Repetitive DNA Part 2 - Transposable Elements

Lecture 1 Recap• Eukaryotic genomes show a broad range of sizes

• Much of this variation is due to repetitive DNA content

• Some repetitive DNA - telomeres, alpha satellites - are functional, structural components of genomes

• Mini- and microsatellites have no known function

• Microsatellites are used in forensic and paternity cases

• Transposable elements are repeats found across the genome

• TEs drive their own replication and are a source of mutation in genomes

Aims & Objectives• Aims: To learn about the two main classes of transposable element, how the classes move within the genome, how TEs interact with their hosts and how TEs are used in research

• Objectives: At the end of this lecture you should know:

• The basic structure and transposition mechanisms of the major TE groups

• The mechanisms by which TEs cause harmful mutations

• The role of TEs in human genetic diseases

• How TEs may promote beneficial mutations

• How TEs are used in research

Transposable Elements• Divided into two major classes based upon their

transposition mechanism

• Class I elements transpose via an RNA intermediate - they are subdivided into:

LTR Retrotransposons (including LTR retroviruses) Non-LTR Retrotransposons SINEs• Transposition is always replicative

• Class II elements transpose as a sequence of DNA, often by a ‘cut-and-paste’ mechanism

• Transposition can be either replicative or non-replicative

Class I TEs

• Class I elements - retroelements - transpose via a RNA element

• Most retroelements have two open-reading frames (ORFs)

• Gag - Encodes proteins which form viral-like particles

• Pol - Encode enzymes involved in replication & transposition

Ty1 virus-like particlesMobile DNA, MH Kazazian, 2011, Pearson Education

HIV-1 virion

Types of Retroelement

LTR Retrotransposon (e.g. Ty1) Retrovirus (e.g. HIV)

Endogenous Retrovirus (e.g. gypsy)Non-LTR Retrotransposon (e.g. L1)

SINE (e.g. Alu)

The central dogma of molecular biology

• DNA is TRANSCRIBED to mRNA• mRNA is TRANSLATED to an amino acid chain = protein

• You will learn much more about this process in detail in the Molecular Biology modules in both 1st and 2nd year.

DNA→RNA→ProteinRetroelements reverse transcribe RNA to double stranded DNA

1. Expression ofelement

RNAelement

2. Reverse transcription

RNA/DNAhybrid element

3. RNA degradation& DNA synthesis

Double stranded DNA element

4. Integration

Active TE Daughter Element

Retrotransposon Lifecycle

Transposition is replicative - copy number increases with the generation of daughter elements

LTR-LTR RecombinationLTR LTR

LTR

LTR

LTR

LTR

Recombinationbetween LTRs

Solo LTR remainsin chromosome

Extrachromosomal element circle

• Retrotransposons have no innate excision mechanism• LTR retrotransposons can be partially excised through recombination• A solo LTR remains in the host chromosome after LTR-LTR recombination• In many genomes (e.g Saccharomyces cerevisiae) solo LTRs outnumber full-length elements

SINEs - the parasite’s parasite

Alu elements (green) in the human genome

• Alu in the human genome transposes using the Pol of L1 (a non-LTR retrotransposon)

Short Interspersed Elements

• Some short retrotransposons do not encode any proteins

• They transpose using the enzymes of other retrotransposons• Shorter than most other TEs - <500bp• Originally transcribed from short RNA molecules• Can create recombination hotspots in the genome (Witherspoon et al., 2009, BMC Genomics 10:530)

• Alu has ~1,500,000 copies in the human genome - 11% of the genome

• Evolved ~90 million years ago from 7SL RNA

• Most copies are inactive

Class II Elements - Transposons

CTATCCCGAT…….GATAGGGCTA

….ATCGGGATAG TAGCCCTATC

• Class II element - transposons - transpose as DNA

• They encode a Transposase (Tnpase) which drives their movement

• Most transposons are flanked by Inverted Terminal Repeats (ITRs)

• ITRs are essential for transposition and bind the Tnpase

• Transposition can be replicative or non-replicative

Cut-and-Paste Transposition

1. Excision 2. Integration

3a. DNA repair withouta template - non-replicative

3b. DNA repair with the sister-chromatid template - replicative

TE 1

TE 1

TE 1

TE 1

TE 2

Transposon Transposition• The cut-and-paste mechanism restricts the increase in copy number compared to retrotransposons

• Unlike retrotransposons, transposons can be excised from the genome• Conserved ITRs are necessary transposition

• Elements with null mutations in their tnpase can still transpose if they have intact ITRs - if there is another source of Tnpase in the cell • Autonomous elements express their own Tnpase for transposition• Non-autonomous elements cannot express Tnpase and require the presence of autonomous elements in the genome in order to transpose

