The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

PLoS ONE

Volumn 8, Issue 10, 2013, Pages

  Tran, Stella ^a,b   Wang, Ying ^a   Lamba, Pankaj ^a,c   Zhou, Xiang ^a   Boehm, Ulrich ^d   Bernard, Daniel J ^a  

^a MCGILL UNIVERSITY   (Canada)

^b UNIVERSITY OF CALIFORNIA   (United States)

^c ST MARY MERCY HOSPITAL   (United States)

^d SAARLAND UNIVERSITY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; LUCIFERASE;

5' UNTRANSLATED REGION; ANIMAL CELL; ANIMAL TISSUE; ARTICLE; CELL CULTURE; CELL IMMORTALIZATION; COMPUTER MODEL; CPG ISLAND; DNA FLANKING REGION; DNA METHYLATION; ENZYME ACTIVITY; FEMALE; FOXL2 GENE; GENE; GENE AMPLIFICATION; GENE EXPRESSION; GENE INTERACTION; GENE MAPPING; GENE SILENCING; GENOMICS; GONADOTROPIN SECRETING CELL; HETEROLOGOUS EXPRESSION; IN VITRO STUDY; MALE; MOUSE; MURINE MODEL; NONHUMAN; NUCLEOTIDE SEQUENCE; PYROSEQUENCING; QUANTITATIVE ANALYSIS; RESTRICTION FRAGMENT; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; TRANSCRIPTION REGULATION;

5' UNTRANSLATED REGIONS; ANIMALS; BASE SEQUENCE; CELL LINE, TRANSFORMED; CPG ISLANDS; DNA METHYLATION; EPIGENESIS, GENETIC; FEMALE; FORKHEAD TRANSCRIPTION FACTORS; GONADOTROPHS; HUMANS; MALE; MICE; MICE, INBRED C57BL; MOLECULAR SEQUENCE DATA; NIH 3T3 CELLS; ORGAN SPECIFICITY; PROMOTER REGIONS, GENETIC; THYROTROPHS;

EID: 84884833872     PISSN: None     EISSN: 19326203     Source Type: Journal    
DOI: 10.1371/journal.pone.0076642     Document Type: Article

References (83)

