Cis ac$ng transcrip$onal elements with nega$ve regulatory func$on

Transcription

1 Cis ac$ng transcrip$onal elements with nega$ve regulatory func$on Laura Elnitski, PhD Na$onal Human Genome Research Ins$tute Na$onal Ins$tutes of Health 2

2 Non coding regulatory signals affec$ng transcrip$on Experimental Design Motif Prediction Functional Characterization 3

3 Silencer and Enhancer Blocker Assay Promoter! LUC Reporter gene! Fold expression! 10! 1! promoter! Enhancer! insertion sites! LUC Fold expression! 10! 1! promoter! + enhancer! Silencer or EB LUC Blocker Fold expression! 10! 1! promoter! + enhancer! silencer! EB! Petrykowska, et al. Genome Research,

4 Chicken β globin Insulator (chs4) 5

5 47 regions from the CFTR locus CR8 CR15 CR18 NR1 NR10 K562 cells Forward orienta$on S Enhancer! Silencer LUC Promoter Orienta$on Cell line CR1 NR4 EB LUC Blocker Enhancer! 6

6 47 regions from the greater CFTR locus Silencer, SV40 EB, SV40 7

7 47 regions from the greater CFTR locus WNT2 WNT2 V$NKX25_01 V$CEBPB_02 V$E47_02 CNC2_PSU B3 CNC2_NIH CNC2R1 Your Sequence from Blat Search del3 delta3.1_f CNC2_F1 del3.1 delta3.2_f delta3.1_r Delta3.3_F Delta3.3_R NHGRI Catalog of Published Genome-Wide Association Studies UCSC Genes Based on RefSeq, UniProt, GenBank, CCDS and Comparative Genomics HMR Conserved Transcription Factor Binding Sites Vertebrate Multiz Alignment & Conservation (17 Species) delta3.2_r Conservation chimp rhesus mouse rat rabbit SINE LINE Repeating Elements by RepeatMasker 8

8 Window Position chr7: ---> Del3.1 Human Mar chr7:116,743, ,743,896 (147 bp) GCAAGGCAGAAAACAGAGAACCATTTGGTGATTCAATATGTCAAGAGGAGTGTGACTTTTTGAACCACACAGCTGGGAGAGCAAACCACCTTTTCACATTGAAGCCCTGCTTGTTTCCGTTTGTCATTCAGTGCTAAAATTTATTA T User Supplied Track Del3.2 Del3.3 Vertebrate Multiz Alignment & PhastCons Conservation (28 Species) Mammal Cons Gaps Human GCAAGGCAGAAAACAGAGAACCATTTGGTGATTCAATATGTCAAGAGGAGTGTGACTTTTTGAACCACACAGCTGGGAGAGCAAACCACCTTTTCACATTGAAGCCCTGCTTGTTTCCGTTTGTCATTCAGTGCTAAAATTTATTA T Chimp GCAAGGCAGAAAACAGAGAACCATTCGGTGATTCAATATGTCAAGAGGAGTGTGACTTTTTGAACCACACAGCTGGGAGAGCAAACCACCTTTTCACATTGAAGCCCTGCTTGTTTCCGTTTGTCATTCAGTGTTAAAATTTATTA T Rhesus GCAAGGCAGAAAACAGAGAACCATTCGGTGATTCAATATGTCAAGAGGAGTGTGACTTTTTGAACCACACAGCTGGGAGAGCAAACCACCTTTTCGCATTGAAGCCCTGCTTGTTTCCGTTTGTCATTCAGTGTTAAAATTTATTA T Mouse GCAAGGCAGAAAGCAGAGAAGCGTTTGGCAATTCAATCTGTCAAGACCAGTGTGACTTTTTGAACCACACAGCTGGGAGAACAGCCTACTTTTCCGCATGGAAGCCTTGCTCATTTCCATGTGCCACTCACGGCTATAGCTTACTGT Rat GCAAGGCAGAAAGCAGAGAAGCGTTTGGCGATTCAATCTGTCAGGACCAGTGTGACTTTTTGAACCACGCAGCTGGGAGAACAGCCTACTTTTCCGAATCGAAGCCTTGCTCGTTTCCATGTGTCACTCATGGCTAGAGCTTACTGT Rabbit GCAAGGCAGAA - - CACAGAACCGTTGGGTGAGTTAGGATGTCAAGCGCAGTGTGACTTTCTGAGCCACACAGCTAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN Hedgehog GCATAGCAGAAAGCAGAGAGTCACTCGGTGATTCAATATGTCAAGACAAGAGTGACTTTTTGAACCACACGGCTGGAAGAACAAACCACCTTTTCGCATCGAAGCCTAGCTTGTTTCCTTTTGTCATTCAGTGCTAAAATTTATTA T Dog GCAAGGCAGAGAACGGAGAGCCATTCAGCGACTCGGTATGTCAAGACGAGTGTGACTTTTTGAACCACACAGCTGGGAGAACAAACCACCTTTTCGCCTTGAAGCCCTGCTTGTTTCCGCCTGTCATTCGGTGCTAAAATTTATTA T Cat ACAAGGCAGAAAACGGAGAGCCAGTCAGTGACTCCATGTGTGAAGACGAGTGTGACTTTTTGAACCACACAGCTGGGAGAACAAACCACCTTTTCGCATTGAAGTCCTGCTTGTTTCCGTTTGTCATTCGTTGCTAAAATTTATTA T Horse GCAAGGCAGAAAACGGAGAGCCATTTGGTGATTCAATATGTCAAGACGAGTGTGACTTTTTGAACCACACAGCTGGGAGAACAAACCACCTTTTCGCATCGAAGCCCTGCTTGTTTCCGTTTGTCATTCAGTGCTAAAATTTATTA T Cow GCAAGGAAGCAAATGGAGAGCCATTTGGTGATTCAATATGTCAAAACGAGTGTGACTTTTTGAACCACGCAGCTGGGAGAACAAACCACCTTTTCGCATCGAAGCCCTACTTGTTTCCGTTTGTCATTCAGTGCTAAAATTTATTA T Armadillo ========================================================================= = ======================================================NAGTGCTAAAATTTATTAT Elephant NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNATTA T Tenrec GAGAGGCAGAACACAGTGCCCCCC GCGATTCGGTCCCTCAGGATGAGTG CCTGTGGAAACACACAG - TGGGA GGACCTTTTTGCATGGAAGTCC - ATATACTGCCGCCTGTCACTCGGTGTTGAAATTTATTAT Opossum TGAAGGCTGAAAACAGACAGCCATTAGGTGATCCAATAGGTCAACAAGAGTGTGACTTTTTGAACCATACATCTGGGAGAACAAACCACCATTTTGCATTGCAGCACTGCTCGTTCCCATTTGTCATTCGGGGCTAAAATTTATTGT Platypus ACAAGACTGAACTCAGAAAGCTGTTAGGTGATTCAATATGTTAAGAAGAGTGTGACTTTTTGAATCATATATCGGGGAAAAAAAATCCCCATTTTGCAGGGAATTTCTGTTCTTTTCGCTTTGCCGCTCAGGGCTAAATTTTGCAA T X_tropicalis ========================================================================= = ======================================================================== = Fold 10 increase 5 0 del_3 SV40_pro HS2_SV40_pro del_3.1_for del_3.2_for del_3.3_for 9

