Imprinting and Epigenetics

Imprinting

• Chromosomes have epigenetic marks from the parent it originated from, which are not reprogrammed in early development
  • Most prominent in placenta and brain tissues, therefore linked to cognitive phenotypes, but not uniform across all tissues
• Azim Surani experimented on mice, transferring male and female pronuclei into unfertilized eggs
  • One paternal and one maternal pronuclei must fuse for live offspring
  • Modified mice genomes to add extra DNA sequences
  • Helped measure methylation transfer from parent to offspring
  • Males didn't pass on DNA methylation, females did
• Half of the DNA should come from each parent
• This is important for X inactivation and genomic imprinting because these genes are parent specific, and only one copy is turned on
• Figure 7.1 demonstrates the difference in a mammalian egg after haploid pronuclei sperm entry; the female pronuclei are larger than the male
• Figure 7.2 summarizes Azim Surani's mouse research: Only surrogates with both maternal and paternal pronuclei survived; others either died or were underdeveloped
• Figure 7.3 summarizes Surani's research, probing the Parent of Origin Effect (POE) through inserted DNA mice, showing parental imprinting caused the offspring to show methyl/unmethyl marks
  • Fathers passed on unmethyl marks, vice versa, independently of the parent's methyl marks, but determined by the parent passing down the inserted DNA

Parent of Origin Effect

• Way for the zygote and daughter cells to differentiate between chromosomes inherited from either parent
• Expression of a gene depends on whether the allele is paternally or maternally derived
• Genes are linked to complex traits, development, and diseases
• IAP retrotransposons are significant
  • Methylation marks aren't removed during the early zygote stage for repression
  • Examples: Axin fused mice and agouti mice genes
  • Upstream DNA methylation marks are passed from parent to offspring
  • RNA is blocked from disrupting neighboring genes by repressing this region
• Similar to imprinted genes, they don't get demethylated when forming a blastocyst

Transgenerational Inheritance

• Definition and significance: the passing on of epigenetic modifications from parent to offspring, which presents as an acquired characteristic
• The mechanism isn't based on DNA methylation alone because it cannot be long-term storage for programming genome
• Acquired traits aren't solely based on DNA sequences but through the epigenetic genome, which changes dynamically in response to the environment, stressors, and nutrition

Hydatidiform Mole

• An egg loses its nucleus but continues being fertilized by sperm, producing a diploid zygote with only paternal copies
• A tumor develops in the uterus because of the nonviable embryo
• Even with 46 chromosomes, no fetus develops due to the need of maternal pronuclei
• All methylation of pronuclei is erased for totipotent cells that can specialize into any cell type except placenta
• In blastocysts, ICM cells become pluripotent, creating embryonic stem cells
• At this stage, new epigenetic modifications are reprogrammed, dividing ES cells before cells specialize into body types

Carlberg section 3.3

• CTCFs function as insulators to insulate closed chromatin from open chromatin
  • Genomic loci separate genes in one chromatin region by binding to TF and enhancers of neighboring chromatin
  • Cohesins and CTCF are the main proteins binding these insulator regions
• Enhancer activity crossover is prevented, inhibiting heterochromatin region spread to active genome parts

