Predicting evolutionarily conserved regions (ECRs) in the Xenopus tropicalis genome using a MultiPipMaker-based bioinformatic strategy

Transkriptio

1 Predicting evolutionarily conserved regions (ECRs) in the Xenopus tropicalis genome using a MultiPipMaker-based bioinformatic strategy OVERVIEW- Method updated and written by Sarah Louie (6/19/08) Initially conceptualized by Hajime Ogino (6/23/07) Laboratory of Robert Grainger Department of Biology University of Virginia 1. Extract genomic sequences of interest from genome assembly data. 2. Build accompanying text files. 3. Generate a multiple genomic sequence alignment. 4. Perform a local realignment of ECR sequences. 5. Identify conserved putative transcription factor binding sites.

2 ECR browser: identify ECRs using premade alignments Pax6 coding region ECRs Slide 1

3 ECR browser misses some ECRs; the Pax6CE1 as an example Pax6CE1 Slide 2

4 Comparison of MultiPipMaker results versusecr Browser and Vista Browser in the detection of the Pax6CE1 enhancer Slide 3

5 Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a global genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Identify short evolutionarily conserved regions (ECRs) and perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using rvista. Slide 4

6 Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a multiple genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 5

7 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. >Go to Slide 6

8 >Go to the genome and position of interest Slide 7

9 >Get orthologous sequences from multiple genomes Get sequence from 1st genome Get orthologous regions in other genomes Slide 8

10 >Get the genomic DNA sequence from the first genome Slide 9

11 >Save the genomic DNA sequence as simple text file >xentro2_dna range=scaffold_399: 'pad=0 3'pad=0 strand=+ repeatmasking=none ATATATATATATATATATATATATATATATATATATATATATATACATGA GAATAGATTTCTATGCGAAGTAATATAATAGATTTCTATACTAAGAATAT ATATATATATATATATATATATGTACATTACATGCAATTCATAACAATCA TCACAACAATAACCTTAAATCCCATTACAGTTCAATTACCCAGAACAATA TTTCCCAAGGTAACTCATCTTTTTATGTGTTATTTTTTCCCCCATTACAT TTTAGTCGAATATATGTATATATTTTGTATTTCTATTTTTTTTGCAAATT GATACAAAGCATATGATAAACAATCACCATAGTTACCAGGGTCCTTAGTG CTAATGAGGTCCCCAGTTGCAGCTCAGTAAATTGAAACTATTTACTGCAT AGAAATGTTTTTTTTTCCCGTTTTCTATTTTTCTCCAAAGCTTGTTTACA CCCCTTATTTTTTTTTTTGTGTGTTATTTGGGCTGTGCTGTACACTGACA CTAGTGCTGGCAGGAGGGGGCTGCTGTAGTTCAAGCTACTGAATGTAGAT AATAGTTTCTCCCCCCCCTCTCTCTGTATTCACTTTAAACCATGAACACA AATAGTCGCTTCCACTTGGACTTTTTTTTAATTTATTTTATATTACAGTT TTAAACTTAACCAAGGGCCACAATAAAGGGCCAGGGAACATGTATAATTC ACGGTTTGTTATGCTATTACTTTTCATTCTTCACAAAAAAAAAAAATAGC AAAAACCCCCTTTCTTGTTATAAATTTCTGGAGGTAATTTTGTTACTGGT GTGAGTTGTTAGGGGTTGTAAAACTACACAACAGCTCGGCAGGGGGCTCA ATTGGAAAGAGACAATTACAATAGTGAAAGCATTGAAGAGAGTTGGTTAA AAAGAAAGAAGGGGTAAAATAATGTAATGGCCTGAAATTTCTCTCCAAAA TGCCCTTTCTGTCTCTTTTATGGCAGCAATGAATATCAAGGCTCTATCGA Slide 10

12 >Convert the genomic DNA sequence to orthologous locations in other genomes of interest (human, mouse, chicken, zebrafish, etc) Slide 11

13 Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a multiple genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 12

14 >Retrieve the exon and underlay files needed for Multipipmaker from PipHelper Slide 13

15 >Download the exon and underlay files Slide 14

16 >Save the exon file as a simple text file Example file for Pax6CE1_Xt containing just the 5 end of coding region : > CE1 > pax exon1 Example file for Pax6CE1_Xt containing all of the Pax6 coding region : < pax UTR exon exon exon exon exon exon exon exon UTR Slide 15

17 >Save the Pax6CE1_Xt underlay file as a simple text file LightCyan Pax6CE LightCyan LightBlue Pax6cds LightBlue LightYellow Pax6intron LightYellow Slide 16

18 >Get the repeat file from RepeatMasker at the ISB Slide 17

19 >Go to Slide 18

20 >Download the repeat text file Slide 19

21 >Save the repeat masker file in simple text Slide 20

22 Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a multiple genomic sequence alignment with high sensitivity using MultiPipMaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 21