1. Rosenfeld MG, Briata P, Dasen J, Gleiberman AS, Kioussi C, et al. (2000) Multistep signaling and transcriptional requirements for pituitary organogenesis in vivo. Recent Prog Horm Res 55: 1-14; discussion: 11036930.
2. Treier M, Gleiberman AS, O'Connell SM, Szeto DP, McMahon JA, et al. (1998) Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 12: 1691-1704. doi:10.1101/gad.12.11.1691. PubMed: 9620855.
3. Crisponi L, Deiana M, Loi A, (2001) The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat Genet 27: 159-166. doi:10.1038/84781. PubMed: 11175783.
4. Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R, et al. (2009) Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell 139: 1130-1142. doi:10.1016/j.cell.2009.11.021. PubMed: 20005806.
5. Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, et al. (2004) The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance. Development 131: 933-942. doi:10.1242/dev.00969. PubMed: 14736745.
6. Uda M, Ottolenghi C, Crisponi L, (2004) Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development. Hum Mol Genet 13: 1171-1181. doi:10.1093/hmg/ddh124. PubMed: 15056605.
7. Fraser IS, Shearman RP, Smith A, Russell P, (1988) An association among blepharophimosis, resistant ovary syndrome, and true premature menopause. Fertil Steril 50: 747-751. PubMed: 3141218.
8. De Baere E, Dixon MJ, Small KW, Jabs EW, Leroy BP, et al. (2001) Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype-phenotype correlation. Hum Mol Genet. doi:10.1591-1600.
9. Udar N, Yellore V, Chalukya M, Yelchits S, Silva-Garcia R, et al. (2003) Comparative analysis of the FOXL2 gene and characterization of mutations in BPES patients. Hum Mutat 22: 222-228. doi:10.1002/humu.10251. PubMed: 12938087.
10. Strømme P, Sandboe F, (1996) Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). Acta Ophthalmol Scand 74: 45-47. PubMed: 8689480.
11. De Baere E, Beysen D, Oley C, Lorenz B, Cocquet J, et al. (2003) FOXL2 and BPES: Mutational Hotspots, Phenotypic Variability, and Revision of the Genotype-Phenotype Correlation. Am J Hum Genet 72: 478-487. doi:10.1086/346118. PubMed: 12529855.
12. Beysen D, De Jaegere S, Amor D, Bouchard P, Christin-Maitre S, et al. (2008) Identification of 34 novel and 56 known FOXL2 mutations in patients with Blepharophimosis syndrome. Hum Mutat 29: E205-E219. doi:10.1002/humu.20819. PubMed: 18642388.
13. Crisponi L, Uda M, Deiana M, Loi A, Nagaraja R, et al. (2004) FOXL2 inactivation by a translocation 171 kb away: analysis of 500 kb of chromosome 3 for candidate long-range regulatory sequences. Genomics 83: 757-764. doi:10.1016/j.ygeno.2003.11.010. PubMed: 15081106.
14. Beysen D, De Paepe A, De Baere E, (2009) FOXL2 mutations and genomic rearrangements in BPES. Hum Mutat 30: 158-169. doi:10.1002/humu.20807. PubMed: 18726931.
15. Beysen D, Raes J, Leroy BP, Lucassen A, Yates JR, et al. (2005) Deletions involving long-range conserved nongenic sequences upstream and downstream of FOXL2 as a novel disease-causing mechanism in blepharophimosis syndrome. Am J Hum Genet 77: 205-218. doi:10.1086/432083. PubMed: 15962237.
16. De Baere E, Fukushima Y, Small K, Udar N, Van Camp G, et al. (2000) Identification of BPESC1, a novel gene disrupted by a balanced chromosomal translocation, t(3;4)(q23;p15.2), in a patient with BPES. Genomics 68: 296-304.
17. Praphanphoj V, Goodman BK, Thomas GH, Niel KM, Toomes C, et al. (2000) Molecular cytogenetic evaluation in a patient with a translocation (3;21) associated with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES). Genomics 65: 67-69. doi:10.1006/geno.2000.6157. PubMed: 10777667.
18. Alao MJ, Lalèyè A, Lalya F, Hans Ch, Abramovicz M, et al. (2012) Blepharophimosis, ptosis, epicanthus inversus syndrome with translocation and deletion at chromosome 3q23 in a black African female. Eur J Med Genet 55: 630-634. doi:10.1016/j.ejmg.2012.07.005. PubMed: 22906557.
19. Schlade-Bartusiak K, Brown L, Lomax B, Bruyere H, Gillan T, et al. (2012) BPES with atypical premature ovarian insufficiency, and evidence of mitotic recombination, in a woman with trisomy X and a translocation t(3;11)(q22.3 q14.1). Am J Med Genet A 158A: 2322-2327.
20. González-González C, García-Hoyos M, Hernaez Calzón R, Arroyo Díaz C, González Fanego C, et al. (2012) Microdeletion found by array-CGH in girl with blepharophimosis syndrome and apparently balanced translocation t(3;15)(q23;q25). Ophthalmic Genet 33: 107-110. doi:10.3109/13816810.2011.634879. PubMed: 22171663.