CTCF-Bound Insulators

• Have two main roles:
  • Forms TADs and prevent cross border enhancer activity
  • Maintain the chromatin architecture of hetero or euchromatin regions, preventing the spread of silenced regions
• DNA methylation in CTCF function affects imprinting
  • ICRs are a subset of insulators that control monoallelic expression of >100 genes
• CTCF role in epigenetic memory:
  • About 30,000 CTCF binding sites exist, but only a few hundred sites control imprinting
  • Sensitive to methylation -- won't bind to highly methylated sites
  • Stabilizes higher order chromatin structures (DNA loops)
    • Small set of unmethylated CTCF binding sites keep proteins throughout the cell cycle (prevents de novo methylation)
  • CTCF mediated chromatin structures represent a heritable epigenetic memory component
• Imprinted genes occur in clusters
  • Some genes in the cluster are expressed from the maternal chromosome, and others are paternally dependent on DNA methylation
  • Chromosome 11p15 region contains protein coding genes (IGF2, KCNQ1, CDKN1C), and nc RNA genes (H19 and KCNQ1OT1) - See Figure 3.4
  • Shares ICRs and ncRNAs for coordinating POE within two ICRs (one unmethylated with CTCF bound, and the other methylated and unbound)
  • Maternal vs paternal controlled cell growth is controlled differently
• Maternal growth is limited; and Paternal is primed for max growth
• Females:
  • Post-implantation: CTCF binding is essential to maintain the hypomethylated state of ICR1 and protect if from methylation in oocytes
  • CTCF obstructs the communication with enhancers in the TSS region IGF2 gene, therefore allowing inititation of H19 transcription
    • Expression of H19, KCNQ1, and CDKN1C
    • Repression of IGF2 and KCNQOT1
• Males:
  • ICR1 is methylated which prevents CTCF binding
  • ICR2 is unmethylated and the effects are reversed
• ICR definition: Imprinting Control Region = Upstream region that controls methylation of DNA and gene expression (varies at each locus)
  • Binds to nearby histones to turn on gene
  • Allows for the addition of proper DNA methylation marks
    • Paternal genes are imprinted = methylated while maternal is not (vice versa)
    • ICR methylation are established to achieve the POE marks
  • Association with CTCFs - aid in maintaining the imprinted gene regions, stabilizing DNA loop structure/chromatin structure
    • Mediate monoallelic expression by restricting enhancer access, therefor maintaining expression

Bruce Cattanach Experiments

• Used mice with abnormalities in inheritance of chromosomes to understand epigenetic differences from either parent
  • Created mice to inherit two copies of a chromosome from one parent
    • Two male copies are larger, which increases the development
    • Two female copies are smaller, which stunts the development
  • Displayed how parental alleles are imprinted and contain different marks that regulate phenotype
• Figure 8.1 shows Cattanach's mice results display differences in mice phenotypically when inheriting 2 copies of one parent's chromosome 11 region, and normal 1:1 copies of the rest of the genome
  • Two maternal copies are smaller and less developed and two paternal copies were larger
• Parent of Origin Effects (POE)
  • Effects on gene expression are dependent on imprinting
• Chromosome 11 as an imprinted chromosome
  • Expression is promoted by paternal copy for placental growth
  • Maternal copy switches the chromosome off to limit growth therefore, balancing the male imperative
• Chromosome 7 (Igf2) and Chromosome 17 (Igf2r)
  • Related to development and growth of offspring (insulin like growth factor - in mice)
  • Igf2 promotes the embryonic growth of offspring, therefore being only expressed in the paternal allele
    • Mutations of this gene on the maternal copy = normal mouse
    • Mutations on paternal copy = smaller/ less developed
  • Igf2r suppresses growth of offspring preventing Igfr2 expression and is only expressed from the maternal copy

Angelman (AS) and Prader-Willi (PW) Syndromes

• Syndromes caused by imprinted genes on chromosome 15 when patients lost a small identical section from one parent when PGCs are not reprogrammed completely
• AS: sever mental underdevelopment, small brain, laughs randomly, and difficulty in speaking
  • Abnormal chromosome inherited from the mother
• PWS: Infants have floppy muscles, struggle when eating, and leads to obesity and mental delays when they eat a lot
  • Abnormal chromosome inherited by father
• Figure 8.2 is the Example of POE effect on chromosome 15
  • Abnormal chromosomes could be passed on from either parent → all children had the same deletion
  • The same deletion can be caused by different parents
• UPD is Uniparental Disomy where one parent passes on both copies of the chromosome that disrupts gene expression, therefore affecting imprinted regions
• In UBE3A an imprinted region of Chromosome 15 is crucial for normal brain function
  • Normally, male is methlyated while female is unmethylated
  • Mutation occurs during gamete production, escaping reprogramming when parents are normal
  • Two male copies are inherited or that both, results in lack of maternal gene = AWS

