12 7 2 David A Ray -3 -8myweb.ttu.edu/daray/posters_etc/DRay_TTU_Nov2014.pdf · TE activity is variable among mammals Analyze and compare the TE landscape/activity and piRNA repertoire

Department of Biological SciencesTexas Tech University

David A Ray-8

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2

7

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The Ray laboratory Genome evolution on a broad taxonomic scale Transposable elements (TEs)

TE dynamics and small RNAs TEs, genomic innovation and miRNAs piRNAs and defense against TEs

Other ongoing projects Questions

Model taxa are great but…▪ Inbreeding▪ Taxonomic diversity▪ ‘Real world’ experience

How applicable are the findings from model taxa to the broader world?▪ Genome-level questions▪ Taxonomically meaningful clades▪ In the ‘real world’

DNA sequences with the ability to relocate within a genome

Pol II or III transcription

Reverse transcription and insertion

1. Usually a single copy or a few ‘master’ copies

2. Transcription to an RNA intermediate

3. Reverse transcription and integration

Double strand break in donor DNA

Transposon w/ ORF

Pol II transcription

Transposase

Translation

Donor

Target

TEs have been implicated in numerous aspects of genome evolution, diversity, functionality.

“A substantial proportion of … eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.” - Mikkelson et al., 2007.




TEs are powerful mutagens and their impact must be reduced….

but they also represent valuable sources of potential innovation.

TEs and miRNAs

TEs and piRNAs

But before I can tell you those stories, I have to tell you this story.

Vesper bats Second most species rich mammalian family (44

genera, ~400 species) World wide distribution Simple, non-descript phenotypes Simmons (1998) – no defining, derived morphological feature

unites Vespertilionidae. Myotis lucifugus, M. davidii, M. brandtii, & Eptesicus

fuscus

Lack & Van De Bussche (2010)Hoofer et al. (2003)

No other mammalian genome so far examined has similar Class II activity levels…

that includes other bat families

human

armadillo

hedgehog

mouse

rabbit

vesper bat

Prosimians

HumanMacaque

100100 6565 4040 MYMY2525~150~150 //

MouseRat

Dog

Vesper bats

Non-vesper bats

00

Marmoset

29 fam.74K copies

11 fam.23K copies

85 fam.284K copies

~ 20 fam.231K copies

25 fam.49K copies

23 fam.47K copies

5 fam. 7K copies

DNA transposons form natural hairpins and are frequently exapted as miRNAs Maruspials – Devor et al. (2009) Primates – Piriyapongsa et al. (2007) Humans – Ahn et al. (2013)

DNA transposons have been recently active in vesper bats DNA transposons have been exapted as miRNAs in other mammals Have DNA transposons been exapted as miRNAs in vesper bats? If so, what impacts are associated? Myotis lucifugus – unavailable Eptesicus fuscus – Another vespertilionid, genome draft released in 2012, TE

landscape is mapped, local

CHAPTER III

E. fuscus M. lucifugus

Genome contributions by TEs, <40 my

To whom to we compare?

No other mammalian genome so far examined has similar Class II activity levels

human

armadillo

hedgehog

mouse

rabbit

vesper bat

Sequenced the small RNA repertoires of dog, horse and Eptesicus.Sequenced the transcriptome from the same taxa.Identified putative miRNAs and their potential target transcripts

323

212

388

341

Novel miRNAsNovel

miRBase

Eptesicus

661

Dog Horsen = 661 n = 535 n = 729

TE derived miRNAsClass I

Class II

Non-TE356

138

165 62

460

13 55

39

635

Dog 184 (2.2/MY) 44 (0.5/MY)Horse 339 (4.1/MY) 56 (0.7/MY)Eptesicus 396 (4.8/MY) 242 (3.0/MY)

miRNAs TE derived

Artibeus & Miniopterus do not contain DNA transposons Pritham & Feschotte (2007), Ray et al. (2007, 2008), Thomas et al. (2011), Pagan et al. (2012)

Have DNA transposons been exapted as miRNAs in vesper bats?

Sho ‘nuff

Target genes identified for 38 bat specific miRNAs

Categorization of GO terms reveals enrichment for:Transcription

• 22 genes, P=0.0000068, FDR=0.01• 11 DNA transposon derived miRNAs

Ubiquitin dependent protein catabolism• 7 genes, P=0.0001, FDR=0.1• 2 DNA transposon derived miRNAs

Some DNA transposon derived miRNAs appear to be functional in regulating transcription

Ex: TE thrust, Epi-Transposon, Horizontal transfer, CASP

DNA transposon derived miRNAs coincides with vespertilionid diversification.

