Genomic Imprinting Flashcards
How are Imprinted genes expressed - generally
Monoallelically in a parent of origin specific manner
Describe imprinted genes
Typically = 2 copies of each gene, one copy of each gene on each homologs, amount of mRNA and expression depends on both copies
IMPrinting = only one or other expressed= parent of origin dictates what allele expressed, only pat or mat
What is imprinting an ex of
Classic epigenetic effect = stably maintained - for a while (not always life long or in every cell), broadly distributed
Describe early evidence for imprinting in mammals - exps
Mouse embryos constructed by pro nuclear transfer exps = need male and female pronuclei to develop
If have 2 fem nuclei = no extra embryonic tissues, partial dev, not viable
If have 2 male nuclei = poor embryonic dev = no viable
Exps = 1980s
Other species can do this = parthenogenesis- NOT mammals
Describe early evidence for imprinting in mammals - do you need both male and female contributions to the embryo
YUHHHHHHHHHH
What does imprinting account for
Non equivalence of maternal and paternal genomes
Newer exp = reveals that imprinting is why cannot use 2 same pronuclei alone
Oocyte nucleus in which 2 maternally silenced genes have been activated = turn these 2 genes on =rescues dev = overcomes lack of imprinting and creates fatherless mice
Where are imprinted genes typically found
In large clusters = tend to form domains or clusters
What are imprinted gene clusters controlled by
Imprinting control regions = ICRs
Region important for whole pattern, specific master control region
Ig-dmr = differentially meyhtlated region - part of ICR
What happens when delete icr
Deletion of icr on paternal allele disrupts the expression pattern for entire cluster of genes
Name and describe functions of imprinted genes - 4
(Share functional characteristics)
1- tend to have high levels of expression in prenatal stages
2- growth and metabolism, neurological development, developed of placenta
3- clinical relevance = genetic disorders involving defective imprinting
4- imprinting is often disrupted in cancer cells - epigenetic disregulation
Describe evolutionary origins of imprinting - emergence
Emergence of imprinting linked to that of the placenta-marsupials (yolk like placenta) and placental mammals have imprinting
MONOTREMEs do not (mammals that do not have placenta)
Describe evolutionary origins of imprinting - parental conflict hypothesis - gen
Imprinted genes reflect competition between males for limited female resources
Describe evolutionary origins of imprinting - parental conflict hypothesis - Mat and pat
If a single female mates with multiple males, males want to ensure their offspring outcompete those from competitor males whereas the female wants to allocate ressources to all offspring evenly - regardless of dad
Males look out for own genetic interest
Describe evolutionary origins of imprinting - parental conflict hypothesis - conclusions
Posits that maternally expressed genes are growth restricting for fetus, opposite for paternally expressed genes - growth promoting (not all genes but most fit the idea)
What is the key epigenetic mark for imprinting
DNA meth
Imprinting starts with dna meth
What are imprinting genes usually associated with
CpG islands that are differentially methylated on maternal and paternal alleles - between (= differetially methylated regions or dmrs) = CORRESPOND TO ICRs defined genetically
How is imprinted established - icrs
By dnmt3a - enzyme, in germ line
Maintained by dmnt1
When is imprinting erased
During germ cell migration to developing gonads, erased at specific stage - during germ cell dev
What else is involved with gene imprinting - mods
Not just dna meth
Histone mods, non coding RNA’s also involved - recent examples of imprinted genes not associated with DMR but instead marked with H3k27 methylation
Describe the 2 instances of global reprogramming of dna meth in dev
Period of global epigenetic remodeling = all and meth removed
Happens when germ cells (pcgs) migrate to genial ridge = E9.5-11.5= all dna meth erased
Imprinting established after this
When does imprinting establishment occur
During germ cell dev after genome wide erasure of dna methylation
DNA CpG methylation erased genome wide in pcgs = diff timings tho for male and female
Describe timing of imprinting
Male = immediately after = E13.5
Female = bit longer, not until embryo post natal = P21
Set own pattern of imprinting - best to do here since erased all other, so can establish patterns
Name/describe the 2 types of imprinting mechanisms involving dna methylation
Maternally methylated
Paternally methylated
How establish methylation = male vs female
describe the MAT types of imprinting mechanisms involving dna methylation
Maternally methylated DMRs and ICRs located at promoters and block promoter function - occurs at CpG islands
describe the PAT types of imprinting mechanisms involving dna methylation
Paternally methylated dmrs and icrs are located between genes and affect enhancer function - more distant
Less studied
Describe Igf2-h19 imprinted domain - gen
Domain with 2 genes = igf2 and h19 next to each other in genome
Mat = enhancer downstream h19 genes, no igf2 but yes h19
Pat = igf2 but no h19 - differential methylation between the 2 genes = h19 not transcribed
