DNA Methylation Flashcards
(37 cards)
1
Q
Programming of the genome is controlled by the epigenome
A
-the epigenome is composed of 2 main components
- Histones
- associated with the DNA
- DNA methylation
- covalently bound to the genome and thus a stable long term signal
2
Q
History of DNA methylation
A
- Rollin hotchkiss discovered methylated DNA in 1948
- found that DNA from certain sources contained a fifth 5-methyl cytosine base
- took almost 3 decades to find a role for it
- in mid 1970s, Harold Weintraub noticed that active genes are low in methyl groups or under methylated
- methylation help repress genes
3
Q
Distribution of DNA methylation
A
- chemical modification of DNA
- can be inherited without sequence change
- common in plants (30%), vertebrates (10%) and fungi
- absent in yeast, flies, nematodes
- occurs predominantly at 5’-CG-3’ (CpG positions)
- in mammals, 60-90% of CpG sites are methylated
- high frequency of CG dinucleotide, CpG islands
- typically 300-3000 base pair in length and hypomethylated
- near approximately 70% of human promoters
- methylation correlated with tissue specific gene expression
4
Q
Methylation of cytosine by DNA methytransferase (DMNT)
A
Cytosine —> 5-methylcytosine (5mc)
-via DNMT
- cytosine methylation maintains inactive-condensed chromatin state
- found in heterochromatin regions
5
Q
Euchromatin vs Heterochromatin
A
- euchromatin
- high histone acetylation
- low DNA methylation
- H3-K4 methylation
- heterochromatin
- low histone acetylation
- dense DNA methylation
- H3-K9 methylation
6
Q
DNMT3L
A
-closely related to DNMT3A/B in structure and critical for methylation but is inactive on its own
7
Q
Types of DNA methylation
A
- Maintenance methylation (DNMT1)
- methylation of newly synthesized DNA strand at positions opposite methylated sites on parent strand (occurs after DNA replication)
- De novo methylation (DNMT3A/B)
- methylation of totally new positions
- changes the pattern of methylation in a localized region of genome during gametogenesis and early development
8
Q
DNA methylation Analysis:
Sodium bisulfite sequencing
A
- methylated cytosine is unaffected
- converts unmethylated cytosine to uracil
- during PCR and subsequent sequencing, the ratio of cytosine and thymine present at each CpG site is quantified and reflects methylation level of that site in genomic DNA
9
Q
Biological function of DNA methylation
A
- transcriptional regulation of cellular genes
- role in mammalian development including imprinting
-heterochromatin formation
10
Q
Essential DNMTs
A
- DNMT1:embryonic lethal
- DNMT2: no obvious effect
- DNMT3A: perinatal death
- DNMT3B: embryonic lethal
- DNMT3L: no imprints
- important in finding methyl groups
- require energy domains
- writers (DNMTS), readers (MBD proteins), erasers (DNA demethylases)
11
Q
Mediators of methylation induced gene silencing
A
-NURD (nucleosome remodelling deacetylase complex)
-bind to methylated DNA and bring complexes in remodeling
-recruiting suppressor complexes
-IL4 and TH2 helpers memory cells
-certain genes have to be expressed/suppressed to remember immune responses
-
12
Q
Pathways for DNMTs to be recruits
A
- PWWP domain
- TGS domain
- MBDs dock into methylated regions
- steric hinderance blocks activating transcription factors
13
Q
MBDs
A
- MECP2
- role in binding to methylated sites
- absent/present in certain species
- categorize different species and how their genes are regulated
- mutations of MBDs cause different diseases
- most die early on (lethal)
- important for differentiation
- methyl binding domains missing can cause certain cancers
- KO of MBDs can cause autism and have deficit in adult neurogenesis and hippocampal function
-
14
Q
Mediators of methylation induced gene silencing
A
- KO or mutation of methyl CpG binding proteins lose attraction of NURD complex and problem of suppressing genes
- genes will be inappropriately expressed
- mental retardation
15
Q
Stable repression of gene expression through development
A
- if you dont want it to be permanently methylated
- like through development
- phosphorylation of MECP2
- undergoes conformational change and loses capacity to bind to repressor complex
- liberation of repressor complex
- TFs are transient, DNA methylation is not
-MECP2 is phosphorylated and causes all suppression complexes to release and gene then expressed
16
Q
How does DNA methylation repress gene transcription
A
- 3 main types
1. Unmethylated (hypomethylated) promoters allows gene transcription
2. Methylated CpGs block binding of TFs- transcription blocked
- Indirect mechanism
- Me-CpG binding proteins also preclude TF binding to the promoter region
- steric hindrance
- transcription blocked
17
Q
Indirect mechanism
A
- CpG islands proximal to promoter regions
- methylation of CpG island upstream of gene provides recognition signals for MeCP components of histone deacetylase complex (HDAC)
- HDAC modifies chromatin isn’t he region of CpG island and inactivates gene
- self reinforcing cycle
- keep heterochromatin closed/silences
- assembly of histone at particular DNA sequence
18
Q
Pharm evidence
A
- Trichostatin A (TSA)
- blocks histone deacetylase activity
- prevents DNA methylation dependent repression
- HDAC inhibitor
- promote acetylation (HAT)
- sodium butyrate
- anticonvulsant
- mimics histone acetylation
- loosen chromatin
19
Q
Essential roles of cytosine methylation in mammals
A
- gene expression
- chromosomal stability
- cell differentiation
- pluripotent -> differentiation (increase in methylation)
- imprinting (inheritance of expression of alleles)
- x inactivation
- carcinogens
- aging
- loss or addition in methyl groups in different age related diseases
20
Q
Methylation during development
A
