Genome-wide analysis of Pax8 binding provides new insights into thyroid functions

BMC Genomics

Volumn 13, Issue 1, 2012, Pages

  Ruiz Llorente, Sergio ^a,b   de Pau, Enrique Carrillo S ^a,c,d   Sastre Perona, Ana ^a   Montero Conde, Cristina ^a,b   Gómez López, Gonzalo ^c   Fagin, James A ^b   Valencia, Alfonso ^c   Pisano, David G ^c   Santisteban, Pilar ^a  

^a UNIVERSIDAD AUTÓNOMA DE MADRID   (Spain)

^b MEMORIAL SLOAN KETTERING CANCER CENTER   (United States)

^c Centro Nacional de Investigaciones Oncológicas   (Spain)

^d RADBOUD UNIVERSITY MEDICAL CENTER   (Netherlands)

Author keywords

Chip seq; CpG island; CTCF; Expression arrays; Pax8; SP1

Indexed keywords

TRANSCRIPTION FACTOR PAX8; TRANSCRIPTION FACTOR SP1; CCCTC-BINDING FACTOR; COTRANSPORTER; PAIRED BOX TRANSCRIPTION FACTOR; PAX8 PROTEIN, RAT; REPRESSOR PROTEIN; SMALL INTERFERING RNA; SODIUM IODIDE SYMPORTER; TRANSCRIPTION FACTOR CTCF;

ANIMAL CELL; APOPTOSIS; ARTICLE; BINDING SITE; CELL ADHESION; CELL DIFFERENTIATION; CELL FUNCTION; CELL MOTION; CELL POLARITY; CELL PROLIFERATION; CHROMATIN ASSEMBLY AND DISASSEMBLY; CHROMATIN IMMUNOPRECIPITATION; CONTROLLED STUDY; CPG ISLAND; DNA STRUCTURE; GENE CONTROL; GENE EXPRESSION REGULATION; GENE TARGETING; GENOME ANALYSIS; IN VIVO STUDY; NONHUMAN; NUCLEOTIDE SEQUENCE; PROMOTER REGION; PROTEIN BINDING; PROTEIN DNA INTERACTION; PROTEIN FUNCTION; RAT; THYROID CELL; THYROID FUNCTION; TRANSCRIPTION REGULATION; ANIMAL; CELL LINE; CYTOLOGY; DRUG ANTAGONISM; GENE SILENCING; GENETIC ASSOCIATION; GENETIC TRANSFECTION; GENETICS; GENOME; HELA CELL; HUMAN; METABOLISM; MICROARRAY ANALYSIS; RNA INTERFERENCE; THYROID GLAND;

ANIMALS; BINDING SITES; CELL DIFFERENTIATION; CELL LINE; CHROMATIN IMMUNOPRECIPITATION; CPG ISLANDS; GENE EXPRESSION REGULATION; GENE SILENCING; GENOME; GENOME-WIDE ASSOCIATION STUDY; HELA CELLS; HUMANS; MICROARRAY ANALYSIS; PAIRED BOX TRANSCRIPTION FACTORS; PROMOTER REGIONS, GENETIC; RATS; REPRESSOR PROTEINS; RNA INTERFERENCE; RNA, SMALL INTERFERING; SP1 TRANSCRIPTION FACTOR; SYMPORTERS; THYROID GLAND; TRANSFECTION;

EID: 84862216535     PISSN: None     EISSN: 14712164     Source Type: Journal    
DOI: 10.1186/1471-2164-13-147     Document Type: Article

References (77)

