Eric D. Rubio

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 beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-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.

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 Lys4, Lys79, and Lys36 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. 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 Lys4, Lys79, and Lys36 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 II 1The abbreviations used are: pol II, RNA polymerase II; IL-2, interleukin 2; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: pol II, RNA polymerase II; IL-2, interleukin 2; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 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 (1Orphanides G. LeRoy G. Chang C.H. Luse D.S. Reinberg D. Cell. 1998; 92: 105-116Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 2Gilbert C. Kristjuhan A. Winkler G.S. Svejstrup J.Q. Mol. Cell. 2004; 14: 457-464Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 3Corey L.L. Weirich C.S. Benjamin I.J. Kingston R.E. Genes Dev. 2003; 17: 1392-1401Crossref PubMed Scopus (114) Google Scholar). 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 (4Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (631) Google Scholar). 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 (5Santisteban M.S. Kalashnikova T. Smith M.M. Cell. 2000; 103: 411-422Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 6Santisteban M.S. Arents G. Moudrianakis E.N. Smith M.M. EMBO J. 1997; 16: 2493-2506Crossref PubMed Scopus (75) Google Scholar, 7Ahmad K. Henikoff S. Mol. Cell. 2002; 9: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). 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 (8Winkler G.S. Kristjuhan A. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3517-3522Crossref PubMed Scopus (210) Google Scholar, 9Kristjuhan A. Walker J. Suka N. Grunstein M. Roberts D. Cairns B.R. Svejstrup J.Q. Mol. Cell. 2002; 10: 925-933Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Indeed, previous studies demonstrated that the yeast Elongator complex is associated with nascent transcripts (2Gilbert C. Kristjuhan A. Winkler G.S. Svejstrup J.Q. Mol. Cell. 2004; 14: 457-464Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In addition to histone acetylation, methylation of H3 lysine residues Lys4, Lys36, and Lys79 also has been reported to play a role in transcript elongation (10Shilatifard A. Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 2003; 72: 693-715Crossref PubMed Scopus (197) Google Scholar, 11Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). In yeast, the Set1, Set2, and Dot1p histone methyltransferase complexes are recruited by the RNA polymerase complex to catalyze methylation of Lys4, Lys36, and Lys79 of histone H3 within the coding region. The recent isolation of the Lys4-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 (12Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3044) Google Scholar). 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 hypomethylated forms in order to stimulate or inhibit transcriptional elongation. In combination, these results suggest that genes regulated at the level of elongation are characterized by changes in histone N-terminal tail modification and/or incorporation of histone variants. Here we have tested these predictions at the mammalian c-myc gene. Antibodies—Antibodies detecting the histone variant H2A.Z were a kind gift from the laboratory of David Tremethick (Australian National University, Canberra) or were purchased from Abcam. Both antibodies specifically immunoprecipitate the histone variant H2A.Z, but not the major H2A form (13Fan J.Y. Gordon F. Luger K. Hansen J.C. Tremethick D.J. Nat. Struct. Biol. 2002; 9: 172-176Crossref PubMed Scopus (3) Google Scholar) (Abcam). Methylated Lys79 (H3)-specific antibodies were generously provided by Dan Gottschling and Fred van Leeuwen (Fred Hutchinson Cancer Research Center, Seattle, WA). Antibodies specific for acetylated (Lys9:Lys14) histone H3 and pan-acetylated H4, methylated (Lys36) histone H3, and RNA polymerase II were purchased from Upstate Biotechnology. Antibodies directed against di- and trimethyl Lys4 (H3), acetylated (Lys5) histone H2A, and acetylated H2B (Lys12:Lys15) were obtained from Abcam. Cell Lines—The human promyelocytic cell line HL60 was grown in RPMI, 10% fetal calf serum. Differentiation was induced by addition of dimethyl sulfoxide (Me2SO) for 2 h at a final concentration of 1.5%. The IL-2-dependent CTLL-2 cell line was grown and induced as described (14Moon J.J. Rubio E.D. Martino A. Krumm A. Nelson B.H. J. Biol. Chem. 2004; 279: 5520-5527Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Chromatin Immunoprecipitation—Chromatin immunoprecipitation (ChIP) experiments were performed as described (15Gombert W.M. Farris S.D. Rubio E.D. Morey-Rosler K.M. Schubach W.H. Krumm A. Mol. Cell. Biol. 2003; 23: 9338-9348Crossref PubMed Scopus (71) Google Scholar). The total amount of antibody used in one chromatin immunoprecipitation was 10 μg. PCR Analysis—DNA