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Epigenetics and gene-editing course notes
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Epigenetics and gene-editing course notes
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Lecture 1 – Smoking and epigenetics – 16-03-2020Cigarette smoking and DNA methylation → implications for chronic obstructive pulmonary disease
(COPD).
Worldwide prevalence of smoking:
• In Nederland roken 22% van de vrouwen. In China 4%.
• In China roken 65% van de mannen. In Nederland 28%.
• Er is te zien dat in Europa meer vrouwen roken, terwijl in Azië veel meer mannen roken.
Chronic obstructive pulmonary disease (COPD) is een verzamelnaam
voor de ziektes:
• Chronic bronchitis: inflammation and excess of mucus. De
vertakking van de luchtpijp (bronchiën) zijn blijvend ontstoken.
Door deze irritatie produceren de slijmvliezen meer slijm dan
normaal. De ontsteking maken het slijmvlies dikker →
doorstroming van lucht moeilijker.
• Emphysema: alveolar membranes break down. Steeds meer
longblaasjes gaan stuk. De wanden raken beschadigt. Zo kan je
moeilijker zuurstof opnemen.
Chronic obstructive pulmonary disease (COPD) → 3th most common cause of death worldwide:
• Constant coughing, ‘smoker’s cough’.
• Shortness of breath while doing activities that used to be easy.
• Excess sputum production.
• Not being able to take a deep breath.
• Wheezing → piepende ademhaling/hijgen.
How many people have COPD?
• Study in five Colombian cities: 8.9% in 2008.
• 100 million people in China (8.6%).
• Three-quarters of COPD cases have their origins in poor lung function pathways beginning in
childhood.
Why do adults get COPD?
• Risk factors for accelerated lung function decline:
o Genes
o Environment
▪ Smoking
▪ Biomass cooking → hout, steenkool, stro, dierlijke uitwerpselen, etc.
▪ Occupational exposure → beroepsmatige blootstelling.
▪ Socioeconomic status → mensen met een lage sociaaleconomische status
hebben vaker COPD. Groep met lager inkomen/opleiding dan gemiddeld.
Epigenetics:
• Epigenetica betekent letterlijk: romdom het DNA.
1
, • Epigenetics is the study of heritable changes in gene expression (active versus inactive genes)
that do not involve changes to the underlying DNA sequence — a change in phenotype
without a change in genotype — which in turn affects how cells read the genes.
• Epigenetica kan beïnvloed worden door leeftijd, omgeving, levensstijl en ziekte.
• At least three systems including DNA methylation, histone modification and non-coding RNA
(ncRNA)-associated gene silencing are currently considered to initiate and sustain epigenetic
change.
Effects of smoking in the lung:
• Inflammaging chronic low-grade systemic inflammation.
• Oxidative stress.
• Accelerated aging senescence and emphysema.
• Steroid resistance.
→ epigenetic effects of smoking.
Effect of smoking on DNA methylation → epidemiology studies in blood cells:
• CpG sites:
o The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed
by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction.
CpG sites occur with high frequency in genomic regions called CpG islands (or CG
islands).
o CpG zijn veelal ongemethyleerd. In veel ziektes worden deze gemethyleerd.
o Er zijn veel minder CpG’s dan verwacht.
o Wanneer cytosine wordt gedeamineerd is het een uracil. Uracil is recognized and
removed → starting the DNA repair process.
o Methyl groep toevoegen → spontane deaminatie verandert dan cytosine in thymine.
Dit kan alleen slecht herstelt worden.
o CG sequenties veranderen zo in TG sequenties.
o Er zijn gebieden in het genoom die CpG eilanden hebben. Dit zijn regio’s met ten
minste 200 baseparen.
o CpG eilanden neigen ernaar om niet gemethyleerd te zijn. Why? The delay in
methylation after replication allows a temporary distinction between the parent and
daughter strands, which aids in DNA proofreading. Spontaneous deamination tends
to turn methylated cytosines into thymines over time.
▪ Parent strands → methylated
▪ Daughter strands → unmethylated
o Genes whose expression is suppressed have methylated CpG sequences →
spontaneous deamination of methylated cytosines → inefficient mismatch repair →
cytosines converted to thymine → deficiency of CpG’s.
o CpG islands in active genes show that parts of the genome have forces acting on
them that select for higher CpG content and less methylation.
o What could these forces protecting CpG islands from mutations be? CpG eilanden die
er nu nog aanwezig zijn: protection = lack of methylation. Because of their action as
promotors (regions of DNA that initiatie gene transcription)! Human promotors have
a high percentage of CpGs. CpG islands are typically near the transcription start site
of genes. Especially common near the transcription start site of housekeeping genes.
Housekeeping genes are genes that are necessary for basic survival functions of the
cell.
o CpGs in promotor regions remain unmethylated in active genes. CpGs in promotor
regions of inactive genes get methylated. The methylation of CpG island promotors
prevent binding of transcription factors. This results in gene silencing.
2
, • Effect size of smoking-related CpG sites reported >3 times in literature.
o Door roken veroorzaakte methylering kan in veel regio's van het menselijk genoom
voorkomen. In bepaalde gengebieden bevindt ongeveer de helft van de aan roken
gerelateerde CpG-sites zich in het lichaam van specifieke genen (GFI1, 2q37.1, AHRR,
F2LR3). Met name was hun effectgrootte gewoonlijk groter dan die van sites die zich
op andere delen bevinden.
