The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication.The available evidence, derived largely from studies with Saccharomyces cerevisiae, supports the idea that transcriptional activator proteins recruit basal transcription machinery and chromatin-modifying enzymes to the promoter (43). In essence, activator proteins increase the local concentration of the transcriptional machinery near the promoter. Most yeast activators bind less than a kilobase upstream of the promoters they activate, and looping of the chromatin between the activator and the promoter accommodates interactions. Consistent with this model, the yeast activators tested work poorly when positioned more than a few hundred base pairs upstream of the promoter (53).Although it explains much of yeast gene activation, the localconcentration model does not adequately explain long-range activation by metazoan enhancers, which are often tens of kilobases from the promoter. Based on theoretical calculations, two sequences separated by several kilobases in DNA, or in various forms of chromatin, do not have a local concentration relative to each other greater than that of the estimated total concentration of all the promoters in a nucleus (46). In addition to these theoretical considerations, it was found experimentally that site-specific recombination between two sites separated by several kilobases in mammalian cells occurs slowly compared to recombination between two sites separated by a kilobase or so (45). Thus, an enhancer located several kilobases from a promoter is not expected to contact the promoter specifically or efficiently if it is dependent on random diffusion alone. Studies with the human ␤-globin locus using two different techniques, however, indicate that an enhancer in the locus control region located some 40 to 60 kb from the globin genes is in close proximity to a globin gene promoter when that promoter is activated (8,56). This suggests that mechanisms exist to facilitate enhancer-promoter contact over large distances.Studies of Drosophila melanogaster have identified sequences that facilitate particular long-range enhancer-promoter interactions. The large interval separating the iab-7 enhancer and the Abd-B gene ...

The mammalian genome depends on patterns of methylated cytosines for normal function, but the relationship between genomic methylation patterns and the underlying sequence is unclear. We have characterized the methylation landscape of the human genome by global analysis of patterns of CpG depletion and by direct sequencing of 3073 unmethylated domains and 2565 methylated domains from human brain DNA. The genome was found to consist of short (<4 kb) unmethylated domains embedded in a matrix of long methylated domains. Unmethylated domains were enriched in promoters, CpG islands, and first exons, while methylated domains comprised interspersed and tandem-repeated sequences, exons other than first exons, and non-annotated single-copy sequences that are depleted in the CpG dinucleotide. The enrichment of regulatory sequences in the relatively small unmethylated compartment suggests that cytosine methylation constrains the effective size of the genome through the selective exposure of regulatory sequences. This buffers regulatory networks against changes in total genome size and provides an explanation for the C value paradox, which concerns the wide variations in genome size that scale independently of gene number. This suggestion is compatible with the finding that cytosine methylation is universal among large-genome eukaryotes, while many eukaryotes with genome sizes <5 x 10(8) bp do not methylate their DNA.

Abnormalities of genomic methylation patterns are lethal or cause disease, but the cues that normally designate CpG dinucleotides for methylation are poorly understood. We have developed a new method of methylation profiling that has single-CpG resolution and can address the methylation status of repeated sequences. We have used this method to determine the methylation status of >275 million CpG sites in human and mouse DNA from breast and brain tissues. Methylation density at most sequences was found to increase linearly with CpG density and to fall sharply at very high CpG densities, but transposons remained densely methylated even at higher CpG densities. The presence of histone H2A.Z and histone H3 di-or trimethylated at lysine 4 correlated strongly with unmethylated DNA and occurred primarily at promoter regions. We conclude that methylation is the default state of most CpG dinucleotides in the mammalian genome and that a combination of local dinucleotide frequencies, the interaction of repeated sequences, and the presence or absence of histone variants or modifications shields a population of CpG sites (most of which are in and around promoters) from DNA methyltransferases that lack intrinsic sequence specificity.

Epigenetic effects in mammals depend largely on heritable genomic methylation patterns. We describe a computational pattern recognition method that is used to predict the methylation landscape of human brain DNA. This method can be applied both to CpG islands and to non-CpG island regions. It computes the methylation propensity for an 800-bp region centered on a CpG dinucleotide based on specific sequence features within the region. We tested several classifiers for classification performance, including K means clustering, linear discriminant analysis, logistic regression, and support vector machine. The best performing classifier used the support vector machine approach. Our program (called HDFINDER) presently has a prediction accuracy of 86%, as validated with CpG regions for which methylation status has been experimentally determined. Using HDFINDER, we have depicted the entire genomic methylation patterns for all 22 human autosomes.DNA methylation ͉ epigenomics ͉ methylation prediction ͉ CpG islands A lthough progress recently has been made toward wholegenome DNA methylation profiling by using molecular techniques, computational epigenomics is still in its infancy (1). Global analyses of DNA methylation have been focused mainly on two themes: the discovery of methylated CpG islands (CGI) and allele-specific cytosine methylation. Computational prediction of CGIs was introduced in 1987 by . They defined CGIs as regions of Ͼ200 bp with GϩC content of Ͼ0.5 and the observed͞expected CpG ratio Ͼ0.6. Takai and Jones (3) later proposed a more stringent definition that requires CGIs to be Ͼ500 bp long, CG content Ͼ55%, and the CpG ratio Ͼ0.65. This latter method is successful in excluding Alu repeats, many of which were annotated as CGIs when the former criteria were used. Matsuo et al. (4) have provided statistical evidence for erosion of mouse CGIs as compared with human ones. They suggested that an accumulation of TpGs and CpAs observed in mouse, presumably due to the higher rate of deamination of the methylated CpGs, results in a lower CpG ratio in mouse. Antequerra and Bird (5) performed comparative analysis on human and mouse and came to a similar conclusion. Yang et al.(6) proposed a computational method to identify genes with significant differences in gene expression between two parental alleles by searching the UniGene database for the presence of monoallelically expressed (or imprinted) genes in the human genome. Wang et al. (7) compared human and mouse sequences for all known imprinted genes and found 15 motifs that are significantly enriched in the imprinted genes. However, currently there is no algorithm that can predict DNA methylation patterns based on the genomic sequence alone. Because almost nothing is known of the mechanisms that target specific sequences for de novo methylation, a key question that arises is whether there are DNA sequences that are more prone or resistant to methylation.To answer this question, we use data that was generated by enzymatic fractionation of 30 Mb of human brain DNA...

We have previously reported the construction and characterization of vindH1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-␤-Dthiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1-deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (tetR), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, vindA14. In the absence of TET, vindA14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of "empty" crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG-and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants.Vaccinia virus is a complex DNA virus characterized by a high degree of genetic and physical autonomy from the host cell. Among the functions of the 200 gene products encoded by the 192-kb genome are those required for the expression of three temporal classes of genes, the replication of viral DNA, and the morphogenesis of nascent virions (27). The last is a complex process which remains poorly understood but is the subject of intense study. Electron microscopy, in conjunction with pharmacological intervention and the study of conditionally lethal mutants, has been useful in generating models of virion assembly (7,9,12,20,24,25,28,31,41,44,47,49,53,55,58,59,62,63). Within the cytoplasm, morphogenesis is concentrated around areas of increased electron density which have been shown to contain viral DNA and the proteins to be encapsidated. Membrane crescents, which appear to elongate and curve until they enclose the electron-dense material within oval immature virions (IV), are the first hallmark of morphogenesis. The origin of these membranes is still a matter ...