Supports the theory - growth vs non growth - mat/pat
What is igf2
Insulin like growth factor expressed fro paternal allele
What is h19
Non coding rna expressed from mat allele
What happens when igf2 ko
Have reduced size at birth but only if get ko allele from father - bc imprinted gene - inherited from mom = no phenotypes since not expressed
What is an enhancer
DNA element that binds sequence specific tfs and activates transcription of genes in same vicinity on chromosome
What does igf2-h19 icr have
Enhancer blocking activity
Ex= normally enhancer can turn on neomycin resistance gene, but if put icr between E and neo = does not work anymore
Insertion of icr region between a heterologous enhancer and promoter prevents enhancer activity
What is an insulator
Insulates gene or promoter from enhancer = basis of how icr establishes pattern of imprinting
What regulates binding of insulator protein ctcf
Methylation of icr
Describe when icr methylated vs not methylated and relationship with ctcf
When icr unmethylated - mat - blocks enhancer from activating igf2 (through ctcff binding)
Methylation of ice in pat prevents ctcf from binding and allows enhancer to work
What is function of ctcf
Important chromosome organizing factor that regulates the formation of loops in chromosomes
Can influence long range, interactions between diff elements on chromosome
Ctcf function very sensitive to methylation as well
Describe methylation of icr and regulation of ctcf on mat
Ctcf binds = what gives you blocking between enhancer and promoter
Enhancer blcoking depends on dna binding protein= ctcf
Turns on h19 but not igf2 since icr region there
Describe methylation of icr and regulation of ctcf on PAT
Icr methylated so ctcf cannot bind anymore
= enhancer turns on igf2 = no enhancer blocking function anymore
What does maternal specific methylation typically do
Typically silences promoters of long non coding rnas located INSIDE protein coding genes
Describe igf2r gene in MAT
Icr in middle of igf2r gene = in coding region of gene
Igf2r = gene encoding igf2 receptor, gets expressed from mat alleles specifically
Methylation occurs here on mat allele
= non coding rna not transcribed since methylated = tgf2r expressed
Describe igf2r gene in PAT
Unmethylated in pat = mtheylation regulates a diff promoter for a non coding rna that gets transcribed within igf2r gene =
Non coding rna transcribed in opp direction and starts in middle of igf2r gene = igf2r cannot be expressed at Same time
Describe kcnq1 gene in MAT And PAT
Mat = me at icr in middle of gene = fine transcription
Pat = Icr = not methylated = antisense rna
Do not know how its affecting other linked genes nearby
What is incRNA
Transcribed antisense to igfr2 and kcnq1
How does IncRNA transcription lead to silencing within imprinted domain
Conserved way = hidden promoters- act of turning on promoter interferes with gene transcription= act of antisense transcription interferes with sense transcription (especially if it crosses the sense promoter)
Rna itself recruits chromatin modification enzymes that silence transcription (like kcnq1ot1 rna recruits prc2 )- in some cases = non coding rna produced has consequences for cell function
Describe kcnq1ot1 rna recruits prc2 - effect of this
Complex = target non coding rna can dictate region = seems to be related to how the effects might be able to spread to other regions of chromosome = rna can interact with and activates other epigenetic remodeling complexes that can spread and act on adjacent genes = some evidence,
One target for this = prc2 complex = non coding rna in this region can dictate fcuntion of prc2 in this region
What context is imprinting established in
Context of different genome wide dna meth patterns in developing germ line
How established differential meth = has to do with dna meth patterns in developing oocyte vs sperm
Histone mods help set up these patterns
Describe oocyte dna me pattern
Low in promoters
Specific enrichment in bodies of genes transcribed in developing oocyte
- maternal icrs = in middle of gene bodies
Oocyte primed by overalls genome methylation pattern
Describe sperm dna me pattern
Less specific
Low in promoters
Present in most/all gene bodies
Present in intergenic regions
Pat icr always in intervening regions, = lots of methylation = some priming established by something that happens in sperm broadly
Where is dna methylation specifically maintained in zygote - describe situation
At icrs in zygote
Have to be maintained for a long time through many cell divisions during embryonic dev
Have to be able to survive global epigenetic remodeling period
DNA methylation erased genome wide but imprinted methylation maintained
When = During pcgs migration- allows imprinting to be established then at zygote to 2/8 cell stage = removes dna meth, not complete since imprinted regions maintained
How is dna methylation specifically maintained at icrs in zygote
Factors that bind to icrs- dna binding protein
H3k9 methylation - important role
Describe zfp57 in imprinting maintenance- gen
DNA binding protein
Binds specifically to when it’s methylated = protects methylation
All found in same place - by chip seq = icrs, sfp57, setdb1, h3k9me3
All found in same place = all set up by zfp57 being able to bind these
Describe zfp57 in imprinting maintenance- specifics = fam? Where binds? Recruits?