• Ds is a non-autonomous element• Ac is an autonomous element

Multiple Choice Questions

Transposition1) Retrotransposons only undergo replicative transpositionsa) True b) False

2) Retrotransposition involves both the replication and degradation of nucleic acid molecules

a) True b) False

3) Non-autonomous transposons encode a functional Transposasea) True b) False

4) SINEs area) Autonomous b) Non-autonomous

Structure1)The terminal repeats of transposons and retrotransposons run in the same orientationa)True b) False

2) Transposition always involve the production of a virus-like particle?a) True b) False

TEs & Natural Selection• In species such as Drosophila and Saccharomyces TE insertions are found at a low frequency in the population

• If TE sites were neutral we would expect to see insertions at low, medium and high frequencies

• This suggests that TEs are harmful to their hosts and evolving under natural selection

• Selection may operate against TEs through a variety of mechanisms

• Genomes have evolved defenses against TE activityDNA methylation - silences expression of TEs RNARNAi - TE transcripts are degraded to prevent translation of proteinsRIP mechanism - introduces null mutations into multicopy elements

• TEs are in a state of genomic conflict with their host

Selection Against Insertion Mutations

TE inserts into the geneExpression knocked outTE provides cryptic splice sitesTE creates a premature stop codonTE provides additional coding DNA

TE inserts adjacent to a geneExpression knocked outExpression up-regulatedExpression down-regulated

Selection Against Ectopic Recombination Mutations

TE 1 TE 2A B C

TE 1 TE 2TE 1 TE 2

TE

TE TE 2A’ B’ C’

TE 1A B C

TE 1 TE 2C B ATE 1 TE 2

A B C

TE 2 TE 1C B A

Deletion

Duplication

Inversion

Selection Against Ectopic Recombination Mutations

TE TE

ChromosomeA

ChromosomeB

TE TE

TranslocatedChromosomes

Translocation

Selection Against The Transposition Process

• The presence of TEs in a genome provides a metabolic burden to the host

• A lot of research has been undertaken to determine which is the dominant mode of selection (e.g Carr et al., 2002, Chromosoma 110: 511-518)

• All three mechanisms probably contribute to the control of TE proliferation

All TEs require the host to express proteins to drive transposition Retrotransposons require the production of RNA daughter

elements Excision of transposons results in double stranded DNA

breaks•The greater the number of TEs the higher the metabolic burden imposed upon the host

Human Genetic Diseases & TEs

• ~80% of visible mutations in Drosophila are caused by TEs

•Only a few hundred active TE copies are present in any human genome - mainly the retrotransposons L1 and Alu

• Approximately 60-70% of the human genome is made from TE DNA

• 0.1% of mutations in humans are caused by TEs

• TE induced mutations can occur in either the parental germline or an individual’s somatic cells

• L1 insertion mutations have been associated with over 30 human genetic diseases - see next slide

• Haemophilia is frequently caused by recurring L1 mutations in the factor VIII gene (Kazazian et al., 1988, Nature 332:164-166)

Chen et al. (2005) Hum Genet 11: 411-427

Human Genetic Diseases & TEs

• Many tumours have L1 mutations in oncogenes (Carreira et al., 2013, FEBS Journal 281: 63-73)

• Recent research has shown that nightime light in shiftwork activates L1

Carreira et al., 2013, FEBS Journal 281: 63-73

• L1 expression is upregulated in most human tumours

• How might this effect tranposition activity?

• Alu activity will increase with the availability of L1 Pol

• Direct link between shiftwork and cancer (de Haro et al., 2014, NAR 42: 7694-7707)

They’re Not All Bad!• TEs can cause beneficial mutations

• TEs can provide coding DNA or regulatory regions for host genes

• Syncytin - human placental gene derived from a retrovirus (Mi et al., 2000, Nature 403: 785-789)

• LTRs provide tissue-specific promoters for some human genes - their influence can affect genes over 50kb away

• Many of the genes which utilise LTRs are involved in reproduction or development

• TEs have been involved in the evolution of insecticide resistance in some species of fly (Rostant et al., 2012, Adv Genet 78: 169-201)

• Many genomic rearrangements caused by TEs are neutral and contribute to reproductive isolation between species

Horizontal Transfer• Closely related families are often present in the

genomes of closely related species• Closely related families are occasionally present in the

genomes of distantly related species

• The P element - a transposon - was discovered in D. melanogaster in the 1970s

• No close relatives of D. melanogaster have P elements

• The distantly related D. willistoni has almost identical P elements

• P jumped from D. willistoni to D. melanogaster in the early 20th Century

• Lab stocks of D. melanogaster caught in the early 20th Century have no P elements