21. D'Haene B, Attanasio C, Beysen D, Dostie J, Lemire E, et al. (2009) Disease-causing 7.4 kb cis-regulatory deletion disrupting conserved non-coding sequences and their interaction with the FOXL2 promotor: implications for mutation screening. PLOS Genet 5: e1000522.
22. Tran S, Zhou X, Lafleur C, Calderon MJ, Ellsworth BS, et al. (2013) Impaired fertility and FSH synthesis in gonadotrope-specific Foxl2 knockout mice. Mol Endocrinol 27: 407-421. doi:10.1210/me.2012-1286. PubMed: 23340250.
23. Justice NJ, Blount AL, Pelosi E, Schlessinger D, Vale W, et al. (2011) Impaired FSHβ Expression in the Pituitaries of Foxl2 Mutant Animals. Mol Endocrinol 25: 1404-1415. doi:10.1210/me.2011-0093. PubMed: 21700720.
24. Ellsworth BS, Egashira N, Haller JL, (2006) FOXL2 in the pituitary: molecular, genetic, and developmental analysis. Mol Endocrinol 20: 2796-2805. doi:10.1210/me.2005-0303. PubMed: 16840539.
25. Asdell SA, (1944) The Genetic Sex of Intersexual Goats and a Probable Linkage with the Gene for Hornlessness. Science 99: 124. doi:10.1126/science.99.2563.124-a. PubMed: 17814292.
26. Vaiman D, Koutita O, Oustry A, Elsen JM, Manfredi E, et al. (1996) Genetic mapping of the autosomal region involved in XX sex-reversal and horn development in goats. Mamm Genome 7: 133-137. doi:10.1007/s003359900033. PubMed: 8835530.
27. Pailhoux E, Vigier B, Chaffaux S, Servel N, Taourit S, et al. (2001) A 11.7-kb deletion triggers intersexuality and polledness in goats. Nat Genet 29: 453-458. doi:10.1038/ng769. PubMed: 11726932.
28. Pannetier M, Renault L, Jolivet G, Cotinot C, Pailhoux E, (2005) Ovarian-specific expression of a new gene regulated by the goat PIS region and transcribed by a FOXL2 bidirectional promoter. Genomics 85: 715-726. doi:10.1016/j.ygeno.2005.02.011. PubMed: 15885498.
29. Moumné L, Dipietromaria A, Batista F, Kocer A, Fellous M, et al. (2008) Differential aggregation and functional impairment induced by polyalanine expansions in FOXL2, a transcription factor involved in cranio-facial and ovarian development. Hum Mol Genet 17: 1010-1019. PubMed: 18158309.
30. Benayoun BA, Caburet S, Dipietromaria A, Bailly-Bechet M, Batista F, et al. (2008) The identification and characterization of a FOXL2 response element provides insights into the pathogenesis of mutant alleles. Hum Mol Genet 17: 3118-3127. doi:10.1093/hmg/ddn209. PubMed: 18635577.
31. Benayoun BA, Batista F, Auer J, Dipietromaria A, L'Hôte D, et al. (2009) Positive and negative feedback regulates the transcription factor FOXL2 in response to cell stress: evidence for a regulatory imbalance induced by disease-causing mutations. Hum Mol Genet 18: 632-644. PubMed: 19010791.
32. Alarid ET, Windle JJ, Whyte DB, Mellon PL, (1996) Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 122: 3319-3329. PubMed: 8898243.
33. Yusta B, Alarid ET, Gordon DF, Ridgway EC, Mellon PL, (1998) The thyrotropin beta-subunit gene is repressed by thyroid hormone in a novel thyrotrope cell line, mouse T alphaT1 cells. Endocrinology 139: 4476-4482. doi:10.1210/en.139.11.4476. PubMed: 9794455.
34. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, et al. (1990) Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5: 1-10. doi:10.1016/0896-6273(90)90028-E. PubMed: 2196069.
35. Buonassisi V, Sato G, Cohen AI, (1962) Hormone-producing cultures of adrenal and pituitary tumor origin. Proc Natl Acad Sci U S A 48: 1184-1190. doi:10.1073/pnas.48.7.1184. PubMed: 13874682.
36. Inoue K, Matsumoto H, Koyama C, Shibata K, Nakazato Y, et al. (1992) Establishment of a folliculo-stellate-like cell line from a murine thyrotropic pituitary tumor. Endocrinology 131: 3110-3116. doi:10.1210/en.131.6.3110. PubMed: 1446645.
37. Yaffe D, Saxel O, (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270: 725-727. doi:10.1038/270725a0. PubMed: 563524.
38. Bernard DJ, (2004) Both SMAD2 and SMAD3 mediate activin-stimulated expression of the follicle-stimulating hormone beta subunit in mouse gonadotrope cells. Mol Endocrinol 18: 606-623. PubMed: 14701940.
39. Klug M, Rehli M, (2006) Functional Analysis of Promoter CPG-Methylation using a CpG-Free Luciferase Reporter Vector. Epigenetics 1: 127-130. doi:10.4161/epi.1.3.3327. PubMed: 17965610.