recovered from immunoprecipitations was analyzed by duplex PCR using gene-specific primers and reference primers (β-globin). The reactions were performed with the Failsafe™ Taq polymerase in a solution containing 1× buffer F (Epicenter) and 10 pmol of each c-myc primer in the presence of [32P]dCTP. Under these conditions, the amplification of fragments was linear. After electrophoresis on a native 6% acrylamide gel, the signals were quantified with the Cyclone Phosphor System. The -fold enrichment in each immunoprecipitation was determined by calculating the ratio of the signal obtained with c-myc primers to that of the reference primer divided by the ratio of signals obtained with the same primers in the input DNA reaction. Primers sequences used to detect immunoprecipitated DNA are available upon request. Nuclear Run-on—Nuclear run-on experiments were performed as described (16Krumm A. Hickey L.B. Groudine M. Genes Dev. 1995; 9: 559-572Crossref PubMed Scopus (190) Google Scholar). RNA Analysis—Nuclease S1 protection assays were performed as described previously (15Gombert W.M. Farris S.D. Rubio E.D. Morey-Rosler K.M. Schubach W.H. Krumm A. Mol. Cell. Biol. 2003; 23: 9338-9348Crossref PubMed Scopus (71) Google Scholar), Northern analysis followed the previously published protocol (14Moon J.J. Rubio E.D. Martino A. Krumm A. Nelson B.H. J. Biol. Chem. 2004; 279: 5520-5527Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Histone Subdomains across Transcribed Regions of Mammalian Genes—The interaction of histone methyltransferase Set1 and Set2 with RNA pol II in yeast requires the transcription elongation complex PAF, suggesting that histone modifications may regulate pol II elongation (11Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 17Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (508) Google Scholar, 18Li B. Howe L. Anderson S. Yates 3rd, J.R. Workman J.L. J. Biol. Chem. 2003; 278: 8897-8903Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 19Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (323) Google Scholar). We set out to define the distribution of the modifications mediated by these complexes across the transcribed region in several mammalian genes. To determine the pattern of histone methylations across the coding region, we used ChIPs. To maximize the resolution of the ChIP experiment, we tested the bcl-2 and bcl-6 oncogenes in which the transcribed portion extends over 35 and 180 kb, respectively (Fig. 1). Both genes are constitutively expressed in the human promyelocytic leukemia cell line HL60 (data not shown). Formaldehyde-cross-linked chromatin prepared from HL60 cells was immunoprecipitated with antibodies specific for methylated Lys4, Lys36, or Lys79 of histone H3. Enrichment of specific gene regions was determined by quantitative duplex PCR in which signals obtained by primer sets were normalized to the signal obtained from the reference primer set. As shown in Fig. 1, the methylation of H3 at residues Lys4, Lys79, and Lys36 of histone H3 is non-uniform over these genes and creates distinct regions characterized by different combinations of histone modifications. For instance, the promoter and the 5′-portion of the transcribed sequences of both the bcl-2 and bcl-6 genes are characterized by Lys4-dimethylated histone H3. However, this modification is undetectable in regions further downstream, consistent with a previous report in which dimethylation of Lys4 was detected in 5′- but not 3′-regions of transcribed genes in chicken erythrocytes (20Schneider R. Bannister A.J. Myers F.A. Thorne A.W. Crane-Robinson C. Kouzarides T. Nat. Cell Biol. 2004; 6: 73-77Crossref PubMed Scopus (607) Google Scholar). By contrast, methylation of lysine 79 is restricted to the transcribed region, and its intensity decreases in the direction 5′ to 3′. The decrease in the level of histone lysine 79 methylation is accompanied by an increase in the level of lysine 36 methylation. Lysine 36 methylation is undetectable in the 5′-region and peaks in the 3′-region (Fig. 1, primer pairs b6-2E to b6-11). These observations demonstrate that transcription elongation complexes encounter distinct chromatin subdomains that differ in the level of specific histone modifications. Histone Modifications and Transcription Elongation—Several studies in and in mammalian cell have shown that RNA polymerase II complexes pause transcription is (16Krumm A. Hickey L.B. Groudine M. Genes Dev. 1995; 9: 559-572Crossref PubMed Scopus (190) Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar, S. J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). The of pol II release from promoter-proximal pause sites to the regulation of c-myc RNA To histone modifications to the release or of pol II within promoter-proximal we have performed ChIP assays using the HL60 cell line in which c-myc RNA are cells are induced to into with is to the of pol II to transcription elongation of the transcription A. T. M. Groudine M. Genes Dev. 6: PubMed Scopus Google Scholar). of by to a decrease in pol II in regions of the pause transcription