- renewing cells in glial cells, stem cells in hippocampus (memory)
- olfactory bulb
- cluster of cells around hair follicles that contain stem cells
- SKIPs
- reduce autoimmunity/rejecting organ tissue from donors
- stem cells to replenish lining of gut
21
Q
Methylated DNA from zygote to adult
A
- increasing methylation as cells differentiate
- regulation of genes
22
Q
Differentiated cells can become totipotent
A
- Stages of nuclear transfer
- the nucleus is removed from and egg and replaces by nucleus from a donor cell
- nuclear equivalence
- differentiated cells maintain potential to generate an entire organism
-reason that dolly died of lung cancer and osteoporosis (age related disorder)
23
Q
De-differentiation
A
- Cloning by nuclear transfer —> regenerate entire organism from transfer of single nucleus
- Induced pluripotent stem cells (iPS) —> expression of 4 genes are sufficient to transform differentiated cells to stem cells
-both processes must involve reprogramming of epigenome
24
Q
Critical CpG sequences in islands near promoters
A
- 4% of all cytosines are methylated
- 70-80% of all CpGs are methylated
25
Methylation in mammalian development
- maintenance and inheritance of tissue specific gene expression
- inhibition of transposone (and other repetitive sequences) gene expression
- prevents transposition
- inhibits DNA recombination between repetitive sequences
- lower probability of genome rearrangements
- genomic imprinting
- inactivation of methylation of a gene on one of a pair of homologous chromosomes
- relatively uncommon but important feature of mammalian chromosomes
26
Genomic imprinting
- developed along with giving live birth
- coincide with placenta
- raising fetal offspring with placenta
- imprinted genes important for placenta - Response to changes of mother and fetus
-no evidence of imprinted genes in birds
27
Genomic imprinting and epigenetic inheritance
- need dosage compensation on genetic material expressed following fertilization
- coevolve with placenta
- if disrupted associated with neurological disorders
- imprinted genes show difference in maternal/paternal lineage
- imprinted gene is switched off/methylated
- important in evolutionary development
- ICE = imprinting centre
28
Autosomal genes with a sex
- result in monoallelic expression
- maternal and paternal genomes not functionally equal
- increase ability to propagate and have offspring
- ICR methylation causes methylation of specific gene and prevents transcription of H19
- if H19 not expressed, upstream regulatory factors are expressed and cause transcription of IGF2
-if not CTCF able to bind to ICR and upstream factors able to bind to promoter silencing of IGF2 gene
29
Insulator model for control of gene expression at H19 and IGF2 imprinted locus
- in humans IGF2 and h19 gene are differentials expressed depending on parent of origin
- ICR between the 2 genes
- the ICR is not methylated on maternal allele
- this allows CTCF protein to bind to ICR and prevent enhancing factors from activating expression of IGF2 gene because the CTCF is blocking acces
- H19 gene is active on this allele
- ICR is methylated on paternal allele
- prevents CTCF protein from binding ICR and allows enhancing factors to activate expression if IGF2 gene because CTCF is not blocking access
- H19 gene is inactive on this allele
30
Imprinting maintained by DNA methylation
- differential methylation is erased in germ line cells at early stage
- can be modified by diet
- low methylated intake associated with lack of ability to methylated DMR groups
- in uterine effects
- insulin growth factors
- placental growth
- postnatal effects
- movement disorders
- lactation
- brain development
- genetic disorders
- epimutations (inappropriate methylation)
- prader willi/angel man syndrome
- I’m somatic cells, imprints are maintained and modified during development
31
Syndrome with DNA methylation
1. Prader-Willi
- deletion or inactivation of genes on paternally inherited chromosome 15
- maternal copy is imprinted and silenced
- associated with growth
2. Angelman syndrome
- deletion or inactivation of genes on maternally inherited chromosome 15
- paternal copy is imprinted and silenced
32
Methylation changes during development
- maternal genome has influence on paternal genome
- decrease in methylation as egg matures
- reestablishment of methylation
- maternal faster in re-establishment
- loss of methylation associated with viral DNA
33
Barr body
- X chromosome inactivation
- chromosome becomes condensated and shut down
- Xist gene in one of X chromosomes is expressed that lead to inactivation
- Xist gene in second chromosome is inactive (DNA methylation) leaving chromosome active
-Cix is an uncategorize gene with contains protein coding genes and 4 genes that express non coding RNA
34
XIC
- X in activation centre
- contain 4 genes that express non coding RNA
- 20kb cDNA with no open reading frame
- results in highly expressed Xist RNA that remained intranuclear, coats the flute Xi, surrounds Barr body’s
-stain for non-coding RNA to find
35
Tsix
- gene located 15kb downstream of Xist
- Xite function as an enhancer for development specific Tsix regulation
- Tsix expresses a 40kb ncRNA transcript antisense to Xist RNA
- before onset of X inactivation
- Tsix is expressed from both X chromosomes (blocks Xist RNA)
- at onset of x inactivation Tsix expression becomes monoallelic
- in Xa, Tsix RNA blocks Xist RNA
- in Xi, Xist RNA coats chromosome to repress Tsix expression
36
Xi unacetylated Histone H4
- inactive X has inactive chromatin
| - unacetylated histone H4
37
Chromatin related disorders
- cis effect
- within
- mutation within sequence
- trans
- across
- mutation in effectors/binding sites