Thus, miRNA formation via DNA transposon activity is a potential explanatory mechanism for the diversification of Vespertilionidae and worth further investigation.

Diversification TE Activity

Million Years Million Years

Perc

ent o

f gen

ome

MyotisEptesicus

Million Years Million Years

Myotis

Eptesicus

25 SpeciesSplit from sister genus ~15 MYA

103-113 SpeciesSplit from sister genus 16.2 MYA

Lineage specific DNA transposon activity in the early vesper bats have been exapted into the miRNA pathway

miRNAs are enriched for involvement in transcription

miRNA exaptation coincides with a period of rapid radiation

Suggests a mechanism to explain rapid diversification

Can a similar pattern be observed in other example taxa?




Some TEs can transfer horizontally to unrelated genomes, thereby invading new ‘habitats’

TEs can cause significant genome instability

How do genomes protect themselves against new TE invasions and TE activity?

piRNAs (PIWI-interacting RNAs)• No defining structural features• ~24-32 nucleotides long• 5’ U bias in first position• Organized in clusters• Expressed in a developmentally regulated mannerPIWI proteins• Members of the Argonaute gene family• RNA binding • Endonuclease / slicer activities • Most abundant in male germ cells• Responsible for gene silencing • Cleaves RNA transcripts • Can methylate DNA• Use piRNAs as guides

How do genomes protect themselves against new TE invasions and TE activity?

Genomic piRNA clusters can act as TE traps, recruiting novel TEs as sources of piRNAs

The ‘ping-pong’ cycle can modulate the strength of the piRNA response so that it matches the TE challenge

Transcription of active TE (sense transcript)

Genomic TE locus

Ping-pong model


Genomic TE locus

PUAGGCAUUGCAUGGGCAUUAAGCUGGAC1°piRNA/MILI complex

1° processing

Ping-pong model


Genomic TE locus


1° processing

Targeting of complementary

transcriptPUAGGCAUUGCAUGGGCAUUAAGCUGGAC

1°piRNA/MILI complex5’..GUCCCGAAAUUGCGUCCAGCUUAAUGCCCAUGCAAUGCCUACGAUGGAACGUCCGCAAGUU..3’

Ping-pong model


Genomic TE locus


1° processing



pGCAAUGCCUAGCAUGGAACGUCCGCAAGU

2°piRNA/MILI complex

1°piRNA/MILI complex5’..GUCCCGAAAUUGCGUCCAGCUUAAUGCCCAUGCAAUGCCUACGAUGGAACGUCCGCAAGUU..3’

Ping-pong model


Genomic TE locus


1° processing





1°piRNA/MILI complex5’..GUCCCGAAAUUGCGUCCAGCUUAAUGCCCAUGCAAUGCCUACGAUGGAACGUCCGCAAGUU..3’ pGCAAUGCCUAGCAUGGAACGUCCGCAAGU

2°piRNA/MIWI2 complex

Nuclear translocation and methylation-induced

TE silencing

Ping-pong model


Genomic TE locus


1° processing






5’..AACUUGCGGACGUUCCAUCGUAGGCAUUGCAUGGGCAUUAAGCUGGACGCAAUUUCGGGAC..3’

5’..GUCCCGAAAUUGCGUCCAGCUUAAUGCCCAUGCAAUGCCUACGAUGGAACGUCCGCAAGUU..3’



pGCAAUGCCUAGCAUGGAACGUCCGCAAGU2°piRNA/MIWI2 complex


TE silencing


transcript

Ping-pong model


Genomic TE locus


1° processing






5’..AACUUGCGGACGUUCCAUCGUAGGCAUUGCAUGGGCAUUAAGCUGGACGCAAUUUCGGGAC..3’


5’..GUCCCGAAAUUGCGUCCAGCUUAAUGCCCAUGCAAUGCCUACGAUGGAACGUCCGCAAGUU..3’



pGCAAUGCCUAGCAUGGAACGUCCGCAAGU2°piRNA/MIWI2 complex


TE silencing


transcript

Ping-pong model

TE activity is variable among mammals Analyze and compare the TE landscape/activity and piRNA

repertoire from three non-model Laurasiatherians Test specific predictions of the ping-pong model piRNAs would be expected to preferentially target younger TEs TEs that are actively transcribed should elicit much more robust

piRNA response

What about DNA transposons, which lack an RNA intermediate? Are they also targeted by piRNAs? Do they feed the ping-pong cycle?

