HHMI HOWARD HUGHES MEDICAL INSTITUTE

Studies of a Human Studies of a Human Retrotransposon

Moran LabD f H G i /HHMIDepartment of Human Genetics/HHMIUniversity of Michigan Medical School

OutlineOutlineBackground• Background

• L1 retrotransposition can create structural i ti i th h variation in the human genome

• L1s can retrotranspose via an alternative h b l DN “l ”pathway by exploiting DNA “lesions”

• Active L1s are quite prevalent in the human population• Conclusions

Transposable Element Derived Repeats in Human DNAp p

Example % of Genome

• Use DNA intermediates– Transposons Mariner ~3%

• Use RNA intermediates– LTR-containing retrotransposons HERVs ~8%g p

– Non-LTR-containing retrotransposons

• Autonomous LINEs ~21%

• Non-Autonomous SINEs ~13%

Lander et al. 2001 (Nature 409, 860-921)

L1 Structure and Retrotranspositionp

ORF1EN RT

ORF23’UTR A

5’ UTR C

Transcription

An

5 'An

Integration by Reverse Transcription

An Active Human LINE-1 Element

ORF1 ORF2ACA

ORF1EN RT

ORF23’UTR An

5’ UTR C

• Comprise ~17% of human DNA; most elements areimmobile. However, some are “full-length” and are capablemm . H w , m f g pof retrotransposition.

• The L1-encoded proteins also can mobilize certain cellularRNA ( Al l U6 RNA d RNA)RNAs (e.g., Alu elements, U6 snRNA, and messenger RNA)

• L1 can act as a mutagen in both germ and somatic cells(~1/250 human mutations result from de novo L1 mediated(~1/250 human mutations result from de novo L1-mediatedretrotransposition events)

Potential Ways L1 Insertions Can yAffect Gene Expression

Hulme et al., Genomic Disorders, 2006

SD SASD SA

Post-insertional Non-allelic Recombination EventsSD SASD SA

pASD SASD SA X

L1 L1

pA

Unequal Non-Allelic HomologousDNA Recombination

SDSD SA SD SASA

ADuplication pA

SD SA

Duplication

pADeletion

Hulme et al., Genomic Disorders, 2006

An Assay for L1 Retrotransposition

SD SAEP R REP-AEN RT C

Transcription

An

SD SA

Splicing

5’ An

SD SA

Splicing

5’ AnReverse Transcription

Integration

An

REP+

REP

An

Moran et al. Cell, 1996

Hoechst

Hoechst

Hoechst

OCT4

TRA 1-60

TRA181Hoechst TRA 1-81

Moran et al., Cell, 1996; Ostertag et al. NAR 2000; Garcia Perez et al. HMG 2007; Beck et al., ARGHR, 2011, Richardson/Garcia Perez, unpublished

A Model for L1 Retrotransposition

Beck et al., ARGHG, 2011

L1 Retrotransposition can L pCreate Structural Variation

i th H Gin the Human Genome

Retrotransposition of the Rescue Vector

ColE1

NEO

An3’

UTR5’

UTR C

No DNA 103 104 105 105

~1/20

Rescue vector/L1.3JM101/L1.3Gilbert et al., Cell, 2002

The Rescue Procedureu u

Extract genomic

Digest DNAgenomic

DNA

Ligate DNA Transform E.coliand select for KanR

Characterizeplasmids & compare g and select for KanR p asm s & compar sequence to HGWD

Gilbert et al., Cell, 2002

Rescued L1 StructuresColE1

NEO

6An3’

UTR5’

UTR C

6817

An

3’UTR

3’

C

C 1773’

UTR

nA3

UTRC

CChimeras 7An

UTR

nA3’

UTR 2C

C

100nA

Gilbert et al., Cell, 2002; MCB 2005

cDNA Mediated Recombination(4 i t f ll h t i d)(4 instances fully characterized)

Endogenous L1Endogenous L1

Endogenous L1

XX

Chimera

Inversion/deletion Chimera with Partial Duplication(2 events)

L1

5’5’An

~115 kb 5’3’

L1 5An

3’RT dissociation Recombination

5’L1

Recombination

5’

5’An

L1 3’3’

5’ An

604-626 bp duplicationResolution

L1 L1

L1 5’L1

5’

An

An

2 bp microhomology

L1s can Retrotranspose via L pan Alternative Pathway by

l iti DNA “l i ”exploiting DNA “lesions”

EN-independent L1 Retrotransposition

SD SA

RTEN C

D205AH230A D702A

REP

WT EN EN RT EN /RT

REP

WT EN- EN- RT- EN-/RT-

CHO K1

4364A4364A

XRCC4-

XR122

Morrish et al. Nature Genetics, 2002; Morrish et al., Nature 2007

Model for EN-independent (ENi) L1 Retrotranspositionp

Wild type L1s• 5’ truncations

’ l ( ) l

EN mutant L1s• 5 ’truncations

3’ P l (A) t il 3’ t ti• 3’ Poly (A) tail• Integrates at EN cleavage

site (5’-TTTT/A-3’)• Variable size TSD

• 3’ Poly (A) tail or 3’ truncation• Integrates at an atypical EN cleavage site• No TSD (frequent deletions of target-

site nucleotides)• Insertions are accompanied by shortInsertions are accompanied by short

sequences that apparently are derivedfrom endogenous cDNAs

• Insertions in DNA-PKcs-deficient CHOcells integrate adjacent to ‘perfect’t l i t

TPRT EN-independent

telomeric repeats

Morrish et al. Nature Genetics, 2002; Morrish et al., Nature 2007; Kopera et al., PNAS, 2011