Beckwith-Wiedemann (BW) and Silver-Russell (SR) Syndromes

• Inheritance of imprinted genes on chromosome 11, p15 locus
• BW: copies of both the male and female are expressed (in females they should only be silenced)
  • Loss of methylation at ICR2 —> expression of KCNQ1OT1 nc RNA on both alleles and repression of CDKN1C
  • Over expression of IGF2 caused by deletions in ICR1 of maternal allele disrupting CTCF binding and loss of H19 expression
    • Overgrowth and high likelihood of kidney tumors
• SR: Both copies of the gene
  • Hypomethylation at ICR1 leads to biallelic expression of h19 and loss of IGF2 expression
  • Undergrowth and asymmetry
• Epigenetic diseases are the same and only have methylation of the DNA as their determinant
• ICRs controlled by methylation include:
  • ICRS bind histones
  • Upstream regions that control gene expression
• Expression and the effect of imprinting varies from tissue to tissue
  • Imprinting happens mainly in the placenta and brain cells
  • High imprinting in brain = post-natal war of sex or offsprings that drain mothers recources

Imprinting

• Figure 8.3 is related to the process where imprints are put (put imprint on, or take imprint off for both egg and sperm production)
• Shows somatic cells with parent imprinted genes that arise from fertilized zygotes
  • DNA methylation marks interrupted by Blimp1 gene stop cells from differentiating marks are removed
  • Primordial germ cell formation with reprogrammed marks produces either sperm or eggs
  • New modifications define cellular identity
• Significance of ICM in Development: The inner cell mass forms embryos by going through early development after the blastocyst phase
• Sequence of sperm fertilization of egg to production of new sperm via PGC, a 5-step process:
  1. Sperm enters the egg and fertilization begins with epigenetic marks
  2. Epigenetic marks are removed, but the imprinting region is not (shortly after fertilization)
  3. Methylation marks are put in place for the specialized ICM cells to differentiate
  4. PGC cells remove epigenetic marks from all regions as they become a somatic pathway
  5. Sperm and egg development is determined through epigenetic marks
• Histone modifications play a role in establishing imprints during gamete production
  • Histones on the sperm genome: protamines
    • Located at ICRs, and shields against reprogramming
    • DNA Compacts DNA for efficient packaging
  • ICRs must shed histone modifiers and permanent DNA methylation to gain a repressive imprint
• Transgenerational inheritance operates slightly differently in Axin fused gene, and in agouti gene in mice
  • Axin fusion uses methylation marks that are retrotransposons, resisting reprogramming (agouti uses retrotransposons and methylation, the mechanism for this is more complex)
  • Transgenerational inheritance is complex, so genes use epigenetic mechanisms to meet their goals
  • The reprogramming mechanism used depends on each gene and cell type, or transgenerational inheritance cannot work

Genomic Reprogramming

• Genomic reprogramming in early development has major impacts, allowing highly differentiated cell types to fuse and form a pluripotent cell

Histone Variants

• Allis Chapter 20 materials in Sakai week 6 contain the following information:
  • Overview Section
  • Section 1 & Figure 1 which shows major domains
    • HFD histone domain is crucial for proper folding
    • Most histones don't have variants
  • 3 & Figure 4: This figure shows the new and old nucleosomes for replication forks
    • Old nucleosomes stay the fork and new are deposited in gaps
    • Leading and lagging strand have CAF1 mediated nucleosome assembly
• Figure 5A : Human neocentromeres lack alpha satellite DNA (alfoid repeats) but have CENP-a and heterochromatin
• Variant incorporation:
  • Sections 6 & Figure 7 - H3 localizes to transcribe regions of Drosophila chromosomes
    • DAPI atin shows DNA bands of a pattern
    • H3.3 localizes to interbands
    • These localizations create RNA Pol II sides
    • Decondenced bands have H3.3 = gene expression
  • Section 7
  • Section 8 & Figure 10 - shows spermatogenesis stages and dependence of sex body formation on H2A.X – enrushced in secbody and
  • Sections 13 & Figure 11 shows Variants of H2A, and inative C
  • Sections 15 & 16

