SCGI W6-8 Flashcards
why do mutations in NHEJ give rise to SCID/CID
DNA DSBs are physiologically induced during immune system development - V(D)J-recombination and class switching recombination (CSR)
V(D)J recombination = process of cutting/pasting DNA within the B and T cell receptor loci to create huge amounts of diversity within the antigen binding part of the receptor
RAG1/2 recognise specific sequences within the B and T cell receptor loci that allow DSB induction
DSBs in the BCR and TCR are predominantly repaired by NHEJ - unrepaired DSBs trigger activation of p53-dependent apoptosis
patients with mutations in NHEK genes often present with severe combined immunodeficiency due to an inability to repair DSBs induced during V(D)J recombination
microcephaly and the NHEJ genes
DNA DSBs can naturally occur during S-phase due to replication fork collapse
increased proliferation increases the chances that replication forks will collapse into DSBs
rapidly dividing neuro-progenitor cells will generate more DSBs that slower replicating cells and such have a higher dependence on DSB repair pathways for their repair
if DSBs aren’t repaired properly in the VZ by HR as the neuro-progenitor cells move into the SVZ and start to differentiate and come out of cycle - NHEJ will be required to repair these breaks - inability ti repair these DSB = trigger p53-dependent apoptosis and neuronal loss
mutations in HR genes and immunodeficiency
loss of ATM - components of the MRN complex/RNF168 don’t result in a major problem with V(D)J recombination
A-T/A-TLC/NBS/NBSLD patients have recurrent spontaneous chromosomal translocations involving immune receptor genes
ATM plays a role in stabilising post-cleavage complexes of coding ends
A-T/NBS/RIDDLE patients experience a reduction in Ab production (IgG/IgA) - inactive defect in CSR
CSR mechanism where B cells switch from IgM to other Ig isotypes
CSR also involves the generation and repair of DNA DSBs
loss/mutation of the ATM-MRN pathways -> CSR dependent DSBs repaired by Alt-EJ -> internal deletions within BCR switch regions + non-productive CSR events - reduction of IgG/A/D/E production
HR gene mutations and microcephaly
during development the neuro-progenitor cells rapidly expand to prodcue a sufficient number of cells to allow the brain to develop properly
genetic mutations that slow the cell cycle of neuro-progenitor cells reduces the total pool of cells required for brain development - results in small brain
severity of the cell cycle defect correlates with the severity of the reduction in head size
DNA DSBs occur naturally during S-phase due to replication fork collapse
increased proliferation inc. chance of replication fork collapse
rapidly dividing neuro-progenitor cells generate more DSBs than slower replicating cells - higher dependence on HR for repair
mutations in ATM and MRE11 and cerebellar degeneration
brain development in A-T/A-TLD patients = normal, cerebellum degenerates once the brain is fully developed
A-T/A-TLD/NBS/NBSLD cells exhibit many common cellular defects
unknown as to why the cerebellum deteriorates
chromatin packaging and signalling
how the chromatin is packaged effects gene expression
signalling enabled the expression/switching off of genes
remodelling of the chromatin allows for altered expression
the nucleosome
nucleosome = core histones: tetramer of H3/4 and dimer of H2A/B above/below
146 bp of DNA wrap around the nucleosome in a left handed superhelix - tethered to the the nucleosome by H1 (linker histone)
the flexible N terminal tail = the point of signalling
the histone tail
signalling occurs via post-translational modifications
- allows for complexes to open/close a region of DNA for expression
flexible and unstructured
post translational modifications
acetylation
methylation
phosphorylation
ADP-ribosylation
ubiquitination
acetylation
occurs on the e- amino group of specific lysins at the amino-terminal end of all CHs
H4 - 4 sites - least-K16 -> K12 & K8 -> K5-most
H3 - 5 sites - least-K14 -> K23 & K27 -> K9 & K18-most
acetyl CoA -> CoA via acetyltransferases [HATs]
acetyl lysine -> lysine via deacetylases [HDACs]
block action of HDACs via Na Butyrate/Na Valproate leads to hyperacetylated histones