23 >Go to Slide 22

24 >Input the number of sequences to be aligned 5 Slide 23

25 >Input the sequence, exon, underlay and repeat files for the base sequence, and just the sequence files from the other genomes Slide 24

26 >Go to the resulting pip.pdf file for the plot: Pax6CE1 Slide 25

27 >Go to the resulting alignment in the acgt.pdf file: Slide 26

28 Example of pip plot for 80 kb surrounding the Pax6 locus with base genome and annotation from the mouse genome: Slide 27

29 -Ogino, unpubl. Slide 28

30 Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a global genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 29

31 >Perform a local realignment of ECR sequences with ClustalW Use VectorNTI or other basic sequence analysis program or SDSC website for the following tasks: 1. Create sequence files 2. Align (with ClustalW at SDSC website) 3. Shade (with BOXSHADE at SDSCwebsite) Slide 30

32 >Go to Slide 31

33 >Add genes to your SDSC Biology Workbench account Slide 32

34 >Perform local alignment with ClustalW, then perform BOXSHADE on the resulting ClustalW alignment Slide 33

35 >Save the BOXSHADE results for phylogenetic footprinting Of Transcription Factor Binding Motif (TFBM) analysis Slide 34

36 Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a global genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 35

37 Scanning for putative transcription factor binding sites- A. Employ the TRANSFAC database ( OR B. Employ user-defined motifs ( Slide 36

38 >Predict TFBMs with the TRANSFAC database at the Gene-regulation website Slide 37

39 >Transfac search results from P-Match Slide 38

40 >Check Position weight matrix and references for TRANSFAC database Slide 39

41 >Enter your own TFBM list and sequences using DNApattern at the RSA tools website Slide 40

42 >Results of user-defined search using DNA-pattern PatID Strand Pattern SeqID Start End matching_seq Score SEQ_START DR - Pax6CE1_Xt_227bp SEQ_END DR - Pax6CE1_Xt_227bp OTX/PITX D Taakcy Pax6CE1_Xt_227bp gtactaatcccaac 1.00 MSX2 D Waatkr Pax6CE1_Xt_227bp attaaaatgacgtc 1.00 MSX2 D Waatkr Pax6CE1_Xt_227bp ttgctaattgagac 1.00 MSX2 R Waatkr Pax6CE1_Xt_227bp attttaatggagag 1.00 TCF/LEF R ctttgww Pax6CE1_Xt_227bp tgggctttgtacacc 1.00 RX D ctaattg Pax6CE1_Xt_227bp gttgctaattgagac 1.00 CRE DR gacgtc Pax6CE1_Xt_227bp aaatgacgtcagct 1.00 SEQ_START DR - pax6ce1_hs_241rev SEQ_END DR - pax6ce1_hs_241rev OTX/PITX D Taakcy pax6ce1_hs_241rev gtgctaatccactc 1.00 OTX/PITX R Taakcy pax6ce1_hs_241rev gccgtaatcttgtt 1.00 OTX/PITX R Taakcy pax6ce1_hs_241rev ttcttaagctttgc 1.00 MSX2 D Waatkr pax6ce1_hs_241rev tcgctaattgagag 1.00 SOX D Wwttgww pax6ce1_hs_241rev gattttttgttaaaa 1.00 PROSPERO D caynnct pax6ce1_hs_241rev cattcactcctcgct 1.00 PROSPERO R caynnct pax6ce1_hs_241rev ctcccacccctccaa 1.00 ETS R mggaw pax6ce1_hs_241rev gcctcggaaagaa 1.00 TCF/LEF R ctttgww pax6ce1_hs_241rev ggctctttgaatcag 1.00 SU(H) D rtgrgar pax6ce1_hs_241rev aggggtgggagaagg 1.00 SIP1 R cacct pax6ce1_hs_241rev ttagcaccttctt 1.00 RX D ctaattg pax6ce1_hs_241rev gtcgctaattgagag 1.00 CRE DR gacgtc pax6ce1_hs_241rev caatgacgtcagtg 1.00 SEQ_START DR - Pax6CE1_Mm_240bp SEQ_END DR - Pax6CE1_Mm_240bp OTX/PITX D Taakcy Pax6CE1_Mm_240bp gtgctaatccactg 1.00 OTX/PITX R Taakcy Pax6CE1_Mm_240bp gccataatcttgtt 1.00 OTX/PITX R Taakcy Pax6CE1_Mm_240bp ctcttaagctttgc 1.00 MSX2 D Waatkr Pax6CE1_Mm_240bp ttgctaattgagag 1.00 SOX D Wwttgww Pax6CE1_Mm_240bp atttttttgttaaaa 1.00 SU(H) D rtgrgar Pax6CE1_Mm_240bp gtgggtgggagaagg 1.00 SIP1 R cacct Pax6CE1_Mm_240bp atctcacctcaga 1.00 RX D ctaattg Pax6CE1_Mm_240bp gttgctaattgagag 1.00 CRE DR gacgtc Pax6CE1_Mm_240bp caatgacgtcagcg Slide 41