1. Farnham PJ. Insights from genomic profiling of transcription factors. Nat Rev Genet 2009, 10(9):605-616. 10.1038/nrg2636, 2846386, 19668247.
2. Santisteban P, Bernal J. Thyroid development and effect on the nervous system. Rev Endocr Metab Disord 2005, 6(3):217-228. 10.1007/s11154-005-3053-9, 16151626.
3. De Felice M, Di Lauro R. Thyroid development and its disorders: genetics and molecular mechanisms. Endocr Rev 2004, 25(5):722-746. 10.1210/er.2003-0028, 15466939.
4. Plachov D, Chowdhury K, Walther C, Simon D, Guenet JL, Gruss P. Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland. Development 1990, 110(2):643-651.
5. Mansouri A, Chowdhury K, Gruss P. Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet 1998, 19(1):87-90. 10.1038/ng0598-87, 9590297.
6. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, Souabni A, Baserga M, Tassi V, Pinchera A, et al. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet 1998, 19(1):83-86. 10.1038/ng0598-83, 9590296.
7. Ohno M, Zannini M, Levy O, Carrasco N, di Lauro R. The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription. Mol Cell Biol 1999, 19(3):2051-2060. 83998, 10022892.
8. Miccadei S, De Leo R, Zammarchi E, Natali PG, Civitareale D. The synergistic activity of thyroid transcription factor 1 and Pax 8 relies on the promoter/enhancer interplay. Mol Endocrinol 2002, 16(4):837-846. 10.1210/me.16.4.837, 11923479.
9. Zannini M, Francis-Lang H, Plachov D, Di Lauro R. Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters. Mol Cell Biol 1992, 12(9):4230-4241. 360331, 1508216.
10. Fraizer GC, Shimamura R, Zhang X, Saunders GF. PAX 8 regulates human WT1 transcription through a novel DNA binding site. J Biol Chem 1997, 272(49):30678-30687. 10.1074/jbc.272.49.30678, 9388203.
11. Deaton AM, Bird A. CpG islands and the regulation of transcription. Genes Dev 2011, 25(10):1010-1022. 10.1101/gad.2037511, 3093116, 21576262.
12. Esposito C, Miccadei S, Saiardi A, Civitareale D. PAX 8 activates the enhancer of the human thyroperoxidase gene. Biochem J 1998, 331(Pt 1):37-40. 1219318, 9512459.
13. Pellizzari L, Fabbro D, Lonigro R, Di Lauro R, Damante G. A network of specific minor-groove contacts is a common characteristic of paired-domain-DNA interactions. Biochem J 1996, 315(Pt 2):363-367. 1217204, 8615801.
14. Czerny T, Schaffner G, Busslinger M. DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev 1993, 7(10):2048-2061. 10.1101/gad.7.10.2048, 8406007.
15. Ho Sui SJ, Mortimer JR, Arenillas DJ, Brumm J, Walsh CJ, Kennedy BP, Wasserman WW. oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes. Nucleic Acids Res 2005, 33(10):3154-3164. 10.1093/nar/gki624, 1142402, 15933209.
16. Planque N, Leconte L, Coquelle FM, Benkhelifa S, Martin P, Felder-Schmittbuhl MP, Saule S. Interaction of Maf transcription factors with Pax-6 results in synergistic activation of the glucagon promoter. J Biol Chem 2001, 276(38):35751-35760. 10.1074/jbc.M104523200, 11457839.
17. Garcia B, Santisteban P. PI3K is involved in the IGF-I inhibition of TSH-induced sodium/iodide symporter gene expression. Mol Endocrinol 2002, 16(2):342-352. 10.1210/me.16.2.342, 11818505.
18. Shih HA, Nathanson KL, Seal S, Collins N, Stratton MR, Rebbeck TR, Weber BL. BRCA1 and BRCA2 mutations in breast cancer families with multiple primary cancers. Clin Cancer Res 2000, 6(11):4259-4264.
19. Thedieck C, Kuczyk M, Klingel K, Steiert I, Muller CA, Klein G. Expression of Ksp-cadherin during kidney development and in renal cell carcinoma. Br J Cancer 2005, 92(11):2010-2017. 10.1038/sj.bjc.6602597, 2361784, 15886705.
20. Di Palma T, Conti A, de Cristofaro T, Scala S, Nitsch L, Zannini M. Identification of novel Pax8 targets in FRTL-5 thyroid cells by gene silencing and expression microarray analysis. PLoS One 2011, 6(9):e25162. 10.1371/journal.pone.0025162, 3179481, 21966443.
21. de Cristofaro T, Di Palma T, Fichera I, Lucci V, Parrillo L, De Felice M, Zannini M. An essential role for Pax8 in the transcriptional regulation of Cadherin-16 in thyroid cells. Mol Endocrinol 2012, 26(1):67-78. 10.1210/me.2011-1090, 22135066.