Sequenced the small RNA repertoires of dog, horse and Eptesicus.Identified putative piRNAsMapped the putative piRNAs and their TE counterparts

EptFus1

0

0.5

1

0 10 20 30 40 50

0

0.5

1

0 10 20 30 40 50

0

0.5

1

0 10 20 30 40 50

DNA

LTR

LINE

SINE

% o

f gen

ome

MY ago

TE activity < 50 mya

Dog Horse Bat

Total 65.8 69.4 18.3Collapsed 5.6 8.4 2.624-32 bp 4.8 7.2 2.0Mapped 2.7 (62%) 4.0 (55%) 1.3 (65%)

Single mappers 2.6 (53%) 3.5 (47%) 1.1 (55%)

0

0.8

1.6

15 17 19 21 23 25 27 29 31 33 35

U G C A

0

0.6

1.2

15 17 19 21 23 25 27 29 31 33 350

1.25

2.5

15 17 19 21 23 25 27 29 31 33 35

x106x106 x105

piRNA length

Leng

th a

bund

ance

Dog Horse Bat

All values in millions

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 200

0.050.1

0.150.2

0.250.3

0 5 10 15 20

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20

5-UACAGGACUACGGAUAGACACGAG-3

3-GACAGCCUGAUCAAAUGUCCUGAU-5

5-CUGUCGGACUAGUUUACAGGACUACGGAUAGACACGAG-3

Overlap length

frequ

ency

BatHorseDog

Primary piRNA

Secondary piRNA

Dog

Horse

Bat

-8

-3

2

7

12

-8

-3

2

7

12

-8

-3

2

7

12

5’ ORF1 ORF2 3’

0

0.5

1

0 20 40

0

0.5

1

0 20 40

0

0.5

1

0 20 40

LINE

% o

f gen

ome

MY ago

LINE activity < 50 mya

piRNAs should map more densely to TEs that have more recent

activity in the genome

Some nice correlations in

some cases but not all

More recently active elements should correlate with increased

ping-pong pairs

TE terminology has been sloppy Accumulation ≠ activity Accumulation = successful insertion of a transcribed

retrotransposon or the successful duplicative integration of a DNA transposon

Activity = rate of transcription

0

0.5

1

0 10 20 30 40 500

0.5

1

0 10 20 30 40 50

0

0.5

1

0 10 20 30 40 50

% o

f gen

ome

SINE accumulation < 50 mya

Generally, the more actively expressed elements are targeted by PIWIs and piRNA. However, Similar levels of SINE expression in dog and horse, yet

higher piRNA densities in horse SINEs Historical/current abundance of TEs may not reflect

current TE expression Instead, abundance of TEs may reflect the differential

success of piRNAs in silencing TEs




The International Crocodilian Genomes Working Group (ICGWG) Director – David Ray Genome Assembly/Analysis – Ed Green Education Outreach – Ed Braun

www.crocgenomes.org Objectives:

Draft genomes of Alligator mississippiensis, Crocodylus porosus, and Gavialis gangeticus

Annotation based on transcriptome data, de novo transposable element analyses, and comparison to other vertebrates

Resources free to the crocodilian and broader genomics communities to facilitate research

Coordinate research associated with the overall project and other researchers to avoid duplication of effort

Full genome assemblies for three crocodilians Among vertebrates, crocodilians have some of the slowest rates

of molecular change

bird

s

frog

s

coel

ocan

th bird

s

mam

mal

s

croc

odili

ans

turt

les

Among vertebrates, crocodilians have some of the slowest rates of molecular change Multiple whole genome alignments that included 23

species’ genomes

Crocodile vs. alligator▪ 93% identity across the genome▪ 90 my divergence▪ 93% identity b/t human and macaque▪ 23 my divergence

Genome pair Percent ID

Alligator, crocodile 92.9%

Crocodile, gharial 95.7%

Alligator, gharial 93.4%

• Evolution of insect TE control• Within an explicit phylogenetic context….

• Do piRNA repertoires track TE landscapes?• Is argonaute gene family evolution related to

historical TE activity?

Graduate students Neal Platt Mike Vandewege Heidi Pagan Christine Lavoie

Postdoctoral associates Meganathan Ramakodi Neal Platt

Co-PIs Federico Hoffmann ICGWG members

http://www.myweb.ttu.edu/darayhttp://www.crocgenomes.org

Documents

12 7 2 David A Ray -3 -8myweb.ttu.edu/daray/posters_etc/DRay_TTU_Nov2014.pdf · TE activity is variable among mammals Analyze and compare the TE landscape/activity and piRNA repertoire