Member of zinc finger family of dna binding proteins
Specifically binds to methylated TGCCGC at icrs (protects methylation at that site, specific sequence found, rich in c and g)
Recruits h3k9 methyltransferase setbdb1 = to protect
You discover a new species of mouse with large ears that talks in a high pitched voice. Propose a way that you could identify the imprinted genes in this species. Assume you will need to use a genome-wide profiling method in adult tissue. What do you think will be the biggest challenge you will encounter?- exp ideas
Create mat and pat and see if passes down
Hardest part = figuring out which is pat or Mat allele
Can do rna seq - but still wont know, finding specific CpG islands in middle of gene - do not know if mat or pat but could guess its an icr and look closer
Describe identification of imprinted genes using genomics - gen
Best bets are either RNA-seq (RNA levels) or whole genome bisulfite sequencing (DNA methylation) BUT…
* Need to be able to experimentally distinguish maternal and paternal alleles at many genes
* Difficult to achieve in human studies
Describe identification of imprinted genes using genomics - exp
Mouse studies involving crosses of two different inbred strains have been the most valuable-in these strains individuals are homozygous for virtually all genetic markers, but the markers are different across strains
Offspring heterozygous for many genetic markers, so can distinguish paternal and maternal alleles by sequence!
Gen = way male or fem contribute - dif, make them diff enough so have markers that can distinguish - use inbred lines = so can distinguish - off spring heterozygous for all the makers = can distinguish since sequence diff
How do you go from genomics to imprinted genes
RNA-seq (RNA levels) or whole genome bisulfite sequencing (DNA methylation) will give you genes that show monoallelic expression (from only the maternal or paternal allele) or monoallelic DNA methylation
How do you know that these patterns arise from imprinting (= something that is really due to parent of origin?)
Mechanism depends on parent of origin so do same exp and switch mom and dad between strains - pattern of imprinting willl be switched = track parent of origin
If imprintin= monoallelically expressed genes should now be expressed from other allele
Name 4 developmental disorders linked to imprinting
Prader willi and angelman syndromes
Beck with wiedemann and silver russel syndromes
Describe clinical phenotype of angelman syndrome
Happy disposition, laughter, widely spaced teeth, wide mouth, stiff upheld arms, broad stance
Describe clinical phenotype of prader willi syndrome
Central obesity, short stature, small hands and feet, mild facial dysmorphism
Describe what PWS and AS are typically caused by - gen
Deletion of entire imprinted domain on chromosome 15 - leads to dev problems, defect in region of chrom
Involves deletions that affects all or most of entire region of chrom 15
Whole bunch of genes expressed in an imprinted way - parent of origin specific way
Genetic = effects at an imprinted region can have very divergent phenotypes dependent on transmission from mom or dad
What specifically causes Angelman syndrome
Maternal transmission of deletion (UBE3A)
What specifically casues prader wili syndrome
Paternal transmission (due to SNORD116)
Describe clinical phenotype of beckwith wiedemann syndrome
Macroglossia - big tongue, ear creases
Neonatal gigantism - wight of babies is in 98th percentile
1000 fold increased chance of developing tumours
What are BW and SR caused by
Effects of multiple diff imprinted genes misexpressed and no genetic cause
Epigenetic errors
Loss of imprinting at kcnq1 and igf2 loci, other imprinted loci can be affected as well
Caused by epigenetic errors that property establish imprinting - basis not known
Why does BW and SR happen?
Some underlying genetic Basis maybe - undiagnosed genetic cause
Maybe environmental factors
(Sporadic)
Describe imprinting and ART
Could art impinge on these mechanism of imprinting
Some studies= expression of imprinted genes affected by art
Some evidence that BWS increases incidence of Art
Potential environmental effects