40. Tran S, Lamba P, Wang Y, Bernard DJ, (2011) SMADs and FOXL2 Synergistically Regulate Murine FSHβ Transcription Via a Conserved Proximal Promoter Element. Mol Endocrinol 25: 1170-1183. doi:10.1210/me.2010-0480. PubMed: 21622537.
41. Oakes CC, La Salle S, Trasler JM, Robaire B, (2009) Restriction digestion and real-time PCR (qAMP). Methods Mol Biol 507: 271-280. doi:10.1007/978-1-59745-522-0_20. PubMed: 18987821.
42. Wen S, Schwarz JR, Niculescu D, Dinu C, Bauer CK, et al. (2008) Functional characterization of genetically labeled gonadotropes. Endocrinology 149: 2701-2711. doi:10.1210/en.2007-1502. PubMed: 18325995.
43. Fortin J, Kumar V, Zhou X, Wang Y, Auwerx J, et al. (2013) NR5A2 regulates Lhb and Fshb transcription in gonadotrope-like cells in vitro, but is dispensable for gonadotropin synthesis and fertility in vivo. PLOS ONE 8: e59058. doi:10.1371/journal.pone.0059058. PubMed: 23536856.
44. Lee KB, Khivansara V, Santos MM, Lamba P, Yuen T, et al. (2007) Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone beta subunit transcription. J Mol Endocrinol 38: 315-330. doi:10.1677/jme.1.02196. PubMed: 17293449.
45. Corpuz PS, Lindaman LL, Mellon PL, Coss D, (2010) FoxL2 Is required for activin induction of the mouse and human follicle-stimulating hormone beta-subunit genes. Mol Endocrinol 24: 1037-1051. doi:10.1210/me.2009-0425. PubMed: 20233786.
46. Coss D, Mellon PL, Thackray VG, (2010) A FoxL in the Smad house: activin regulation of FSH. Trends Endocrinol Metab 21: 562-568. doi:10.1016/j.tem.2010.05.006. PubMed: 20598900.
47. Bernard DJ, Tran S, (2013) Mechanisms of activin-stimulated FSH synthesis: the story of a pig and a FOX. Biol Reprod 88: 78. doi:10.1095/biolreprod.113.107797. PubMed: 23426431.
48. Ghochani Y, Saini JK, Mellon PL, Thackray VG, (2012) FOXL2 is involved in the synergy between activin and progestins on the follicle-stimulating hormone beta-subunit promoter. Endocrinology 153: 2023-2033. doi:10.1210/en.2011-1763. PubMed: 22294749.
49. Lamba P, Fortin J, Tran S, Wang Y, Bernard DJ, (2009) A novel role for the forkhead transcription factor FOXL2 in activin A-regulated follicle-stimulating hormone beta subunit transcription. Mol Endocrinol 23: 1001-1013. doi:10.1210/me.2008-0324. PubMed: 19324968.
50. Lamba P, Wang Y, Tran S, Ouspenskaia T, Libasci V, et al. (2010) Activin A regulates porcine follicle-stimulating hormone beta-subunit transcription via cooperative actions of SMADs and FOXL2. Endocrinology 151: 5456-5467. doi:10.1210/en.2010-0605. PubMed: 20810560.
51. Gardiner-Garden M, Frommer M, (1987) CpG islands in vertebrate genomes. J Mol Biol 196: 261-282. doi:10.1016/0022-2836(87)90689-9. PubMed: 3656447.
52. Samlowski WE, Leachman SA, Wade M, Cassidy P, Porter-Gill P, et al. (2005) Evaluation of a 7-day continuous intravenous infusion of decitabine: inhibition of promoter-specific and global genomic DNA methylation. J Clin Oncol 23: 3897-3905. doi:10.1200/JCO.2005.06.118. PubMed: 15753459.
53. Suzuki-Ishigaki S, Numayama-Tsuruta K, Kuramasu A, Sakurai E, Makabe Y, et al. (2000) The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region. Nucleic Acids Res 28: 2627-2633. doi:10.1093/nar/28.14.2627. PubMed: 10908316.
54. Ou JN, Torrisani J, Unterberger A, Provençal N, Shikimi K, et al. (2007) Histone deacetylase inhibitor Trichostatin A induces global and gene-specific DNA demethylation in human cancer cell lines. Biochem Pharmacol 73: 1297-1307. doi:10.1016/j.bcp.2006.12.032. PubMed: 17276411.
55. Han T, Shang D, Xu X, Tian Y, (2012) Gene expression profiling of the synergy of 5-aza-2'-deoxycytidine and paclitaxel against renal cell carcinoma. World J Surg Oncol 10: 183. doi:10.1186/1477-7819-10-183. PubMed: 22950635.
56. Razin A, (1998) CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J 17: 4905-4908. doi:10.1093/emboj/17.17.4905. PubMed: 9724627.
57. Cameron EE, Bachman KE, Myöhänen S, Herman JG, Baylin SB, (1999) Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 21: 103-107. doi:10.1038/5047. PubMed: 9916800.
58. Kuck D, Caulfield T, Lyko F, Medina-Franco JL, (2010) Nanaomycin A Selectively Inhibits DNMT3B and Reactivates Silenced Tumor Suppressor Genes in Human Cancer Cells. Mol Cancer Ther 9: 3015-3023. doi:10.1158/1535-7163.MCT-10-0609. PubMed: 20833755.
59. Dodge JE, Okano M, Dick F, Tsujimoto N, Chen T, et al. (2005) Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem 280: 17986-17991. PubMed: 15757890.
