Abstract
Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the α-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid β (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402
Antes TJ, Namciu SJ, Fournier RE, Levy-Wilson B (2001) The 5′ boundary of the human apolipoprotein B chromatin domain in intestinal cells. Biochemistry 40, 6731–6742
Arnold R, Maueler W, Bassili G, Lutz M, Burke L, et al. (2000) The insulator protein CTCF represses transcription on binding to the (gt)(22)(ga)(15) microsatellite in intron 2 of the HLA-DRB1(*)0401 gene. Gene 253, 209–214
Bell AC, Felsenfeld G (1999) Stopped at the border: boundaries and insulators. Curr Opin Genet Dev 9, 191–198
Bell AC, West AG, Felsenfeld G (1999) The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98, 387–396
Bell AC, West AG, Felsenfeld G (2001) Insulators and boundaries: versatile regulatory elements in the eukaryotic genome. Science 291, 447–450
Bode J, Schlake T, Iber M, Schubeler D, Seibler J, et al. (2000) The transgeneticist’s toolbox: novel methods for the targeted modification of eukaryotic genomes. Biol Chem 381, 801–813
Bode J, Goetze S, Ernst E, Huesemann Y, Baer A, et al. (2003) Architecture and utilization of highly-expressed genomic sites. In: Gene Transfer and Expression in Mammalian Cells, Makrides S, ed. (Amsterdam: Elsevier) pp 551–572
Bonifer C, Vidal M, Grosveld F, Sippel AE (1990) Tissue specific and position independent expression of the complete gene domain for chicken lysozyme in transgenic mice. EMBO J 9, 2843–2848
Borrelli E, Heyman R, Hsi M, Evans RM (1988) Targeting of an inducible toxic phenotype in animal cells. Proc Natl Acad Sci U S A 85, 7572–7576
Brand AH, Breeden L, Abraham J, Sternglanz R, Nasmyth K (1985) Characterization of a “silencer” in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer. Cell 41, 41–48
Brown AJ, Goldsworthy SM, Barnes AA, Eilert MM, Tcheang L, et al. (2003) The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J Biol Chem 278, 11312–11319
Chernov IP, Akopov SB, Nikolaev LG, Sverdlov ED (2002) Identification and mapping of nuclear matrix-attachment regions in a one megabase locus of human chromosome 19q13.12: long-range correlation of S/MARs and gene positions. J Cell Biochem 84, 590–600
Chernov IP, Akopov SB, Nikolaev LG (2004) Structure and function of nuclear matrix associated regions (S/MARs). Bioorg Khim 30, 3–14
Chung JH, Whiteley M, Felsenfeld G (1993) A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74, 505–514
de Laat W, Grosveld F (2003) Spatial organization of gene expression: the active chromatin hub. Chromosome Res 11, 447–459
Emery DW, Yannaki E, Tubb J, Nishino T, Li Q, et al. (2002) Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma-globin gene silencing in vivo. Blood 100, 2012–2019
Filippova GN, Fagerlie S, Klenova EM, Myers C, Dehner Y, et al. (1996) An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol Cell Biol 16, 2802–2813
Goetze S, Baer A, Winkelmann S, Nehlsen K, Seibler J, et al. (2005) Performance of genomic bordering elements at predefined genomic loci. Mol Cell Biol 25, 2260–2272
Heng HH, Krawetz SA, Lu W, Bremer S, Liu G, et al. (2001) Re-defining the chromatin loop domain. Cytogenet Cell Genet 93, 155–161
Jurka J (2004) Evolutionary impact of human Alu repetitive elements. Curr Opin Genet Dev 14, 603–608
Kalos M, Fournier RE (1995) Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain. Mol Cell Biol 15, 198–207
Kazazian HH Jr, Goodier JL (2002) LINE drive. retrotransposition and genome instability. Cell 110, 277–280
Kellum R, Schedl P (1991) A position-effect assay for boundaries of higher order chromosomal domains. Cell 64, 941–950
Kellum R, Schedl P (1992) A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol 12, 2424–2431
Kent WJ (2002) BLAT—The BLAST-Like Alignment Tool. Genome Res 12, 656–664
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, et al. (2002) The human genome browser at UCSC. Genome Res 12, 996–1006
Laimins L, Holmgren-Konig M, Khoury G (1986) Transcriptional “silencer” element in rat repetitive sequences associated with the rat insulin 1 gene locus. Proc Natl Acad Sci USA 83, 3151–3155
Magdinier F, Yusufzai TM, Felsenfeld G (2004) Both CTCF-dependent and -independent insulators are found between the mouse T cell receptor alpha and Dad1 genes. J Biol Chem 279, 25381–25389
Mukhopadhyay R, Yu W, Whitehead J, Xu J, Lezcano M, et al. (2004) The binding sites for the chromatin insulator protein CTCF map to DNA methylation-free domains genome-wide. Genome Res 14, 1594–1602
Nagaya S, Yoshida K, Kato K, Akasaka K, Shinmyo A (2001) An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells. Mol Genet Genomics 265, 405–413
Ohlsson R, Renkawitz R, Lobanenkov V (2001) CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet 17, 520–527
Orlando V (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem Sci 25, 99–104
Oshiman K, Motojima K, Mahmood S, Shimada A, Tamura S, et al. (1991) Control region and gastric specific transcription of the rat H+,K(+)-ATPase alpha subunit gene. FEBS Lett 281, 250–254
Recillas-Targa F, Valadez-Graham V, Farrell CM (2004) Prospects and implications of using chromatin insulators in gene therapy and transgenesis. Bioessays 26, 796–807
Renan MJ, Reeves BR (1987) Chromosomal localization of human endogenous retroviral element ERV1 to 18q22-q23 by in situ hybridization. Cytogenet Cell Genet 44, 167–170
Sambrook J, Russell DW (2001) Molecular Cloning. A laboratory manual, 3rd edn (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press)
Tybulewicz VL, Crawford CE, Jackson PK, Bronson RT, Mulligan RC (1991) Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65, 1153–1163
Wasco W, Bupp K, Magendantz M, Gusella JF, Tanzi RE, et al. (1992) Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid beta protein precursor. Proc Natl Acad Sci USA 89, 10758–10762
Wei GH, Liu de P, Liang CC (2005) Chromatin domain boundaries: insulators and beyond. Cell Res 15, 292–300
West AG, Fraser P (2005) Remote control of gene transcription. Hum Mol Genet 14 (Spec No). 1, R101–111
Acknowledgments
The authors are grateful to Eugene Snezhkov for advice, Viktor Potapov and Nadejda Skaptsova for oligonucleotide synthesis, Dmitry Didych for participation in ChIP experiments, and Boris Glotov for critical reading of the manuscript. We are also indebted to Anne Olsen (Lawrence Livermore National Laboratory, USA) for cosmid clones of the FXYD5-COX7A1 locus. The work was supported by INTAS (project 01-0279), the Scientific School program of the Russian Federation President (project NSh 2006.2003.4), and the program of the Russian Academy of Sciences on Molecular and Cellular Biology.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Akopov, S.B., Ruda, V.M., Batrak, V.V. et al. Identification, genome mapping, and CTCF binding of potential insulators within the FXYD5-COX7A1 locus of human Chromosome 19q13.12. Mamm Genome 17, 1042–1049 (2006). https://doi.org/10.1007/s00335-006-0037-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00335-006-0037-3