Carlberg/Molnar Box 4.1

• Has all box material including properties of canonical versus new variants
• Canonical: (H2A, H2B, H3, H4)
  • Most histones assemble into nucleosomes after replication to pack new DNA
• Variants: (H2A.B, macroH2A, CENP-A, H2A.B)
  • They are incorporated independent of synthesis
• H2A.B leads to less compact and more accessible chromatin
• H2A.Z and H.3.3 containing nucleosomes are less stable than normal, and can be found depleted in nucleosome regions of active promoters acting as “place holders"
• Variable composition of nucleosomes directly influences gene expression

Coursera Video

• Know 3 Variant examples:

1. CENP-A: Is linked to centromere formation to create neocentromeres with no repeats
   • H3A CENPA can recognize these repeats as an epigenetic mark
   • Plays histone strength role
2. H2A.X: Plays a role in DNA repair - differing C position to serine at 139, and with DSB double strands.
   • yH2A.x recruits DNA restoration proteins and epigenetic factors to break DNA
3. macro H2A: Is inked to inactive chromosomes and is an gene expression inhibitor
   • caries within each cell of facultative heterochromatin with a transcriptional silencer

• RC versus RI varies significantly with replication
  • Coupled histone variants come before the need and may cause an excess during S phase
  • H2A variant (DNA repair) is critical to restoration.

Twins

• Definition of MZ twins model of human epigentics
• MZ twins created from one egg mean they are identical
  • phenotypic defects by effects the epigenome
    • stress, nutrition, and the environment
• Terms for rate definitions
  • Twins phenotypically different when rate defers
    • MZ twin
      • fraternal %25

Twins and disorders

• disorders environmental
• genetic change
• Humans good
  • data hard
• Models good for human
  • study span easier
• Definition in mouse breeding
  • identical are epigenetic
• switch with retrotransposon methylation
• not in humans for body weight

Twin Experimentation.

• tests with Dmnt3a and Trim 28
• winter from long term test
• epigenetic effect from fetus?
  • dutch when German took it
  • nutrients
• nutrients over time lead to health
• epigenome mech
• the above with phenotypes
• outcome with agouti
  • A plastic BPA
    • mice DNA change
    • coated
• nutrition
  • 3rd part is the story
  • Pick a pair the diff, effect mech

Nutrition and Epignome

• Epigenetic from "genetic learn:
• "health genes"
• adaption of nutrition explain
• from a generaiton
• Example, Holland winter
  • generational
• generational malnutrition
• can it be the uterus
• goal and out
• big period - sperm based enivroment - passed
• what is
• epigenetics and obesity
• diet (agouti
• what is the epigenetics diet
• nutri mole, for dietary
• micro
• figures to understand the process

How Diet Help

• diet to affect - as ligand the factors can - by modulate with factor
• stimi signls factors on and up
• vitamin B donor acts with groups
• embrys coding effect from that
  • low lead to birth bad
  • tumor high
• write it
• what good with modifers

1. active state state
2. inactive state
3. heter effect
4. cell activity
5. affect what you need help
   • in what amount

• is the metabolite important or not
• genes to help
• cell is good like that
• what the TET and that

More on Diets

• Alpha subs to all
• block to all
• hypo for more
• too many tumor - no enzyme to help
• make better cell state after
  • help modify
• what metabolism is calling for the cell

Chromatin Structure

• modifier
  • after after that
• where did they eat at
• index methylation diet good
• mark to cell modulator
• green for more
• inactive state = hyperchromatin
• and can the test
• tissue to help fix you
• fat to the tissue