9 47 regions from the greater CFTR locus 15 of 47 regions had NRE ac$vity 12 of 15 were dependent on orienta$on NRE func$on differs with promoter iden$ty CFTR 10

10 NREs within the CFTR locus 11

11 A Mo$f detec$on and tes$ng B * C 12

12 Chicken Insulator (chs4) MEME E value 1.9 x 10 6

13 Genomic Prevalence (X 10 4 ) 19 bp mo$f is depleted in CpG island promoters and 5 UTRs, but not non CpG island promoters or intergenic regions

14 Co localiza$on of func$onal regions and open chroma$n 7 regions have DNAse I HS /FAIRE data consistent with open chroma$n 15 FAIRE: Giresi et al 2006 Open chroma$n: Boyle et al 2008

15 Scale chr7: 5C long distance interac$on data Lajoie, B.R., van Berkum, N.L., Sanyal, A. and Dekker, J. (2009) 100 kb User Supplied Track K562 CTCF BO K562 Raw 1 CAV1 CAV1 CAV2 CAV2 CAV1 CAV1 CAV1 100 _ 0 _ 100 _ 1 _ Conservation CR1 UCSC Genes Based on RefSeq, UniProt, GenBank, CCDS and Comparative Genomics ENCODE Open Chromatin, Duke/UNC/UT ENCODE Open Chromatin, UT ChIP-seq Base Overlap Signal (CTCF in K562 cells) ENCODE Univ. Washington DNaseI Hypersensitivity by Digital DNaseI ENCODE UW Digital DNaseI Raw Signal - 1st (in K562 cells) Vertebrate Multiz Alignment & Conservation (17 Species) mouse rat rabbit dog armadillo elephant opossum chicken x_tropicalis tetraodon 16

16 Summary NREs func$on as conserved or non conserved regions to ex$nguish strong enhancer ac$vity Func$on in transient transfec$on experiments, indica$ng that protein interac$ons are responsible Implicate proteins other than CTCF in EB ac$vity Involved in long distance interac$ons 17

17 Acknowledgments ENCODE Consor$um Elnitski Lab NHGRI Hanna Petrykowska Adam Woolfe Sasha Scog Job Dekker UMass Tyra Wolfsberg NHGRI 18