and of RNA polymerase II complexes within the promoter region (Fig. In to HL60 the of c-myc gene expression in the line CTLL-2 is regulated at the level of transcription is from the cell the of RNA pol II complexes to the c-myc promoter is (Fig. The addition of to CTLL-2 cells a of c-myc expression within The level c-myc expression for To determine the of the to elongation with changes in histone we performed ChIP experiments with antibodies specific for acetylated or methylated lysine residues on histone H3 Lys4, Lys4, Lys79, and and for pan-acetylated Enrichment of histone modifications within specific c-myc gene regions was determined by quantitative duplex with previous results (15Gombert W.M. Farris S.D. Rubio E.D. Morey-Rosler K.M. Schubach W.H. Krumm A. Mol. Cell. Biol. 2003; 23: 9338-9348Crossref PubMed Scopus (71) Google Scholar), a level of histone H3 and acetylation is both within the promoter region and in the 5′-portion of transcribed However, histone acetylation is within H3 and the of the transcription elongation and across the c-myc gene in HL60 cells (Fig. results were obtained in cells that were induced to for h (data not shown). This indicates that the regulation of transcription elongation at the c-myc gene is not directly to histone acetyltransferase activity of all or a of histones to elongation. We analyzed the pattern of histone methylation of the c-myc gene. ChIP assays using antibodies that the and histones distinct subdomains within a gene and and The promoter and the transcribed region of the c-myc gene are in Lys4-dimethylated histone H3. H3, a modification previously to active transcription H. Schneider R. Bannister A.J. J. J. Kouzarides T. 2002; PubMed Scopus Google Scholar), was in both the promoter and transcribed sequences and with the pattern of Lys4-dimethylated H3. In contrast, methylation of Lys79 in histone H3 was detected within transcribed histone H3 was restricted to the of the transcribed further observations at the bcl-2 and bcl-6 genes. To the histone methylation pattern directly with transcription the level of histone methylation in HL60 cells was with that in Lys4 di- and as as Lys79 and Lys36 methylation are by the in pol II (Fig. the tested histone methylation are of transcriptionally active not with gene activity or RNA pol II in mammalian with previous observations F. R.A. K. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar), the methylation of N-terminal for an suggesting that the of transcription requires the of methyltransferase complexes with the elongating RNA polymerases. The elongation of transcription complexes may of the of histone In to HL60 the IL-2-dependent cell line CTLL-2 down-regulates c-myc expression through a decrease in pol II to the c-myc promoter by of transcription elongation of promoter-proximal transcription CTLL-2 cells not of the of in the of cells by increases RNA pol II upon addition of IL-2, as shown by the results of run-on and ChIP experiments (Fig. and not shown). the in pol II associated with these conditions, the of Lys4, Lys36, and Lys79 methylation in the promoter region and in the transcribed regions of the c-myc gene not (Fig. by the of these methylation and the changes are within the of In combination, these suggest that histone modifications by histone or methyltransferases in distinct regions but are not to regulate the to transcription elongation. Transcription and Histone studies have that histone variants are important of transcriptional activity (5Santisteban M.S. Kalashnikova T. Smith M.M. Cell. 2000; 103: 411-422Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 7Ahmad K. Henikoff S. Mol. Cell. 2002; 9: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, J.R. F. R. Tremethick D.J. PubMed Scopus Google Scholar, C. D. K. Dev. 2002; PubMed Scopus Google Scholar, M.M. Mol. Cell. 2002; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). The histone variant H2A.Z in has been detected at both transcribed and analysis in the yeast genes that are either or regulated in the of M. Cell. 2003; Full Text Full Text PDF PubMed Scopus (492) Google Scholar). The interaction of with which the transcription elongation suggests that H2A.Z also may play a role in transcription elongation N.J. M.C. N. C. Ryan O.W. H. R.A. J. Tong A. Canadien V. Richards D.P. Emili A. Buratowski S. Greenblatt J.F. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). studies of nucleosomes demonstrated that the incorporation of H2A.Z results in a local of the which is to with the of histone Tremethick D.J. Luger K. Nat. Struct. Biol. 2000; PubMed Scopus Google Scholar). studies using that this not result in an of the it the interaction of the dimer with the (H3/H4)2 tetramer Tremethick D.J. Luger K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). In and ChIP studies have that yeast and to the of into transcriptionally active M. Cell. 2003; Full Text Full Text PDF PubMed Scopus (492) Google Scholar). Indeed, a role of H2A.Z in the of poised for transcriptional activation (13Fan J.Y. Gordon F. Luger K. Hansen J.C. Tremethick D.J. Nat. Struct. Biol. 2002; 9: 172-176Crossref PubMed Scopus (3) Google Scholar). To the role of H2A.Z in the regulation of the mammalian c-myc we performed ChIP assays with chromatin from both and CTLL-2 cells using an antibody specific for the of H2A.Z (Fig. The results demonstrate that H2A.Z with the c-myc promoter in both and CTLL-2 H2A.Z the promoter region to the of which we have shown to part of the c-myc of the transcription (15Gombert W.M. Farris S.D. Rubio E.D. Morey-Rosler K.M. Schubach W.H. Krumm A. Mol. Cell. Biol. 2003; 23: 9338-9348Crossref PubMed Scopus (71) Google Scholar). consistent with the that H2A.Z the of this histone variant the c-myc gene H2A.Z has been as one of the in cellular complexes H. Y. G. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). This suggests that its through its with H2A.Z in the c-myc gene In addition to the c-myc promoter region, the coding region also with H2A.Z, with the of the of the c-myc gene Fig. transcriptional activation of the c-myc gene H2A.Z within the coding H2A.Z within the promoter region In contrast, the level of acetylated H2B during transcriptional activation the c-myc gene suggesting that the of H2A.Z is not to a of nucleosomes, as previously in a in yeast Shibata Y. B. Strahl B.D. Nat. 2004; PubMed Scopus Google Scholar). In the of H2A.Z with an increase in the form of major histone H2A within the transcribed region. The increase in H2A with a decrease in H2A.Z suggests that the histone H2A the histone variant H2A.Z during of with a role of H2A.Z in the of transcriptional the coding region of the constitutively transcribed gene not with H2A.Z, this histone variant is in the promoter region of (Fig. This that the exchange of c-myc transcription is not the of the of histone H2A cells the of the cell Both the modification of histones and the incorporation of histone variants into nucleosomes to the and of in and a for the histone variant H2A.Z in gene activation (5Santisteban M.S. Kalashnikova T. Smith M.M. Cell. 2000; 103: 411-422Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, J. M. M.A. Genes Dev. PubMed Scopus Google Scholar, M.S. S. J.W. J.L. A.J. J. Biol. 2004; Scholar). However, of chromatin in and of in have an enrichment of H2A.Z in D. L. P. Tremethick D.J. EMBO J. 2003; PubMed Scopus Google Scholar, M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). studies of nucleosomal in a role for the histone variant H2A.Z in the of and the of chromatin domains. H2A.Z in both transcription and the of that H2A.Z is from the transcribed region but not from the promoter region suggests that the histone variant H2A.Z has a its with the promoter region, it active genes and in a transcriptionally poised the same the incorporation of H2A.Z into the coding region transcriptional through the nucleosomal which in decreases both transcription elongation and of These the previously reported results that H2A.Z a interaction with the tetramer at the same is in transcriptionally active regions (5Santisteban M.S. Kalashnikova T. Smith M.M. Cell. 2000; 103: 411-422Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, Tremethick D.J. Luger K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The of H2A.Z on transcription may on its to facilitate the of within the transcribed region the of chromatin by (13Fan J.Y. Gordon F. Luger K. Hansen J.C. Tremethick D.J. Nat. Struct. Biol. 2002; 9: 172-176Crossref PubMed Scopus (3) Google Scholar). However, the of is not with the presence of H2A.Z in the promoter regions of both the c-myc and the genes. of histone H2A.Z N-terminal modifications may provide important this The incorporation of H2A.Z into chromatin of the c-myc coding region may catalytic protein complexes that of the yeast complex M.S. S. J.W. J.L. A.J. J. Biol. 2004; Scholar). the catalytic exchange of nucleosomal H2A for the H2A.Z variant in yeast N.J. M.C. N. C. Ryan O.W. H. R.A. J. Tong A. Canadien V. Richards D.P. Emili A. Buratowski S. Greenblatt J.F. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. W.H. S. C. Science. 2004; PubMed Scopus Google Scholar). However, the of variant forms of histones with the major form of histone H2A is not to specific chromatin remodeling may also result from chromatin remodeling the passage of RNA pol The elongation of RNA pol II through nucleosomes is to the of H2A-H2B dimers V. Bondarenko V. M. Studitsky V.M. Mol. Cell. 2002; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, V.M. M. M. G. Biochem. Sci. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). in and in studies have a role of the complex FACT in the and the of a histone H2A-H2B dimer during transcription (4Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (631) Google Scholar, A. J. E.D. T. S. Reinberg D. Science. 2003; 301: PubMed Scopus Google Scholar). the that transcriptional activity the exchange of histone variants in nucleosomes may the in of FACT histone exchange activity in We are to David Dan and Fred van Leeuwen for antibodies and to and Henikoff for of this

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.