RNA polymerase II holoenzyme
turn a gene off temporarily/permanently
interaction and combination of post translational modifications that occur both on the histone and on the DNA
RNA polymerase II holoenzyme
turn a gene off temporarily/permanently
interaction and combination of post translational modifications that occur both on the histone and on the DNA
acetylation effect on chromatin
acetylation increases accessibility of TFs to DNA by opening up chromatin
chromatin closed - transcriptionally inactive, low levels of acetylation
chromatin open - transcriptionally active, high levels of acetylation
euchromatin = acetylated
heterochromatin = hypoacetylated
methylation
occurs on specific lysine residues
methyltransferases
1/2/3 methyl groups added to each e-amino group
methylation occurs on CHs histones H3 and H4 ONLY and on H1
- H3 methylated at lysine 4/9/27
- H4 methylated at lysine 20
- H1 methylated at lysine 26
methylation effect on chromatin and DNA
methylation at specific lysine residues on H3 signals DIFFERENT complexes
active chromatin - high levels of acetylation, high levels of H3K4 methylation, DNA unmethylated
heterochromatin - methylation at H3K9 signals HP1 to bind, spreading of HP1 coated nucleosomes silences heterochromatin
inactive chromatin - low levels of acetylation, high levels of H3K27 methylation, DNA methylated
phosphorylation
in vivo proteins can be phosphorylated at 2 types of amino side chain
- O-phosphate linkage [phosphate for a OH group] e.g. serine threonine tyrosine
- N-phosphate linkage e.g. lysines histidines arginines
phosphorylation: protein kinase - ATP -> ADP
removal of phosphate: protein phosphatase
histone phosphorylation
binds to the dyad axis where the DNA wraps around the nucleosome + crosses over
H1
- 3 serine residues [S17/S169/S185]
- 2 threonine residues [T134/T151]
H3
- 2 serine residues [S10/S28]
position of phosphorylation alters the stability of H1 binding to the nucleosome
when are histones phosphorylated
highest level of histone phosphorylation observed at mitosis - H3 and H1
also occurs on
- H3 as cells are stimulated from quiescence to growth by GFs
- only present on a small number of genes (e.g. immediate early genes)
different combinations of histone modifications and their outcomes
condensation caused by high levels of
- H3K9 methylation
- H3K27 methylation
- H3S10/S28 phosphorylation
opening cased by high levels of
- H3K9 acetylation
- H3S10 phosphorylation
- H3K27 acetylation
same post translational modification on a residue, the adjacent modification is the riving force for the change in transcriptional activity
enzyme families that modify histone tails
AcetylCoA - acetylation (lysine)
S-adenosyl methionine - methylation (lysine, arginine)
ATP - phosphorylation (serine)
the epigenetic code
changes to DNA and its associated proteins that can alter gene expression without altering the DNA sequence
- altering DNA methylation
- histone modification
single-nucleosome code/multi-nucleosome code
histone acetylation
acetylation
- intermediary metabolism
de-acetylation
- environmental stress
- intermediary metabolism
- signalling pathways
- therapeutic inhibitors
epigenetic modifications: histone modifications
a combination of different molecules can attach to the tails of histone proteins
alter the activity of the DNA wrapped around them
epigenetic modifications: DNA methylation
methyl marks added to certain DNA bases repress gene activity
epigenetic modifications: micro RNAs
RNA molecules that are not translated into protein but still functional
methylation of cytosine in DNA
5th DNA base
occurs on CpG islands
- CpG dinocleotides are palindromic
difference in heterochromatin and euchromatin DNA methylation and histone modifications
euchromatin
- high histone acetylation
- low DNA methylation
- H3-K4 methylation
heterochromatin
- low histone acetylation
- dense DNA methylation
- H3-K9 methylation
maintenance of methylation
DNA methyltransferases (Dnmts) are essential
maintenance: Dnmt1 - puts on the mirror methylation onto the daughter strand
Dnmt3A/B responsible for new methylation
methylation of DNA from a zygote to an adult
totipotent - devoid of DNA methylation
pluripotent - some regions are methylated (turn off genes that code for extra-embryonic tissues)