A i L1 Q i Active L1s are Quite Prevalent in Human GenomesPrevalent in Human Genomes

LINE-1 and Human Structural VariationLINE 1 and Human Structural Variation• Data from the human genome reference (HGR)

– The average human genome is estimated to contain ~80-100 retrotransposition-competent L1s

– Six highly active elements comprised >80% of activitySix highly active elements comprised >80% of activity(Lander et al. 2001, Brouha et al. 2003)

• Differential presence of retrotransposons is responsible for somehuman structural variation (Sheen et al 2000 Myers et al 2002 Boissinot et al 2004 Korbelhuman structural variation (Sheen et al. 2000, Myers et al. 2002, Boissinot et al. 2004, KorbelJO et al. 2007, Xing et al. 2009, Iskow et al., 2011; Ewing et al., 2011; Huang et al., 2011; Witherspoon et al., 2011and others)

• ATLAS (Badge RM et al. 2003)

– Low coverage of genome--allowed identification of L1s that are absentin the HGR

5’ UTR ORF1 ORF2 ACA

Principle of ATLAS

R RRR

An5’ UTR ORF1 ORF2

EN RT C

RB5PA2 RB3PA2

ACA

DigestionDigestion

Linker LigationLinker Ligation

L1

Linker LigationLinker Ligation

PCR (fill in)PCR (fill in)

Extension from L1 primer

L1

DenaturationDenaturation

Hairpin formationno amplification

Exponential amplificationX

***

Linear Amplificationwith labeled oligo

Fractionate by gel electrophoresis

* with labeled oligo

Badge et al., AJHG, 2003

ATLAS Patterns Segregate in a CEPH Family

MW FF FM F D S DD D D D S SS M MM MF C1 C2 C3 C4

500 b512 bp

MW

500 bpA

400 bpB

300 b300 bpC

D

3 of 7 full-length L1s were ‘hot’ for retrotranspositionBadge et al., AJHG, 2003

Hypothesis: L1s present at low allele f i i th b lk f frequencies comprise the bulk of

retrotransposition activity in humans

CollaboratorsEvan Eichler Evan Eichler

(University of Washington/HHMI)

Richard Badge Richard Badge (University of Leicester)

Christine Beck

Identifying Dimorphic LINE-1sFosmid Clones

HGR

~40Kb

~40Kb <40Kb >40Kb

~40Kb~40KbLINE-1?

Concordant Region Insertion in Fosmid Deletion in Fosmid

400050006000

Allele Specific Hybridization

LINE-1 Insertion in Fosmid?

Fosmid Fingerprint and

1650

3000

4000

2000

Allele Specific Hybridization

Southern Blotting

3’UTREN RT CORF15’UTR

Full-Length Dimorphic LINE-1s

g pSouthern Blot Direct Fosmid Sequencing/ATLAS

An

CO

G

Tuzun et al., Nat Genet, 2005; Kidd et al., Nature, 2008; Beck et al., Cell, 2010

ACA

Results of the Assay: ABC10

• Geographically diverse set of six individuals• Subtractive set of L1 dimorphisms• 37/68 are active in the cell culture assay (~32% in introns)• L1s frequently use polyadenylation signals located in 3’ genomic L1s frequently use polyadenylation signals located in 3 genomic

flanking sequences, leading to the retrotransposition of non-L1 sequences (also see Holmes et al., Nature Genet, 1994; Moran et al., Science, 1999)

Beck et al., Cell, 2010

Genomic Locations of L1 insertions

Beck et al., Cell, 2010

I

LRE3

RP

II

III

IV

V(pre-Ta)

Beck et al., Cell, 2010

A General Model for L1 Amplification in Human Genomes

Transduction sequence-for example RP, LRE3, or

pre-TAAn

An AnAn An

An An

Truncation inactivates the

majority of new L1s

An An

An An

An An An An

An An

Mutation or poorMutation or poor genomic context can inactivate new L1s

Successive Amplification

Beck et al., Cell, 2010

Conclusions• L1 retrotransposition can create structural

i ti i th h variation in the human genome• L1s can retrotranspose via an alternative pathway

b l DN “l ”by exploiting DNA “lesions”• Active L1s are underrepresented in the current

genome reference sequence and are quite prevalent in the human population (similar data f m th B ns D in nd K i n l bs)from the Burns, Devine, and Kazazian labs)

• L1s are a major source of inter-individual (and h i t i di id l) ti i tiperhaps intra-individual) genetic variation

Remaining Questionsg Q

• What cell types accommodate L1 • What cell types accommodate L1 retrotransposition?

• What host factors act to aid and/or restrict L1 • What host factors act to aid and/or restrict L1 retrotransposition?

• Do environmental factors impact L1 • Do environmental factors impact L1 retrotransposition?

Acknowledgements b ( ) b ( )Moran Lab (present)

Aurélien DoucetDiane Flasch

ll l

Moran Lab (past)Reid Alisch

Jyoti AthanikarBilly Giblin

Peter LarsonNancy Leff

Huira Kopera

Jyoti AthanikarRichard BadgeChristine Beck

José Luis Garcia-PerezNicolas Gilbertp

Nancy LeffYing Liu

Tomoichiro MiyoshiJohn Moldovan

Nicolas GilbertShuen HonAmy Hulme

Deanna KulpaTammy Morrish

Mitsuhiro NakamuraSandy Richardson

Tammy MorrishSheila Lutz

Wei Wei

CollaboratorsFunding

NIH, Keck Foundation, University of Michigan

Richard BadgeEvan EichlerFred Gage

Haig Kazazian y gCancer Center, HHMI, ACS

Haig KazazianJoAnn SekiguchiThomas StamatoGuillermo Taccioli