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Mapping mobile DNA elements: Sources of human genetic diversity and disease.OverviewThe (junk) GenomeLINEs are dynamic genetic sequences.Major epigenetic remodeling takes place in germ cell and embryo development.Retrotransposons can act as insertional mutagens “Accidental Discoveries” of LINEs as Agents of DiseaseTumor suppressors are down-regulated in neoplasia in the context of broad genome hypomethylationSlide 8Slide 9Where are the transposons? TIP-chip strategyLINE mapping strategy: Vectorette PCRSlide 12Slide 13Slide 14The T(a)LINE TIP-Chip is a robust genotyping toolGaussian Distribution of Background and Foreground ValuesModeling Peak ShapeLISA LINE Insertion Signal AnalysisSlide 19Finding novel T(a)LINE insertionsSummaryFuture DirectionsAcknowledgementsMapping mobile DNA elements: Sources of human genetic diversity and disease.Kathleen H. Burns, M.D., [email protected] Hopkins Department of PathologyOverviewI. The Mobile Genome: Transposable elements in human diversity & disease. II. Finding a needle in a haystack - one new mobile element insertion in a million. III. Novel microarray-based transposon mapping technology and data analysisIV. Future directionsThe (junk) GenomeData from Jasinska & Krzyzosiak, FEBS Letters (2004).55% of the human genome is repetitive45% of the human genome is comprised of interspersed repeatsnon-repetitivetandem repeats21% of the human genome is comprised of long interspersed LINEsLTRs (HERVs)SINEs (Alu)LINEs are dynamic genetic sequences.Multiplication by “copy-and-paste”AAAAA5’ UTR ORF1 ORF23’ UTRASPMajor epigenetic remodeling takes place in germ cell and embryo development.PGCs BlastocystFertilizationextraembryonic tissueepiblastFrom Burns & Matzuk, “Rewriting and its Risks” in Preimplantation Embryo Development, Knobil and Neill’s Physiology of Reproduction, 3rd Ed., 2006.TotalGenome MethylationZygoteRetrotransposons can act as insertional mutagens“Accidental Discoveries” of LINEs as Agents of Disease LINELINEcompromise transcriptional elongation splicing disruption“gene breaking”/pre-mature polyALINEregulatory/epigenetic effectsdisrupted ORFDisrupted Genes Disorder Insertion Site Inserted Elements ReferenceCYBBCGD Exon L1 Ta Meischl et al. (2000)Intron Brouha et al. (2002)F8Haemophilia A Exon L1 Ta Kazazian et al. (1988)L1 preTa Kazazian et al. (1988)F9Haemophilia B Exon L1 Ta Li et al. (2001)Mukherjee et al. (2004)HBBthalassemiaExon L1 Ta Divoky et al. (1996)Intron Kimberland et al. (1999)Tumor suppressors are down-regulated in neoplasia in the context of broad genome hypomethylationModified from Melki & Clark (2002). Normal Cell Cancer CellHypermethylation of tumor suppressorsOverall hypomethylationTumor Evidence for LINE hypomethylation Reference breast cancer 5’ flanking sequences of hypomethylated L1Hs elements isolated by MSP iPCR Alves, et al. 1996.chronic myeloid leukemia (blast phase)methylation-specific PCR of primary samples; hypomethylation associated with ↑ BCR-ABL mRNA, resistance to tyrosine kinase inhibitorsRoman-Gomez, et al. 2006. chronic lymphoid leukemiaprimary specimens analyzed by HpaII digest and Southern blot Dante, et al. 1992.colorectal adenocarcinoma compared to neighboring normal colon; alternate MSI progression pathwayEstecio, et al. 2007. hepatocellular carcinoma hepatocellular carcinomas compared to surrounding, cirrhotic liver; Hpa II restriction enzyme digestTakai, et al. 2000.pancreatic endocrine and carcinoid tumorscompared to surrounding tissue; LINE hypomethylation correlates with lymph node metastasis, cytogenetic aberrationsChoi, et al. 2007.prostate cancer compared to surrounding tissue; hypomethylation associated with Gleason grade, clinical stage, and cytogenetic abnormalitiesSantourlidis, et al. 1999; Schultz, et al. 2002; Cho, et al. 2007.urothelial carcinoma appreciated by Southern blot or MSP-PCR in most specimensNeuhausen, et al. 2006.Miki, et al. Cancer Research 1992.BglII PstI MspI HindIII EcoRIChr5:112203758-112203784 A known case of colon cancer associatedwith APC mutation by a somatic LINE insertionchr4; human genome sequencing projectReducing the haystack:Seeing the transcriptionally active T(a)LINE subsetBoissinot & Furano, 2005.AAAAAAACAL1fossil L1 elementspolymorphic L1 elementsWhere are the transposons? TIP-chip strategyTiling Array:masked featuremicroarray slideL1 T(a)LINE mapping strategy: Vectorette PCRR1R2R3R1 R1R2R3R32kbLINE mapping strategy: Vectorette PCREnzyme Total Coverage1 9 7 8 6 8 6 7 5 5 923456Covering 3 billion base pairs of the human genomeTotal Length = 29-- 7 3 3 5 4 5 5 4 16-- -- 1 3 4 5 1 3 1 21-- -- 1 3 2 -- 0 0 4 25-- -- 1 3 1 -- 0 0 -- 28-- -- 0 -- 1 -- 0 0 -- 29Comparison to the reference genomelocation of a T(a)LINE in the referencePartial overlap of mapped T(a)LINEs with the human genome reference6,932,52711,641,35011,863,05633,335,61634,746,98543,049,91845,334,45649,615,81954,160,94456,745,09563,148,05665,272,70967,180,02472,523,64573,829,56175,459,36175,866,87976,328,40576,340,93877,583,29880,989,51485,496,58594,744,09898,150,743111,115,626118,453,435119,776,693120,225,018120,498,092124,905,894129,910,188140,342,653141,399,674143,237,654150,255,511154,398,622154,583,236PM S DPM S DP MS DInsertion carrierInferred heterozygoteNo insertionThe T(a)LINE TIP-Chip is a robust genotyping toolPMSDGaussian Distribution of Background and Foreground Valueslog2 scaleReference peakOther peaksNoise estimateFrequencyLN scaleModeling Peak Shape0 1 2 3 4 56 7 8 9 10 11 12 13 32Strong signalWeak signalAbsent signalKilobases1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2121 states x 2 peak directions = 42 peak states + 1 background state = 43 statesLISALINE Insertion Signal AnalysisRanking of 34 known L1 insertionsignals on the X chromosomeReference (Ta)L1are polymorphicAbsentPresentPM S DPM S DFinding novel T(a)LINE insertions6,932,52711,641,35011,863,05633,335,61634,746,98543,049,91845,334,45649,615,81954,160,94456,745,09563,148,05665,272,70967,180,02472,523,64573,829,56175,459,36175,866,87976,328,40576,340,93877,583,29880,989,51485,496,58594,744,09898,150,743111,115,626118,453,435119,776,693120,225,018120,498,092124,905,894129,910,188140,342,653141,399,674143,237,654150,255,511154,398,622154,583,236UpDnDnUpH H A P P P P P A P A P PLINE methylation is relaxed in germ cells and preimplantation embryos as well as in some types of cancer. • LINE insertions arising in the former can


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Bloomberg School BIO 624 - Burns

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