multipotent - acquiring more DNA methylation (turn off different lineages)
unipotent - specific gene expression profile (majority of genome silenced by DNA methylation)
pluripotent cells most CpG islands will be unmethylated, unipotnet cells most CpG islands will be methylated
de-methylation of the paternal pronucleus
occurs at the 1 cell stage embryo
in embryogenesis
male genome demethylation occurs as the gametes fuse
female genome demethylation takes longer and is complete by the morula stage
genomic imprinting
some genes are expressed only from the maternal genome and some are only from the paternal genome
estimated that around 80 genes are imprinted - found on several different chromosomes
epigenetic genomic imprinting
modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilisation - “parent of origin” gene expression
affects the expression but NOT transmission of alleles
2 gene copies present
1 gene copy active = functional haploidy
genomic imprint - the unequal expression of the maternal and paternal alleles of a gene
epigenetic genomic imprinting: Igf2 and H19
insulin growth factor 2 (Igf2) paternally expressed
H19 (a non-coding RNA) maternally expressed
regulated by a cis-acting, differentially methylated region (DMR) = an imprinting control region (ICR)
epigenetic genomic imprinting: Igf2 and H19 mechanisms
paternal DMRs methylated = H19 OFF and Igf2 ON
- DMR1 is a silencer inactivated by methylation
- DMR2 is an enhancer that is activated by methylation
maternal CMRs hypo-methylated = H19 ON and Igf2 OFF
- CTCF binds DMR
- downstream enhancer engaged for H19 expression
- DMR1 silencer of Igf2 active
- DMR2 enhancer of Igf2 is inactive
genomic imprinting in mammals
parent of origin gene expression imprints must be correctly set in the gametes every generation
failure to reset correctly leads to developmental disorders in the next generation
prader-willi syndrome
mostly sporatic
deletion at 15 q11-q13
obesity, short, small hands/feet, unusual facial features, mild mental retardation
compulsive overeaters
angelman syndrome
speech impairment
movement/balance disorder
behavioural uniqueness
excitable personality
maternal chromosome deletion UBE 3A
roles of DNA methylation
transcriptional silencing
protecting the genome from transposition
genomic imprinting
X inactivation
tissue specific gene expression
complexity of human disease
the following contribute to the disease phenotype
genetic mutation/alteration
environmental factors
imprinting and epigenetics
lifestyle factors
inherited diseases
2 types
- multigenic diseases
- monogenic diseases
multigenic diseases
mutations in multiple genes
arise from less severe mutations in multiple genes
any one particular mutation may not result in a disease phenotype: only when they are combined does the disease phenotype occur
established by analysing function interactions between candidate genes - epistasis
monogenic diseases
mutations in single gene
e.g. cystic fibrosis (CFTR)/ataxia telangiectasia (A-T/ATM)
caused by more severe mutations in single genes
tend to be rare due to selective pressure against deleterious mutations
may be caused by recessive mutations and therefore escape selective pressure
monogenic inherited diseases and the DNA damage response
lost of different sources of DNA damage
- IR/X-rays/chemotherapy
- MMS/EMS/chemo
- MMC/cisplatin
- metabolism/chemo
- CPT
developmental diseases focus around
- brain development
- growth development
- premature ageing
monogenic inherited diseases and the DNA damage response examples
fanconi anaemia (FA)
seckel syndrome (SS)
Ataxia-Telagiectasia (A-T)
Ataxia-Telagiectasia-like disease (ATLD)
Nijemgen breakage syndrome (NBS)
Nijemgen breakage syndrome-like disorder (NBSLD)
werner syndrome (WS)
bloom syndrome (BS)
fanconi anaemia
bone marrow failure
skeletal defects
genome instability (chromosomal aberrations)
myeloid leukaemia
seckel syndrome
growth retardation
dwarfism
microcephaly
mental retardation
genome instability (chromosomal aberrations)
ataxia-telangiectasia
genome instability (chromosomal breakage)
T/B cell leukaemia/lymphoma
ataxia-telangiectasia-like disease
neurodegeneration
ataxia
genome instability (chromosomal breakage)
