L8: Tandem repeats & Repeat expansion disorders Flashcards
What makes up the majority of our genome?
Repetitive DNA that includes TEs and related sequences (44%)
What are the two main categories of repeated elements?
tandem repeats and dispersed repeats
What are the three types of tandem repeats?
Tandem paralogues
Satellite DNA
rDNA
What are the three types of satellite DNA?
(Macro)Satellites
Minisattelites
Microsattelites
What are the four types of dispersed repeats?
Paralogues
Transposons
tDNAs
Retro(pseduo-)genes
How can transposons be further categorised?
Class I:
LINEs
SINEs
LTR retrotransposons
Class II:
DNA transposons
What is the difference between the different satellites?
Their motif length:
(Macro)Satellites: (>100bp)
Minisattelites (10- 100bp)
Microsattelites (1-9bp)
What is the max length of satellite DNA?
They are very large arrays of repetitive DNA – each repeat typically kilobases long. Satellite DNA can extend over megabases of DNA but its maximum length is unknown
Give some other names for tandem repeats
Variable number tandem repeats VNTR (most frequently used when referring to minisatellites)
- Simple repeat
- Short tandem repeat (STRs)
- Simple sequence repeats (SSRs)
(most frequently used when referring to microsatellites)
Where are macro satellites most commonly found?
They are most commonly found at centromeres and in heterochromatin (cytologically dense material that is typically found at centromeres and telomeres)
Typically, how long are minisatellites and how often do they repeat?
Range in length from 10-60 base pairs, typically repeated 5-50 times.
How many minisatelites are there in the human genome?
More than 1000 locations in the human genome
How variable are minisatellites?
Highly variable between individuals in terms of repeat length
Why would a sequence be classified as a mini satellite?
Repeat sequence is 10-100, repeats 5-50 times and the number of repeats varies between people but the motif itself does not.
Typically, how long are microsatellites and how often do they repeat?
- Motifs range from one to ~six base pairs
- Motif typically repeated 5-50 times
What are the most common motif lengths for microsattelites and why?
Often 3-6 so no frame shift
How variable are microsatellites?
- Very high mutation rate compared to other regions in the genome
- Highly variable between individuals
What are the consequences of this variability in microsatellies?
Often pathological
In what ways can tandem repeats vary
- Tandem repeats are highly variable in the number of repeat copies
- They vary between individuals but can also vary within an individual – for example in different cell types or tissues
When was DNA fingerprinting first developed and how was it done?
Developed in the 1980s, Originally used restriction enzymes to fragment DNA and then a southern blot to detect fragment length
What do modern DNA fingerprinting techniques utilise?
PCR
What structural effects can short tandem repeats have on DNA?
STRs are able to form secondary DNA structures such as G quadruplexes. Tandem repeats are very complimentary and are likely to repeat these secondary structures
G-quadruplex (G4) structures are only one of many (ten or more) non-B-form DNA secondary structures analysed to date. Briefly describe three well-studied structures
Z-DNA: In contrast to standard B-form DNA (B-DNA), Z-DNA is a left-handed helix. Z-DNA motifs (that is, sequences that form Z-DNA in vitro) are tracts of alternating purines and pyrimidines. Negative supercoiling stabilizes the formation of Z-DNA under physiological salt conditions130, and it is hypothesized that Z-DNA relieves transcription-induced torsional stress
Cruciform structures: Negative supercoiling can also cause B-DNA to adopt a four-armed, cruciform secondary structure. These structures require ≥6-nucleotide inverted repeats (cruciform motif) to form, and such motifs are located near replication origins, breakpoint junctions and promoters in diverse organisms
Triplex DNA: Three-stranded triplex DNA occurs when single-stranded DNA forms Hoogsteen hydrogen bonds in the major groove of purine-rich double-stranded B-DNA. Triplexes in which the third strand is antiparallel to the DNA duplex can form at physiological pH, and these structures are stabilized by negative supercoiling
What secondary structures can form in CAG, CTG, and CCG repeats?