43 >Use the Feature Map for visual comparison of predicted TFBMs in each sequence Slide 42

44 Overlay TFBM search results on ECR alignment to identify predicted binding sites that are also conserved in Xenopus Pax6CE1 Hs Pax6CE1 Mm Pax6CE1 Xt CRE (1) CGGAGCCCCCTTCCCCACCC------AATGACGTCAGTGGCATTCACTCC (1) CGAAGCCCCCATCCCCACCC------AATGACGTCAGCGGCATTCATGCC (1) CGCAGCCCCCCCCCCCTCTCCATTAAAATGACGTCAGCTGTATACA-TCA Pax6CE1 Hs (45) TCGCTCCTTGCCTTTTCTGATTTTTTG-TTAAAACTTTTCTCCGCAGCGA Pax6CE1 Mm (45) AGGCTCCTAGCTTCTTCTGATTTTTTTGTTAAAACTTTTCTCTGAGGTGA Pax6CE1 Xt (50) T--CTCCCTCCCCCTTATGATTTTTTT-TTT----TTTTCTCTAAAACTT Pax6CE1 Hs (94) GATGGGGGTTGGAGGGGTGGGAGAAGGAGCTGATTCAAAGAGC-CAGTGC Pax6CE1 Mm (95) GATGGGGGTTGT--GGGTGGGAGAAGGGGCTGATTGAAAGAGC-CAGTGC Pax6CE1 Xt (93) TTTTT--TTTTGTCGGGAGGGTGTA CAAAGCCCACTCTGC Pax6CE1 Hs (143) CCTGGGGGGAAATAGCAACAAGATTACGGCTGCTTCTTTCCGAGGCAAAG Pax6CE1 Mm (142) CCTGGGGGGAAATAGCAACAAGATTATGGCTGCCTCTTTCTTAGGCAAAG Pax6CE1 Xt (131) CCTAGGGAGAAATAGCAACATGATTAGAGCTTCT-----CGGAGCAAAGG OTX/PITX Rx/MSX Pax6CE1 Hs (193) CTTAAGAAGGTGCTAATCCACTCGGTCGCTAATTGAGA---GGTTGTAAA Pax6CE1 Mm (192) CTTAAGAGGGTGCTAATCCACTGGGTTGCTAATTGAGA---GGTTGTAAA Pax6CE1 Xt (176) CATTATTTCGTACTAATCCCAACAGTTGCTAATTGAGACTTGCTTGGAAA Pax6CE1 Hs (240) AA Pax6CE1 Mm (239) AA Pax6CE1 Xt (226) AA Slide 43

45 References Ivan Ovcharenko, Marcelo Nobrega, Gabriela Loots, Lisa Stubbs. ECR Browser: a Tool for Visualizing and Accessing Data from Comparisons of Multiple Vertebrate Genomes. Nucleic Acids Research (2004) 32: W280-W286. Ivan Ovcharenko, Gabriela Loots, Ross Hardison, Webb Miller, Lisa Stubbs. zpicture: Dynamic Alignment and Visualization Tool for Analyzing Conservation Profiles. Genome Research (2004) 14: Kleinjan DA, van Heyningen V. Long-range control of gene expression: emerging mechanisms and disruption in disease. Am J Hum Genet Jan;76(1):8-32. Epub 2004 Nov 17. Lettice LA, Hill AE, Devenney PS, Hill RE. Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly. Hum Mol Genet Apr 1;17(7): Epub 2007 Dec 21. Ogino H, Fisher M, Grainger RM. Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification. Development Jan;135(2): Scott Schwartz, Zheng Zhang, Kelly A. Frazer, Arian Smit, Cathy Riemer, John Bouck, Richard Gibbs, Ross Hardison, and Webb Miller. PipMaker---A Web Server for Aligning Two Genomic DNA Sequences. Genome Research (2000) Vol. 10, Issue 4; Scott Schwartz, Laura Elnitski, Mei Li, Matt Weirauch, Cathy Riemer, Arian Smit, Eric Green, Ross Hardison, NISC Comparative Sequencing program, Webb Miller. MultiPipMaker and Supporting Tools: Alignments and Analysis of Multiple Genomic DNA sequences. Nucleic Acids Research (2003) 31: Scott Schwartz, W. James Kent, Arian Smit, Zheng Zhang, Robert Baertsch, Ross C. Hardison, David Haussler, and Webb Miller. Human-Mouse Alignments with BLASTZ. (2003) 13: ; originally published online Dec 30, 2002; Genome Res.