston 1998). Dynamic changes in chromatin structure can facilitate or prevent the access of the transcription machinery to nucleosomal DNA, leading to transcription regulation. Recent studies have revealed two mechanisms by which chromatin structure can be altered. One mechanism involves multisubunit protein complexes that use the energy derived from ATP hydrolysis to alter the structure of, or 'remodel', nucleosomes (for review, see Tsukiyama and Wu 1997;Kadonaga 1998;Varga-Weisz and Becker 1998;Travers 1999). The other mechanism involves covalent modification of nucleosomes, in particular acetylation of the core histone tails and methylation of DNA (for review, see Grunstein 1997;Struhl 1998;.Since the discovery of histone acetylation by Allfrey et al. (1964), a general correlation between histone acetylation and gene activity has been established (Hebbes et al. 1988). The enzymes that catalyze histone acetylation and deacetylation have been identified (Brownell et al. 1996;Taunton et al. 1996). Several transcriptional coactivators have histone acetyltransferase (HAT) activity, whereas several transcriptional corepressors have histone deacetylase activity (for review, see Grunstein 1997; Kuo and Allis 1998; Struhl 1998). In addition, mutagenesis studies with Gcn5 and Rpd3, the prototypical histone acetyltransferase and deacetylase, respectively, confirmed the long-speculated role of histone acetylation and deacetylation in transcription regulation (Kadosh and Struhl 1998a;Wang et al. 1998). Moreover, Rpd3/Sin3-dependent repression has been shown to be directly associated with the deacetylation of lysine 5 of histone H4 in the promoters of UME6-regulated genes (Kadosh and Struhl 1998b;Rundlett et al. 1998). However, how cor...

Nanog, Sox2, and Oct4 are transcription factors all essential to maintaining the pluripotent embryonic stem cell phenotype. Through a cooperative interaction, Sox2 and Oct4 have previously been described to drive pluripotent-specific expression of a number of genes. We now extend the list of Sox2-Oct4 target genes to include Nanog. Within the Nanog proximal promoter, we identify a composite sox-oct cis-regulatory element essential for Nanog pluripotent transcription. This element is conserved over 250 million years of cumulative evolution within the eutherian mammals. A Nanog proximal promoter-EGFP (enhanced green fluorescent protein) reporter transgene recapitulates endogenous Nanog mRNA expression in embryonic stem cells and their differentiated derivatives. Sox2 and Oct4 interaction with the Nanog promoter was confirmed through mutagenesis and in vitro binding assays. Electrophoretic mobility shift assays indicate that the Sox2-Oct4 heterodimer forms more efficiently on the composite element within Nanog than the similar element within Fgf4. Using chromatin immunoprecipitation, we show that Oct4 and Sox2 bind to the Nanog promoter in living mouse and human embryonic stem cells. Furthermore, by specific knockdown of Oct4 and Sox2 mRNA by RNA interference in embryonic stem cells, we provide genetic evidence for a link between Oct4, Sox2, and the Nanog promoter. These studies extend the understanding of the pluripotent genetic regulatory network within which the Sox2-Oct4 complex are at the top of the regulatory hierarchy.Nanog is a homeobox-containing transcription factor with an essential function in maintaining the pluripotent cells of the inner cell mass and in the derivation of embryonic stem cells (ESCs) 1 from these (1). Furthermore, overexpression of Nanog is capable of maintaining the pluripotency and self-renewing characteristics of ESCs under what normally would be differentiation-inducing culture conditions (2). Concomitant with this essential function in pluripotent cell maintenance is its restricted expression pattern. Nanog transcripts first appear in the inner cells of the morula prior to blastocyst formation (1, 2), are restricted to the inner cell mass in the blastocyst (3), and are no longer detectable at implantation. Expression of Nanog reappears in the proximal epiblast at embryonic day 6 and remains restricted to the epiblast as development progresses (4). The factors controlling expression of this gene have yet to be described. The POU domain-containing Oct4 and the HMG domaincontaining Sox2 are two other transcription factors known to be essential for normal pluripotent cell development and maintenance (5, 6). Although both have independent roles in determining other cell types (6, 7), at least part of their function in pluripotent cells is via a synergistic interaction between the two to drive transcription of target genes. Currently known targets of Sox2-Oct4 synergy are Fgf4, Utf1, and Fbx15, as well as Sox2 and Pou5f1 (the gene encoding Oct4) themselves (8 -13). Each of these targe...