22. Zacchi P, Stenmark H, Parton RG, Orioli D, Lim F, Giner A, Mellman I, Zerial M, Murphy C. Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells. J Cell Biol 1998, 140(5):1039-1053. 10.1083/jcb.140.5.1039, 2132691, 9490718.
23. Muller T, Hess MW, Schiefermeier N, Pfaller K, Ebner HL, Heinz-Erian P, Ponstingl H, Partsch J, Rollinghoff B, Kohler H, et al. MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity. Nat Genet 2008, 40(10):1163-1165. 10.1038/ng.225, 18724368.
24. Min J, Zaslavsky A, Fedele G, McLaughlin SK, Reczek EE, De Raedt T, Guney I, Strochlic DE, Macconaill LE, Beroukhim R, et al. An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB. Nat Med 2010, 16(3):286-294. 10.1038/nm.2100, 2903662, 20154697.
25. Bianco AC, Kim BW. Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest 2006, 116(10):2571-2579. 10.1172/JCI29812, 1578599, 17016550.
26. Nowak RB, Fischer RS, Zoltoski RK, Kuszak JR, Fowler VM. Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens. J Cell Biol 2009, 186(6):915-928. 10.1083/jcb.200905065, 2753162, 19752024.
27. Liu C, Papewalis C, Domberg J, Scherbaum WA, Schott M. Chemokines and autoimmune thyroid diseases. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme 2008, 40(6):361-368. 10.1055/s-2008-1073153, 18418812.
28. Min HS, Choe G, Kim SW, Park YJ, Park do J, Youn YK, Park SH, Cho BY, Park SY. S100A4 expression is associated with lymph node metastasis in papillary microcarcinoma of the thyroid. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2008, 21(6):748-755.
29. Ying S, Dong S, Kawada A, Kojima T, Chavanas S, Mechin MC, Adoue V, Serre G, Simon M, Takahara H. Transcriptional regulation of peptidylarginine deiminase expression in human keratinocytes. J Dermatol Sci 2009, 53(1):2-9. 10.1016/j.jdermsci.2008.09.009, 19004619.
30. Riepe FG. Clinical and molecular features of type 1 pseudohypoaldosteronism. Horm Res 2009, 72(1):1-9.
31. Montero-Conde C, Martin-Campos JM, Lerma E, Gimenez G, Martinez-Guitarte JL, Combalia N, Montaner D, Matias-Guiu X, Dopazo J, de Leiva A, et al. Molecular profiling related to poor prognosis in thyroid carcinoma. Combining gene expression data and biological information. Oncogene 2008, 27(11):1554-1561. 10.1038/sj.onc.1210792, 17873908.
32. Stuart ET, Haffner R, Oren M, Gruss P. Loss of p53 function through PAX-mediated transcriptional repression. EMBO J 1995, 14(22):5638-5645. 394679, 8521821.
33. Dehbi M, Pelletier J. PAX8-mediated activation of the wt1 tumor suppressor gene. EMBO J 1996, 15(16):4297-4306. 452155, 8861958.
34. Scouten WT, Patel A, Terrell R, Burch HB, Bernet VJ, Tuttle RM, Francis GL. Cytoplasmic localization of the paired box gene, Pax-8, is found in pediatric thyroid cancer and may be associated with a greater risk of recurrence. Thyroid 2004, 14(12):1037-1046. 10.1089/thy.2004.14.1037, 15650356.
35. Scacheri PC, Davis S, Odom DT, Crawford GE, Perkins S, Halawi MJ, Agarwal SK, Marx SJ, Spiegel AM, Meltzer PS, et al. Genome-wide analysis of menin binding provides insights into MEN1 tumorigenesis. PLoS Genet 2006, 2(4):e51. 10.1371/journal.pgen.0020051, 1428788, 16604156.
36. Yang A, Zhu Z, Kapranov P, McKeon F, Church GM, Gingeras TR, Struhl K. Relationships between p63 binding, DNA sequence, transcription activity, and biological function in human cells. Mol Cell 2006, 24(4):593-602. 10.1016/j.molcel.2006.10.018, 17188034.
37. A M, A AP, AS M, M B, Z M, M H. Evaluation of apoptosis-related genes; Fas (CD94), FasL (CD178) and TRAIL polymorphisms in Iranian multiple sclerosis patients. J Neurol Sci 2011,
38. Illingworth RS, Bird AP. CpG islands-'a rough guide'. FEBS Lett 2009, 583(11):1713-1720. 10.1016/j.febslet.2009.04.012, 19376112.
39. Illingworth RS, Gruenewald-Schneider U, Webb S, Kerr AR, James KD, Turner DJ, Smith C, Harrison DJ, Andrews R, Bird AP. Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet 2010, 6(9). 2891719, 20585624.
40. Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, et al. Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes. Genome Res 2006, 16(1):55-65. 1356129, 16344560.
41. Cawley S, Bekiranov S, Ng HH, Kapranov P, Sekinger EA, Kampa D, Piccolboni A, Sementchenko V, Cheng J, Williams AJ, et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 2004, 116(4):499-509. 10.1016/S0092-8674(04)00127-8, 14980218.
42. Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 2009, 458(7235):223-227. 10.1038/nature07672, 2754849, 19182780.
43. Maunakea AK, Nagarajan RP, Bilenky M, Ballinger TJ, D'Souza C, Fouse SD, Johnson BE, Hong C, Nielsen C, Zhao Y, et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 2010, 466(7303):253-257. 10.1038/nature09165, 20613842.
44. Ohler U, Liao GC, Niemann H, Rubin GM. Computational analysis of core promoters in the Drosophila genome. Genome Biol 2002, 3(12). RESEARCH0087.
45. Lim CY, Santoso B, Boulay T, Dong E, Ohler U, Kadonaga JT. The MTE, a new core promoter element for transcription by RNA polymerase II. Genes Dev 2004, 18(13):1606-1617. 10.1101/gad.1193404, 443522, 15231738.
46. Juven-Gershon T, Hsu JY, Theisen JW, Kadonaga JT. The RNA polymerase II core promoter - the gateway to transcription. Curr Opin Cell Biol 2008, 20(3):253-259. 10.1016/j.ceb.2008.03.003, 2586601, 18436437.
47. Jin VX, Singer GA, Agosto-Perez FJ, Liyanarachchi S, Davuluri RV. Genome-wide analysis of core promoter elements from conserved human and mouse orthologous pairs. BMC Bioinforma 2006, 7:114.
48. Di Palma T, D'Andrea B, Liguori GL, Liguoro A, de Cristofaro T, Del Prete D, Pappalardo A, Mascia A, Zannini M. TAZ is a coactivator for Pax8 and TTF-1, two transcription factors involved in thyroid differentiation. Exp Cell Res 2009, 315(2):162-175. 10.1016/j.yexcr.2008.10.016, 19010321.
49. Lanctot C, Cheutin T, Cremer M, Cavalli G, Cremer T. Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat Rev Genet 2007, 8(2):104-115. 10.1038/nrg2041, 17230197.
50. Phillips JE, Corces VG. CTCF: master weaver of the genome. Cell 2009, 137(7):1194-1211. 10.1016/j.cell.2009.06.001, 3040116, 19563753.
51. Li L, Davie JR. The role of Sp1 and Sp3 in normal and cancer cell biology. Ann Anat 2010, 192(5):275-283. 10.1016/j.aanat.2010.07.010, 20810260.
52. De Leo R, Miccadei S, Zammarchi E, Civitareale D. Role for p300 in Pax 8 induction of thyroperoxidase gene expression. J Biol Chem 2000, 275(44):34100-34105.
53. Grasberger H, Ringkananont U, Lefrancois P, Abramowicz M, Vassart G, Refetoff S. Thyroid transcription factor 1 rescues PAX8/p300 synergism impaired by a natural PAX8 paired domain mutation with dominant negative activity. Mol Endocrinol 2005, 19(7):1779-1791. 10.1210/me.2004-0426, 15718293.
54. Pasca di Magliano M, Di Lauro R, Zannini M. Pax8 has a key role in thyroid cell differentiation. Proc Natl Acad Sci U S A 2000, 97(24):13144-13149. 10.1073/pnas.240336397, 27192, 11069301.
55. Robson EJ, He SJ, Eccles MR. A PANorama of PAX genes in cancer and development. Nat Rev Cancer 2006, 6(1):52-62. 10.1038/nrc1778, 16397527.
56. Arnaldi LA, Borra RC, Maciel RM, Cerutti JM. Gene expression profiles reveal that DCN, DIO1, and DIO2 are underexpressed in benign and malignant thyroid tumors. Thyroid 2005, 15(3):210-221. 10.1089/thy.2005.15.210, 15785240.
57. Rossi DL, Acebron A, Santisteban P. Function of the homeo and paired domain proteins TTF-1 and Pax-8 in thyroid cell proliferation. J Biol Chem 1995, 270(39):23139-23142. 10.1074/jbc.270.39.23139, 7559458.
58. Buttiglieri S, Deregibus MC, Bravo S, Cassoni P, Chiarle R, Bussolati B, Camussi G. Role of Pax2 in apoptosis resistance and proinvasive phenotype of Kaposi's sarcoma cells. J Biol Chem 2004, 279(6):4136-4143.
59. Schebesta A, McManus S, Salvagiotto G, Delogu A, Busslinger GA, Busslinger M. Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function. Immunity 2007, 27(1):49-63. 10.1016/j.immuni.2007.05.019, 17658281.