Hairpins of the As Ts or Cs, meaning in this structure they have no pairing. This can happen in both odd and even repeats although the pattern differs slightly in odd repeats.
What secondary structure can form in GAA repeats?
Triple helice formed by (GAA)n repeats
What secondary structure can form in CCG repeats?
Tetraplex structures; G4s- unwinding this structure takes a lot of force
Name three models for STR repeat expansion
- Replication slippage model
- Double strand break repair model
- Transcription mediated model
Describe the replication slippage model
During replication of a repeat-containing sequence, the replication machinery may pause on the lagging strand, due to secondary structures or other kinds of lesions.
Partial unwinding of the lagging strand may lead to replication slippage when replication restarts, giving rise to an expansion or a contraction of the repeat tract, depending on what strand (template or newly synthesised strand) slippage occurred.
Alternatively, partial unwinding of the lagging strand may lead to lesion bypass by homologous recombination with the sister chromatid, also leading to contractions or expansions of the repeat tract
Describe the double stranded break model
Following a DSB, gene conversion is initiated by strand invasion, forming a “D-loop.” (two strands of a double-stranded DNA molecule are separated for a stretch and held apart by a third strand of DNA)
DNA synthesis within the repeat tract may be faithful or associated with slippage. After capture of the second end of the break, DNA synthesis of the second strand may also be faithful or associated with slippage.
Slippage events will lead to expansions of the repeat tract or to contractions if slippage occurs on the template strand.
Alternatively, after capture of both ends followed by DNA synthesis, the two newly synthesized strands may unwind and anneal with each other in frame or out of frame, leading to expansions or contractions of the repeat tract, however it is more biased to expansion than contraction.
Describe transcription mediated repair
Transcription through CAG·CTG repeats promotes the formation of slipped-strand structures, which subsequently stall RNA polymerase (RNAP) and lead to recruitment of the nucleotide excision repair (NER) machinery. Transcription-coupled NER removes the portion of the transcribed strand containing the RNAP-blocking hairpin; the resulting gap is filled in using the non-transcribed strand (NTS) as a template. Depending on the location of loops on the NTS relative to the removed hairpin, the repair event will either expand or contract the trinucleotide repeat.
Why are STRs difficult to study using PCR?
The underlying properties of STRs make them difficult to study using PCR based methods, they show up in laddering fragments during gel electroporesis
What else can be problematic about studying STRs?
Mapping/ sequencing of STRs is also problematic, As the similarity between two copies of a repeat increases, the confidence in any read placement within the repeat decreases.
What is making the studying of STRs easier?
New technologies like PacBio and Nanopore are making the study of tandem repeats more feasible as they can read a whole long strand of DNA
Besides telomers and centromeres, where are tandem repeats often found in the vicinity of?
Tandem repeats are frequently found in the vicinity of genes; 10% to 20% of coding and regulatory sequences in eukaryotes contain an unstable repeat tract. 15% of human promoters have a TR in close proximity
Name three things tandem repeat variation has been linked to in different species
- Rapid variation in microbial cell surface
- Tuning of internal molecular clocks in flies
- Dynamic morphological plasticity in mammals.
Where is there a particularly high density of microsattelites in the human genome?
Transcriptional start sites
What relationship is seen in these TRs and TSS? (2)
There is a correlation between TR polymorphism and gene expression variation. Genes with a promoter-associated TR showed significantly higher variation in both expression and DNA methylation levels. This effect was more pronounced for genes with highly polymorphic promoter TRs- More variability in TRs, more variability in gene expression and methylation.
What functional consequences have been observed for this TR-gene relationship?
Some TR lengths correlate with quantitative traits: Correlation shown between specific features of the dog snout (curvature and length) with the ratio of length between two TRs in Runx-2, a gene that regulates bone formation
Give five potential mechanisms for gene expression regulation by TRs
- Overlap with regulatory protein binding sites
- Chromatin structure
- Z-DNA formation
- Spacing of promoter elements
- RNA structure
What relationship is seen with repeats and TFs?