• Effect sizes of smoking-induced methylation for current smokers and former smokers
compared to non smokers → quitting smoking helps: former smokers catch up with non-
smokers with respect to DNA methylation.
• F2RL3 methylation as a biomarker of current and lifetime smoking exposure: population
based cohort southwest Germany:
o F2RL3: coagulation factor II (thrombin) receptor-like 3.
o Current smokers have lower methylation of F2RL3 in peripheral blood
cells.
• Dose-response relationships between smoking behavior and F2RL3 methylation
intensity: population based cohort in southwest Germany.
o Dose-response relationship between number of cigarettes (intensity of
smoking) and F2RL3 methylation intensity. Hoe meer sigaretten, hoe
minder methylatie. Eerst sterke afname van methylatie, daarna langzaam.
o Dose-response relationship between cumulative dose of smoking (pack-
years) and F2RL3 methylation intensity. Hoe meer pakjaren, hoe minder
methylatie.
o Dose-response relationship between time since quitting and F2RL3
methylation intensity. Methylatie neemt weer toe nadat je gestopt bent.
• F2RL3 methylation is a strong predictor of mortality, including cardiovascular,
cancer and other mortality:
o Lower methylation intensity is associated with:
▪ Higher all-cause mortality, higher cardiovascular mortality, higher
cancer mortality and higher mortality from other causes.
Smoking and DNA methylation → studies in airway epithelial cells:
• SPDEF hypomethylation after air-liquid interface (ALI) culture of primary
bronchial epithelial cells in COPD:
o Control: residual tracheal (achtergebleven trachea) and main stem
bronchial tissue from transplant donors.
o COPD: bronchial tissue harvested from transplant recipient lungs of
patients with GOLD stage IV COPD.
o Pseudostratified epithelium → eenlagig epitheel → cilitated cells and
goblet cells.
• SPDEF dictates goblet cells (slijmbekercellen) differentiation and mucus
production.
o Mucus producing cell: IL-13 → IL13-Ralfa → STAT6 wordt
gefosforyleerd → SPDEF → MUC5AC → mucus productie.
o Schematic representation of the ALI culture model. PBECs from
control 7–17 and COPD 6–16 were seeded onto transwell inserts
and grown to confluence. Thereafter, apical medium was
removed to create an ALI. Cells were harvested after 14 days for
RNA and DNA analysis
• More mRNA expression of MUC5AC and SPDEF in COPD.
• SPDEF hypomethylation in COPD.
3
, o Schematic representation of the promoter region of the SPDEF gene, outlining the
putative binding sites for the most relevant transcription factors (STAT6, GFI, NKX2-
1/NKX3-1, SMAD, and FOXA1/FOXA2) as analyzed. The transcription start site was
shown as +1. CpGs are indicated as vertical bars. DNA methylation status of 15 CpGs
was analyzed.
o In the absence of IL-13, COPD-derived
ALI-cultured cells displayed higher SPDEF
expression than control-derived ALI
cultures, whereas no difference was
found for FOXA2 expression. This was
accompanied with hypomethylation of
CpG number 6 in the SPDEF promoter.
o Aberrant DNA methylation of SPDEF is
one of the factors underlying mucus
hypersecretion in COPD, opening new
avenues for epigenetic-based inhibition of
mucus hypersecretion.
Take home messages:
• Smoking changes DNA methylation.
• Methylation is dose-dependent, cell-type dependent, gene-specific and reversible.
• Waar kan een epigenetische markering, zoals methylatie voor gebruikt worden? Dat kan als
biomarker, maar ook als target voor epigenetische editing. De een is voor diagnostiek. De
ander voor therapeutische doeleinden.
• Silencing of SPDEF methylation by EE (epigenetic editing) reduced mucus production in a cell
line.
P A P E R : DNA M ET H Y L A T I O N C HA N GE S O F WH O L E BL O O D C EL L S I N R E S P ON S E T O
A C T I V E SM O K I N G E XP O S U R E I N AD U L T S
The three most frequently reported CpG sites (genes) in whole blood samples were AHRR, F2RL3 and
GPR15, followed by other loci within intergenic regions 2q37.1. There are evidences for the use of
blood DNA methylation patterns as biomarkers of smoking exposure for research and clinical
practice. In particular, it provides a reservoir for constructing a smoking exposure index score
which could be used to more precisely quantify long-term smoking exposure and evaluate the risks
of smoking induced diseases.
• Using methylation markers for a refined quantification of smoking exposure and predicting
the risks of smoking related diseases.
PAPER: BI O M AR K E R S O F E X P O S U R E AN D E F F E C T I N T H E L U N GS OF S M O K E R S ,
N O N SM O K E R S A N D EL E C T R O N I C C I G A R ET T E U S E RS
There were statistically significant differences among never-smokers, e-cig users, and smokers for
inflammatory cell counts and cytokines. The e-cig users had values intermediate between smokers
and never-smokers, with levels for most of the biomarkers more similar to never-smokers. For
differential gene expression and DNA methylation, e-cig users also more like never-smokers; many of
these genes corresponded to smoking-related pathways. Differentially methylated genes were
correlated with changes in gene expression, providing evidence for biological effects of the
methylation associations. These data indicate that e-cigs are associated with less toxicity than
cigarettes for smoking-related pathways.
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