Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human ␤-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the ␤-globin locus. We also identified a new looping interaction in K562 cells between the ␤-globin Locus Control Region and the ␥-␦-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis-and trans-interaction networks of genomic elements and for the study of higher-order chromosome structure.

Cohesin is required to prevent premature dissociation of sister chromatids after DNA replication. Although its role in chromatid cohesion is well established, the functional significance of cohesin's association with interphase chromatin is not clear. Using a quantitative proteomics approach, we show that the STAG1 (Scc3/ SA1) subunit of cohesin interacts with the CCTC-binding factor CTCF bound to the c-myc insulator element. Both allele-specific binding of CTCF and Scc3/SA1 at the imprinted IGF2/H19 gene locus and our analyses of human DM1 alleles containing base substitutions at CTCF-binding motifs indicate that cohesin recruitment to chromosomal sites depends on the presence of CTCF. A large-scale genomic survey using ChIP-Chip demonstrates that Scc3/SA1 binding strongly correlates with the CTCF-binding site distribution in chromosomal arms. However, some chromosomal sites interact exclusively with CTCF, whereas others interact with Scc3/SA1 only. Furthermore, immunofluorescence microscopy and ChIP-Chip experiments demonstrate that CTCF associates with both centromeres and chromosomal arms during metaphase. These results link cohesin to gene regulatory functions and suggest an essential role for CTCF during sister chromatid cohesion. These results have implications for the functional role of cohesin subunits in the pathogenesis of Cornelia de Lange syndrome and Roberts syndromes.cohesion ͉ transcription ͉ insulator ͉ centromere ͉ metaphase

Insulator elements and matrix attachment regions are essential for the organization of genetic information within the nucleus. By comparing the pattern of histone modifications at the mouse and human c-myc alleles, we identified an evolutionarily conserved boundary at which the c-myc transcription unit is separated from the flanking condensed chromatin enriched in lysine 9-methylated histone H3. This region harbors the c-myc insulator element (MINE), which contains at least two physically separable, functional activities: enhancer-blocking activity and barrier activity. The enhancer-blocking activity is mediated by CTCF. Chromatin immunoprecipitation assays demonstrate that CTCF is constitutively bound at the insulator and at the promoter region independent of the transcriptional status of c-myc. This result supports an architectural role of CTCF rather than a regulatory role in transcription. An additional higher-order nuclear organization of the c-myc locus is provided by matrix attachment regions (MARs) that define a domain larger than 160 kb. The MARs of the c-myc domain do not act to prevent the association of flanking regions with lysine 9-methylated histones, suggesting that they do not function as barrier elements.

Fibroblast growth factor (FGF)-10 plays an important role in regulating growth, differentiation, and repair of the urothelium. This process occurs through a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the epithelium (urothelium). In situ hybridization analysis demonstrated that (i) fibroblasts of the human lamina propria were the cell type that synthesized FGF-10 RNA and (ii) the FGF-10 gene is located at the 5p12-p13 locus of chromosome 5. Recombinant (r) preparations of human FGF-10 were found to induce proliferation of human urothelial cells in vitro and of transitional epithelium of wild-type and FGF7-null mice in vivo. Mechanistic studies with human cells indicated two modes of FGF-10 action: (i) translocation of rFGF-10 into urothelial cell nuclei and (ii) a signaling cascade that begins with the heparin-dependent phosphorylation of tyrosine residues of surface transmembrane receptors. The normal urothelial phenotype, that of quiescence, is proposed to be typified by negligible levels of FGF-10. During proliferative phases, levels of FGF-10 rise at the urothelial cell surface and/or within urothelial cell nuclei. An understanding of how FGF-10 works in conjunction with these other processes will lead to better management of many diseases of the bladder and urinary tract.

The post-translational modification of histones and the incorporation of core histone variants play key roles in governing gene expression. Many eukaryotic genes regulate their expression by limiting the escape of RNA polymerase from promoter-proximal pause sites. Here we report that elongating RNA polymerase II complexes encounter distinct chromatin landscapes that are marked by methylation of lysine residues Lys 4 , Lys 79 , and Lys 36 of histone H3. However, neither histone methylation nor acetylation directly regulates the release of elongation complexes stalled at promoter-proximal pause sites of the c-myc gene. In contrast, transcriptional activation is associated with local displacement of the histone variant H2A.Z within the transcribed region and incorporation of the major histone variant H2A. This result indicates that transcribing RNA polymerase II remodels chromatin in part through coincident displacement of H2A.Z-H2B dimers and incorporation of H2A-H2B dimers. In combination, these results suggest a new model in which the incorporation of H2A.Z into nucleosomes down-regulates transcription; at the same time it may act as a cellular memory for transcriptionally poised gene domains. RNA pol II1 encounters nucleosomes and other multiprotein complexes during transcription. Conceptually, any mechanism that destabilizes the DNA-histone interaction or removes part or all of the histone octamer potentially increases the elongation efficiency of RNA polymerases. Protein complexes such as FACT, Elongator, and Swi/Snf have been proposed to alleviate barriers that are imposed by chromatin (1-3). For instance, the protein complex FACT assists RNA polymerase during elongation by altering the nucleosomal conformation and destabilizing the interaction between the H2A/H2B dimer and the (H3/H4) 2 tetramer (4). After the transcription complex has passed the nucleosome, FACT participates in the reassembly of the nucleosome. In addition to the FACT-mediated catalytic remodeling of nucleosomes, the post-translational modification of histones, and/or the incorporation of histone variants also may facilitate the passage of the transcription complex through nucleosomes (5-7). For example, the identification of the acetyltransferase activity in the Elp3 subunit of the RNA pol II-associated Elongator complex suggests that histone acetylation is an important component in the regulation of transcription elongation (8,9). Indeed, previous studies demonstrated that the yeast Elongator complex is associated with nascent transcripts (2). In addition to histone acetylation, methylation of H3 lysine residues Lys -specific histone demethylase LSD1 suggests a model in which RNA polymerase II elongation is regulated by the antagonistic action of histone methyltransferases and histone demethylases (12). Alternatively, a replication-independent histone exchange pathway in which variant forms of histones are incorporated during active transcription may provide an opportunity to continuously exchange histones with either hyper-or hypomethyl...