Examples of Horizontal Transfer Between Closely Related Species

Chimpanzee Sooty Mangabey

D. willistoni D. melanogaster

Horizontal Transfer• Horizontal transfer is a mechanism which allows TEs to

invade new a host• The mechanisms which facilitate horizontal transfer are

unknown - viruses have been shown to be involved in some cases (Gilbert et al., 2014, Nature Communications 5: 3348)

• For horizontal transfer to be successful an active TE must integrate into the germline

• Horizontal transfer can be limited if the TE interacts with host factors during transposition

• A TE in a new genome faces fewer host defences and may transpose at a high rate until such defences have evolved

• The contributions of vertical and horizontal transfer are not known in TE evolution

• Species-specific TE complements show that horizontal transfer is not so common that it homogenizes TEs across species

TEs As A Research Tool• Molecular biology research has been revolutionised by

the use of TEs as a research tool• TEs - including retroviruses - can be used to introduce genes into a chromosome - transgenics

• Introduced TEs can be used to ‘knock-out’ genes

• Other, non-TE, transgenic techniques are available

• Microbes were the first organisms to be genetically modified• Bacteria are now modified by transgenics to produce insulin, clotting factors and human growth hormone to treat human diseases• The role of TEs in transgenics will be expanded upon in Applied Molecular Genetics in the Final Year

P-Element Transgenesis

Plasmid containing a modified P-element (black). The P-element contains a ~6kb fragment of Drosophila genomic DNA containing the gene rosy

Rubin & Spradling, 1982, Science 218: 348-353 • Rubin and Spradling inserted the wild type rosy gene into Drosophila using a modified P-element present in a plasmid• They used flies with a rosy mutation• The plasmid was injected into mutant fly embryos

• The P-element transposed into the fly chromosomes and recovered the wild type eye phenotype• The recovery was stable as it was inherited in the chromosomes

Enhancer TrapsHost GeneEnhance

r

Host GeneEnhancer

Insertion of non-autonomousenhancer trap reporter gene

Why can’t we use a TE which has autonomous copies present in the genome?

Reporter gene expression driven by host gene enhancer

18-wheeler in a developing Drosophila head

Why do we need a marker gene?

Piggy-Bac Transgenesis

Corby-Harris et al., 2010, PLoS Pathogens 6: e1001003

• piggy-Bac has begun to replace the P element as a choice of vector for transgenics

An of example of piggy-Bac in transgenics

• Corby-Harris and colleagues introduced a piggy-Bac system into Anopheles mosquitoes

• Originally identified in the Cabbage Looper moth

• The pBac element contains a marker gene (DsRed) and a kinase transgene (Akt)• Over-expressed Akt retards the development of malaria parasites in Anopheles

• Anopheles life expectancy is also reduced

Multiple Choice Questions

Selection & Mutation1)Low frequency insertions indicate TEs are evolving under natural selection.a)True b) False

2) Selection against insertion mutations involves genomic re-arrangements.a)True b) False

3) Nightlights activating which TE are associated with tumour formation? a)Alu b) Ty1 c) L1 d) gypsy

4) Translocations always result in deleterious mutations True b) False

5) Insertion mutations always result in deleterious mutations True b) False

6) Mammal genomes employ DNA methylation against TEs - how does this work?a)Introduces premature stop codonsb) Targets TEs for excision by restriction enzymesc) Silences expression of TE transcripts

Multiple Choice Questions

Horizontal Transfer1)In which direction did the P element undergo horizontal transfer?a) D. melanogaster to D. willistoni b) D. willistoni to D. melanogaster

2) HIV2 was transferred to humans from which primate?a) Chimpanzee b) Sooty mangabey c) Allen’s swamp monkeyTransgenics1)Which type of gene shows a transgene construct has integrated into a host genome?a) Reporter gene b) Marker gene

2) Enhancer traps show tissue-specific expression patterns of genesa)True b) False

3) Transgenes can be used to recover wild type phenotypes in mutant organismsa) True b) False

Summary• Eukaryotic genomes contain many forms of retrotransposon

- all of which undergo reverse transcription to generate daughter elements• Retrotransposons have no excision mechanism, but LTR elements can be partially lost through recombination

• Transposons move as DNA, but transposition does not always result in an increase in copy number

• TEs cause mutations through a variety of mechanisms

• TE mutations are often deleterious, but can be beneficial

• TE mutations are associated with numerous human diseases

• Modified TEs are a powerful tool in molecular biology research

• Horizontal transfer allows TEs to jump between species and invade new hosts