60. Albelda SM, Oliver PD, Romer LH, Buck CA. EndoCAM: a novel endothelial cell-cell adhesion molecule. J Cell Biol 1990, 110(4):1227-1237. 10.1083/jcb.110.4.1227, 2116064, 2182647.
61. Cali G, Gentile F, Mogavero S, Pallante P, Nitsch R, Ciancia G, Ferraro A, Fusco A, Nitsch L. CDH16/Ksp-Cadherin Is Expressed in the Developing Thyroid Gland and Is Strongly Down-Regulated in Thyroid Carcinomas. Endocrinology 2011,
62. de Cristofaro T, Di Palma T, Fichera I, Lucci V, Parrillo L, De Felice M, Zannini M. An Essential Role for Pax8 in the Transcriptional Regulation of Cadherin-16 in Thyroid Cells. Mol Endocrinol 2011,
63. Fusco A, Berlingieri MT, Di Fiore PP, Portella G, Grieco M, Vecchio G. One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes. Mol Cell Biol 1987, 7(9):3365-3370. 367981, 3670314.
64. Costamagna E, Garcia B, Santisteban P. The functional interaction between the paired domain transcription factor Pax8 and Smad3 is involved in transforming growth factor-beta repression of the sodium/iodide symporter gene. J Biol Chem 2004, 279(5):3439-3446.
65. Landa I, Ruiz-Llorente S, Montero-Conde C, Inglada-Perez L, Schiavi F, Leskela S, Pita G, Milne R, Maravall J, Ramos I, et al. The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors. PLoS genetics 2009, 5(9):e1000637. 10.1371/journal.pgen.1000637, 2727793, 19730683.
66. Vilain C, Rydlewski C, Duprez L, Heinrichs C, Abramowicz M, Malvaux P, Renneboog B, Parma J, Costagliola S, Vassart G. Autosomal dominant transmission of congenital thyroid hypoplasia due to loss-of-function mutation of PAX8. J Clin Endocrinol Metab 2001, 86(1):234-238. 10.1210/jc.86.1.234, 11232006.
67. Courey AJ, Holtzman DA, Jackson SP, Tjian R. Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell 1989, 59(5):827-836. 10.1016/0092-8674(89)90606-5, 2512012.
68. Klenova EM, Nicolas RH, Paterson HF, Carne AF, Heath CM, Goodwin GH, Neiman PE, Lobanenkov VV. CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms. Mol Cell Biol 1993, 13(12):7612-7624. 364833, 8246978.
69. Lee TI, Johnstone SE, Young RA. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc 2006, 1(2):729-748. 10.1038/nprot.2006.98, 3004291, 17406303.
70. Salmon-Divon M, Dvinge H, Tammoja K, Bertone P. PeakAnalyzer: genome-wide annotation of chromatin binding and modification loci. BMC Bioinforma 2010, 11:415.
71. Flicek P, Amode MR, Barrell D, Beal K, Brent S, Chen Y, Clapham P, Coates G, Fairley S, Fitzgerald S, et al. Ensembl 2011. Nucleic Acids Res 2011, 39(Database issue):D800-806. 3013672, 21045057.
72. Fujita PA, Rhead B, Zweig AS, Hinrichs AS, Karolchik D, Cline MS, Goldman M, Barber GP, Clawson H, Coelho A, et al. The UCSC Genome Browser database: update 2011. Nucleic Acids Res 2011, 39(Database issue):D876-882. 3242726, 20959295.
73. Machanick P, Bailey TL. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 2011, 27(12):1696-1697. 10.1093/bioinformatics/btr189, 3106185, 21486936.
74. Gupta S, Stamatoyannopoulos JA, Bailey TL, Noble WS. Quantifying similarity between motifs. Genome Biol 2007, 8(2):R24. 10.1186/gb-2007-8-2-r24, 1852410, 17324271.
75. Perona R, Montaner S, Saniger L, Sanchez-Perez I, Bravo R, Lacal JC. Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins. Genes Dev 1997, 11(4):463-475. 10.1101/gad.11.4.463, 9042860.
76. Al-Shahrour F, Minguez P, Tarraga J, Medina I, Alloza E, Montaner D, Dopazo J. FatiGO +: a functional profiling tool for genomic data. Integration of functional annotation, regulatory motifs and interaction data with microarray experiments. Nucleic Acids Res 2007, 35(Web Server issue):W91-96. 1933151, 17478504.
77. Medina I, Carbonell J, Pulido L, Madeira SC, Goetz S, Conesa A, Tarraga J, Pascual-Montano A, Nogales-Cadenas R, Santoyo J, et al. Babelomics: an integrative platform for the analysis of transcriptomics, proteomics and genomic data with advanced functional profiling. Nucleic Acids Res 2010, 38(Web Server issue):W210-213. 2896184, 20478823.