Mapping mobile DNA elements: Sources of human genetic diversity and disease.

Kathleen H. Burns, M.D., Ph.D.kburns@jhhmi.edu

Johns Hopkins Department of Pathology

Overview

I. 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 analysis

IV. Future directions

The (junk) Genome

Data from Jasinska & Krzyzosiak, FEBS Letters (2004).

55% of the human genome is repetitive

45% of the human genome is comprised of interspersed repeats

non-repetitive

tandem repeats

21% of the human genome is comprised of long interspersed LINEs

LTRs (HERVs)

SINEs (Alu)

LINEs are dynamic genetic sequences.Multiplication by “copy-and-paste”

AAAAA5’ UTR ORF1 ORF2 3’ UTR

ASP

Major epigenetic remodeling takes place in germ cell and embryo development.

PGCs BlastocystFertilization

extraembryonic tissue

epiblast

From Burns & Matzuk, “Rewriting and its Risks” in Preimplantation Embryo Development, Knobil and Neill’s Physiology of Reproduction, 3rd Ed., 2006.

TotalGenome

Methylation

Zygote

Retrotransposons can act as insertional mutagens“Accidental Discoveries” of LINEs as Agents of Disease

LINE LINE

compromise transcriptional elongation splicing disruption

“gene breaking”/pre-mature polyA

LINE

regulatory/epigenetic effectsdisrupted ORF

Disrupted Genes Disorder Insertion Site Inserted Elements Reference

CYBB CGD Exon L1 Ta Meischl et al. (2000)

Intron Brouha et al. (2002)

F8 Haemophilia A Exon L1 Ta Kazazian et al. (1988)

L1 preTa Kazazian et al. (1988)

F9 Haemophilia B Exon L1 Ta Li et al. (2001)

Mukherjee et al. (2004)

HBB thalassemia Exon L1 Ta Divoky et al. (1996)

Intron Kimberland et al. (1999)

Tumor suppressors are down-regulated in neoplasia in the context of broad genome hypomethylation

Modified from Melki & Clark (2002). Normal Cell Cancer Cell

Hypermethylation of tumor suppressors

Overall hypomethylation

Tumor 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 inhibitors

Roman-Gomez, et al. 2006.

chronic lymphoid leukemia primary specimens analyzed by HpaII digest and Southern blot Dante, et al. 1992.

colorectal adenocarcinoma compared to neighboring normal colon; alternate MSI progression pathway Estecio, et al. 2007.

hepatocellular carcinoma hepatocellular carcinomas compared to surrounding, cirrhotic liver; HpaII restriction enzyme digest

Takai, et al. 2000.

pancreatic endocrine and carcinoid tumors

compared to surrounding tissue; LINE hypomethylation correlates with lymph node metastasis, cytogenetic aberrations

Choi, et al. 2007.

prostate cancer compared to surrounding tissue; hypomethylation associated with Gleason grade, clinical stage, and cytogenetic abnormalities

Santourlidis, et al. 1999; Schultz, et al. 2002; Cho, et al. 2007.

urothelial carcinoma appreciated by Southern blot or MSP-PCR in most specimens Neuhausen, et al. 2006.

Miki, et al. Cancer Research 1992.

BglII PstI MspI HindIII EcoRI

Chr5:112203758-112203784

A known case of colon cancer associatedwith APC mutation by a somatic LINE insertion

chr4; human genome sequencing project

Reducing the haystack:Seeing the transcriptionally active T(a)LINE subset

Boissinot & Furano, 2005.

AAAAAAACAL1

fossil L1 elements

polymorphic L1 elements

Where are the transposons? TIP-chip strategy

Tiling Array:masked feature

microarray slide

L1 T(a)

LINE mapping strategy: Vectorette PCR

R1

R2

R3

R1 R1

R2

R3R3

2kb

LINE mapping strategy: Vectorette PCR

Enzyme Total Coverage

1 9 7 8 6 8 6 7 5 5 9

2

3

4

5

6

Covering 3 billion base pairs of the human genome

Total 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 -- 29

Comparison to the reference genome

location of a T(a)LINE in the reference

Partial overlap of mapped T(a)LINEs with the human genome reference

6,932,52711,641,35011,863,05633,335,61634,746,98543,049,91845,334,45649,615,81954,160,94456,745,09563,148,05665,272,70967,180,02472,523,64573,829,56175,459,36175,866,87976,328,40576,340,938

77,583,29880,989,51485,496,58594,744,09898,150,743

111,115,626118,453,435119,776,693120,225,018120,498,092124,905,894129,910,188140,342,653141,399,674143,237,654150,255,511154,398,622154,583,236

P M S D P M S DP M

S D

Insertion carrier

Inferred heterozygote

No insertion

The T(a)LINE TIP-Chip is a robust genotyping tool

P

M

S

D

Gaussian Distribution of Background and Foreground Values

log2 scale

Reference peak

Other peaks

Noise estimateFre

quen

cy

LN scale

Modeling Peak Shape

0 1 2 3 4 5 6 7 8 9 10 11 12 13 32

Strong signalWeak signal

Absent signal

Kilobases

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

21 states x 2 peak directions = 42 peak states + 1 background state = 43 states

LISALINE Insertion Signal Analysis

Ranking of 34 known L1 insertionsignals on the X chromosome

Reference (Ta)L1are polymorphicAbsent

Present

P M S D P M S D

Finding novel T(a)LINE insertions

6,932,52711,641,35011,863,05633,335,61634,746,98543,049,91845,334,45649,615,81954,160,94456,745,09563,148,05665,272,70967,180,02472,523,64573,829,56175,459,36175,866,87976,328,40576,340,938

77,583,29880,989,51485,496,58594,744,09898,150,743

111,115,626118,453,435119,776,693120,225,018120,498,092124,905,894129,910,188140,342,653141,399,674143,237,654150,255,511154,398,622154,583,236

Up

Dn

Dn

Up

H H A P P P P P A P A P P

LINE methylation is relaxed in germ cells and preimplantation embryos as well as in some types of cancer.

• LINE insertions arising in the former can cause heritable disease.• Relaxed LINE silencing may have an underappreciated roles in

tumor progression.

We have developed a comprehensive mapping method for the most active LINEs in the modern genome. This is a robust method for genotyping known insertions and identifying novel insertions.

• This has immediate pertinence to understanding common polymorphisms and heritable disease.

• The approach is expected to have special utility in comparing tumor and normal DNA.

Summary

Applications of total genome transposon profiling in cancer research.

• Comprehensive T(a)LINE mapping to compare tumor and normal tissue.• Correlate findings with markers of epigenetic T(a)LINE silencing.• Development of HERV-K mapping strategies.• Transposon mapping for identifying causes of familial cancer susceptibility

syndromes.

Nimblegen HD2 platformTotal probes 2.1 millionProbe length 50 - 75mer (ChIP-chip whole genome tiling)Feature size 13μm x 13μmArray size 62mm x 14mm Slide size 1” x 3” (25mm x 76mm) glass

Future Directions

Center for High Throughput Biology• Jef Boeke, Ph.D. & the Boeke Lab• Cheng Ran Huang • Tejas Niranjan

Department of Oncology• Curt Civin, M.D. & the Civin Lab

Institute of Genetic Medicine• Dave Valle, M.D.

Acknowledgements

Funding:• Burroughs Wellcome Foundation• National Cancer Institute • Sol Goldman Pancreatic Cancer Research Center• Goldhirsh Brain Tumor Research Foundation