60. Oka M, Rodić N, Graddy J, Chang LJ, Terada N, (2006) CpG sites preferentially methylated by Dnmt3a in vivo. J Biol Chem 281: 9901-9908. doi:10.1074/jbc.M511100200. PubMed: 16439359.
61. Kim GD, Ni J, Kelesoglu N, Roberts RJ, Pradhan S, (2002) Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases. EMBO J 21: 4183-4195. doi:10.1093/emboj/cdf401. PubMed: 12145218.
62. Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, et al. (2002) DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 416: 552-556. doi:10.1038/416552a. PubMed: 11932749.
63. Chen T, Ueda Y, Dodge JE, Wang Z, Li E, (2003) Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol 23: 5594-5605. doi:10.1128/MCB.23.16.5594-5605.2003. PubMed: 12897133.
64. Bird AP, Wolffe AP, (1999) Methylation-induced repression--belts, braces, and chromatin. Cell 99: 451-454. doi:10.1016/S0092-8674(00)81532-9. PubMed: 10589672.
65. El-Osta A, Wolffe AP, (2000) DNA methylation and histone deacetylation in the control of gene expression: basic biochemistry to human development and disease. Gene Expr 9: 63-75. PubMed: 11097425.
66. Jones PA, Takai D, (2001) The Role of DNA Methylation in Mammalian Epigenetics. Science 293: 1068-1070. doi:10.1126/science.1063852. PubMed: 11498573.
67. Hoivik EA, Aumo L, Aesoy R, Lillefosse H, Lewis AE, et al. (2008) Deoxyribonucleic acid methylation controls cell type-specific expression of steroidogenic factor 1. Endocrinology 149: 5599-5609. doi:10.1210/en.2008-0104. PubMed: 18653709.
68. Hoivik EA, Bjanesoy TE, Mai O, Okamoto S, Minokoshi Y, et al. (2011) DNA Methylation of Intronic Enhancers Directs Tissue-Specific Expression of Steroidogenic Factor 1/Adrenal 4 Binding Protein (SF-1/Ad4BP). Endocrinology 152: 2100-2112. doi:10.1210/en.2010-1305. PubMed: 21343250.
69. Watt F, Molloy PL, (1988) Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. Genes Dev 2: 1136-1143. doi:10.1101/gad.2.9.1136. PubMed: 3192075.
70. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, et al. (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 19: 187-191. doi:10.1038/561. PubMed: 9620779.
71. Ng HH, Zhang Y, Hendrich B, Johnson CA, Turner BM, et al. (1999) MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet 23: 58-61. doi:10.1038/12659. PubMed: 10471499.
72. Sarraf SA, Stancheva I, (2004) Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol Cell 15: 595-605. doi:10.1016/j.molcel.2004.06.043. PubMed: 15327775.
73. Zhang Y, Ng HH, Erdjument-Bromage H, Tempst P, Bird A, et al. (1999) Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev 13: 1924-1935. doi:10.1101/gad.13.15.1924. PubMed: 10444591.
74. Wade PA, Gegonne A, Jones PL, Ballestar E, Aubry F, et al. (1999) Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat Genet 23: 62-66. doi:10.1038/12664. PubMed: 10471500.
75. Wade PA, (2001) Methyl CpG-binding proteins and transcriptional repression*. BioEssays 23: 1131-1137. doi:10.1002/bies.10008. PubMed: 11746232.
76. Flatau E, Bogenmann E, Jones PA, (1983) Variable 5-methylcytosine levels in human tumor cell lines and fresh pediatric tumor explants. Cancer Res 43: 4901-4905. PubMed: 6883340.
77. Smiraglia DJ, Rush LJ, Frühwald MC, Dai Z, Held WA, et al. (2001) Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies. Hum Mol Genet 10: 1413-1419. doi:10.1093/hmg/10.13.1413. PubMed: 11440994.
78. Kawai J, Hirose K, Fushiki S, Hirotsune S, Ozawa N, et al. (1994) Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning. Mol Cell Biol 14: 7421-7427. PubMed: 7935456.
79. Antequera F, Boyes J, Bird A, (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 62: 503-514. doi:10.1016/0092-8674(90)90015-7. PubMed: 1974172.
80. Denamur E, Chehab FF, (1995) Methylation status of CpG sites in the mouse and human CFTR promoters. DNA Cell Biol 14: 811-815. doi:10.1089/dna.1995.14.811. PubMed: 7545404.
81. Pennacchio LA, Ahituv N, Moses AM, Prabhakar S, Nobrega MA, et al. (2006) In vivo enhancer analysis of human conserved non-coding sequences. Nature 444: 499-502. doi:10.1038/nature05295. PubMed: 17086198.
82. Kothary R, Clapoff S, Darling S, Perry MD, Moran LA, et al. (1989) Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. Development 105: 707-714. PubMed: 2557196.
83. Nobrega MA, Ovcharenko I, Afzal V, Rubin EM, (2003) Scanning Human Gene Deserts for Long-Range Enhancers. Science 302: 413. doi:10.1126/science.1088328. PubMed: 14563999.