Repeats surrounding TF binding sites increase TF binding, More repeats, higher TF binding; did not have to match binding site
When do repeat expansion disorders (REDs) occur?
REDs occur when a simple repeat expands over a certain threshold. Different diseases have different pathogenic ranges. Some disorders have a pre pathogenic range where its not healthy, you might have some symptoms but it is not in the pathogenic range
Name four repeat exapnsion disorders
Friedrich’s ataxia
Fragile X
Huntington’s
Spinocerebellar ataxia
Name four pathogenic mechanisms of REDs
- Gene silencing
- RNA binding protein sequestration
- Toxic gain of function
- Repeat associated Non-AUG (RAN) translation
How can gene silencing come about?
The expanded repeat causes a significant reduction in the expression of a nearby gene. This can happen through the formation of secondary structures or through the induction of epigenetic changes: methylation, promote the formation of heterochromatin etc
Name two diseases in which repeat expansions are associated with gene silencing
- Friedrich’s ataxia (When CGG over 200Bp, methylation occurs and it can silence genes )
- Fragile X syndrome
How can TRs result in RNA binding protein sequestration?
Transcripts containing the repeat expansion bind with greater affinity to RNA binding proteins (RBP) than those without. By binding to the RBP, they reduce the available RBP for functional transcripts
What disease is associated with RBP sequestration?
Myotonic Dystrophy type 1 (DM1): The DM1 pathogenic state includes the sequestration of Muscleblind (MBNL) proteins by toxic DM Protein Kinase (DMPK) transcripts, CUG RNA-Binding Protein Elav-Like Family Member 1 (CELF1) protein activation, and fetal-like splicing patterns.
How may there be a toxic gain of function?
In Huntington’s disease and other polyglutamine (polyQ) disorders, mutant proteins containing a long polyQ stretch are well documented as the trigger of numerous aberrant cellular processes that primarily lead to degeneration and, ultimately, the death of neuronal cells. However, mutant transcripts containing expanded CAG repeats may also be toxic and contribute to cellular dysfunction.
Name two ways that repeats can expand (as a trend)
Intergenerational (anticipation)- can expand over generations
Somatic (mosaicism)- can expand with age
What is meant by antagonistic pleiotropy?
When a gene or genomic element can have multiple roles which are both beneficial and detrimental to an organism
Describe one case of antagonistic pleiotropy
- Sickle cell anaemia - Homozygous: blood can’t carry oxygen; Heterozygous: bad at carrying oxygen but resistance to malaria- quite high in African regions
- Huntington’s disease
How could STRs have evolved?
Kept around because they’re useful but can also lead to pathology after a threshold
Describe a RED in NOTCH2NL
NOTCH2NLC & neuronal intranuclear inclusion disease (NIID): a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. It can also result in essential tremor and a large range of disease phenotypes.
Individuals can present with repeat expansion, some purer, some with interruptions. This can determine what kind of disease phenotypes based on the length and interruptions in the repeat. The phenotypes are linked to the size and purity of the repeat.
How do the repeat types correlate with phenotypes?
Generally, the repeat size of muscle weakness-dominant is largest, and parkinsonism-dominant is smallest. Dementia-dominant and essential tremor-dominant usually have a purer GGC repeat.
What paradoxical trend can be seen in repeats in NIID? What disease is this similar to?
Individuals with a repeat number over a certain threshold do not get ill – instead there is evidence of silencing once the repeat reaches a certain threshold; Similarities with FXS/FTAXS
What is FXS/FTAXS?
Fragile X syndrome/ Fragile X-associated tremor/ataxia syndrome
How prevalent is fragile X syndrome?
Fragile X syndrome affects ~1 in 7000 males
What are some common physical symptoms of fragile x?
Prominent, broad forehead
Large ears
Long face
Strabismus (squint)
Prominent Jaw, Dental
Crowding high arched palate
Murmer/ mitral valve prolapse
Hollow chest
Hypotonia/ Joint Laxity
Scoliosis
Macro-orchidism
What cognitive symptoms present with fragile X syndrome?
Autism
Intellectual disability
What is FXS caused by?
Fragile X Syndrome is caused by a CGG repeat expansion in the FMR1 5’UTR
What is the normal FMR1 gene responsible for? What is the normal amount of repeats?
5-40 CGG repeats
Brain development, dendritic function
Regulation of protein-translation in neurons
What is seen when there are too many repeats? How many is too many?
> 200 CGG repeats
Induces hypermethylation and a loss of the FMR1 protein
Fragile X phenotype
Abnormal dendritic development
No translational inhibition
Like NIID, there are a number of molecular phenotypes, what do these correspond to?
Healthy: x30 CGG- normal amount of FMRP
Premutation: x50-200 CGG- More transcripts, lower amount of FMRP (?)
Methylated, full mutation: >x200 CGG: No transcripts
Name two disorders arising from premutation length alleles
Fragile X-Associated Primary Ovarian Insufficiency (FXPOI)
Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS)
What are the symptoms of Fragile X-Associated Primary Ovarian Insufficiency (FXPOI)?
- Causes infertility & early menopause in adult women
- Women stop having menstrual cycles before 40 years of age
- Higher risk of having children with FXS
What are the symptoms of Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS)?
- Neurodegenerative disorder of the nervous system
- Can cause tremors and problems with walking, balance, memory and mood disorders
How frequent is Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS)?
More common in men (X-inactivation)
1 in 450 men have the mutation
1 in 3000 men over 50 affected
What is Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) through to be caused by?
Thought to be caused by toxic RNA product
What is another molecular phenotype not mentioned previously?
People who have the full mutation but it is not methylated and so it behaves like the premutation phenotype- even higher transcripts, lower protein. Some individuals escape methylation at the expanded repeat
What is seen in mouse models?
Mice do not gain methylation at the expanded repeat but still get ataxia symptoms
What did these findings lead Grace towards considering?
Is there a primate specific mechanism responsible for the methylation of the FMR1 5’UTR repeat expansion?
How is Grace looking to investigate this methylation?
Using unmethylated (UME) carriers she is investigating the cause of the CGG methylation in FXS.
What did Grace look at regarding these repeats?
She noted that KZNFs can bind disease associated simple repeats and that KZNFs are recruiters of histone and DNA modifying enzymes.
She then broadened the candidate list to include a range of epigenetic modifiers such as DNA-methyltransferases, gene promoter binding KZNFs, methylcytosine dioxygenases, histone demethylases and histone methyltransferases.
(Their observations kick started collaborations with FXS experts in Rotterdam and Rome)
What did RNA-Sequing transcripts reveal?
RNA-Sequencing revealed truncated transcripts unique to the UME carrier- UME stringtie transcripts. They also observed a change in the transcriptional landscape; there was increased expression in FMR1 carriers.
What else did they see regarding their earlier considerations?
Surprisingly we saw some differences in the expression of epigenetic modifiers, this was backed up by results from the second biological replicate from Italy. TET3 was dysregulated in both carriers- UME carriers have significantly reduced TET3 expression levels. qPCR backed this up in a third biological replicate
So, is TET3 a candidate? Why?
TET3 is a strong candidate:
Robust evidence for a downregulation of TET3:
* Across 3 different cell lines
* Using both qPCR and RNA-seq
What is the relevance for FMR1 methylation?
The TET enzyme family are able to convert 5mC to 5hmC; they can associate to the chromatin using a methylation sensing domain. Their core catalytic domain can sense (CG) domains with substrate preserence and can undergoe catalytic activity. 5hmC is a stable form of methylation found in the brain
