what is genomic imprinting
expression of specific genes from either only maternal or only paternal allele (not both)
levels of epigenetic regulation (3)
- DNA methylation: on/off switch (methylation → off)
- Histone modifications: modification of tail → tight/loose packaging → affects accessibility for DNA transcription
- miRNA regulation: after transcription, fine-tuning
levels of epigenetic regulation (3)
- DNA methylation: on/off switch (methylation → off)
- Histone modifications: modification of tail → tight/loose packaging → affects accessibility for DNA transcription
- miRNA regulation: after transcription, fine-tuning
DNA methylation:
- enzymes
- CpG island vs gene body methylation
- de novo methyltransferases: high affinity for unmethylated CpGs e.g. DNMT 3a/b
maintenance methyltransferases: high affinity for hemi-methylated CpGs e.g. DNMT 1 - CpG island (in promoter) -> gene silencing
Gene body -> gene expression
DNA methylation: mechanisms of gene silencing
Direct: methylation group prevents transcription factor binding to promoter
Indirect: methyl-CpG-binding domain (MBD) proteins bind to methylation group -> recruitment of epigenetic modifying proteins -> repressive chromatin remodelling activity -> gene switched off
example of DNA methylation and histone modification coupling
Methyl-CpG-binding domain proteins (MBD) may have SET domain (containing HMTs) which directly binds and methylates histone tails → gene repression
miRNA epigenetic regulation: mechanism
miRNA regulation occurs after transcription (vs histone/DNA methylation which occurs before)
- Pri-miRNA (primary form) expressed from genes
- Drosha cleaves pri-RNA into pre-miRNA
- Exportin 5 recognises overhang and exports from nucleus
- Dicer cleaves hairpin loop
- Strands unwind and dissociate from each other
- miRNA associates w other proteins to form RNA-induced Silencing Complex (RISC)
a. miRNA complementary to specific region within gene ∴ allows RISC to bind to this region and block ribosome translating of that mRNA
b. RISC also targets and causes degradation of mRNA (via de-adenylation)
examples of epigenetic changes in cancer (2)
repeated essay question
- Inactivation of apoptotic pathways; DNA methylation of pro-apoptotic proteins e.g. DAPK (death associated protein kinase)
- Aberrant promoter CpG-island methylation (hypermethylation) → tumour suppressor silencing e.g. BRCA1 in breast cancer
- Loss of methylation (hypomethylation) → activation of oncogenes e.g. HOX11 proto-oncogene in leukemias
- UV light → ↑production of pyrimidine dimers → ↑p53 mutations (more common in methylated cytosine)
clinical uses of epigenetics (4)
- DNMT inhibitors: incorporated into DNA but cannot be methylated ∴ methylation marks not copied to new strands in DNA replication (leukaemia)
- Histone deacetylase (HDAC) inhibitors: binds to catalytic domain of HDAC, chelates zinc ion and inhibits deacetylation process (lymphoma)
- Biomarkers e.g. BRCA1 promoter methylation associated w better response to PARP (poly ADP ribose polymerase) inhibitors
- Epigenetic Editing using CRISPR: CRISPR allows targeting of modifications to specific genes
challenges with drug delivery
The Dutch hunger winter
2017 SAQ
Adults born to mothers who were poorly nourished during early gestation:
↑early onset of coronary artery disease
↑prevalence of intra-abdominal obesity in men
programming: effects of culture
failure to activate embryonic genome in culture was overcome by modifying culture media
culture (mice) -> low birth weight
programming: fetal outcomes of ART
- increased imprinting disorders e.g. Angelman syndrome
- increased methylation in IVF babies vs natural conception (placenta and cord blood)
- low birth weight, congenital abnormalities
however effects may be due to decreased fertility of ART pts
programming: effects of in utero environment on fetus (3)
- Stress → hypermethylation
- Sub-optimal in-utero environment → impaired β-cell function and T2DM in rats
- Mother w diabetes; high glucose (glucose can be transported across placenta, insulin cannot) → ↑fetal insulin production → too much insulin after birth → hypoglycaemia
Also risk of diabetes later in life
fetal programming by glucocorticoids (5)
Hyperglycaemia Hypertension Obesity Increased fear + anxiety Compromised lung function
what prevents high levels of cortisol in fetus during 1st trimester (3)
importance of this?
- cortisol-binding globulin (maternal circulation)
- placental 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2): deactivates cortisol to cortisone
- high GR levels in fetus
allows growth (vs differentiation w high levels of cortisol)
how do changes in cortisol occur in the 3rd trimester?
important of this?
fetal GR is downregulated -> decreased -ve feedback -> increased cortisol
allows tissue differentiation e.g. alveolarisation in lungs -> surfactant production (prevent respiratory distress syndrome)
define:
- totipotent
- pluripotent
- multipotent
- Totipotent: capacity for form an entire organism e.g. zygote
- Pluripotent: able to form all body’s cell lineages, including germ cells (but not placenta) e.g. hESCs
- Multipotent: can form multiple cell types that constitute an entire tissue or tissues e.g. haematopoietic stem cells
how are embryonic stem cells derived
complement anti-human serum Ab destroys trophoectoderm cells → isolation of ICM cells
types of stem cells (3)
- somatic stem cells: multipotent e.g. haematopoeitic stem cells
- embryonic stem cells: pluripotent
- iPSCs: pluripotent
advs and disadvs of somatic stem cells
advantages 1. can be autologous -> no rejection 2. ready to be transplanted disadvantages: 1. slow growth 2. limited availability 3. low yield 4. low plasticity: only differentiate into few cell types
methods for assessing pluripotency of stem cells (2)
- in vitro: stain embryoid body for markers of 3 germ cell layers
- in vivo: ES cells injected into tetraploid blastocyst and transferred to surrogate mother
Offspring genetically identical to mice from which mES cells cultured, as original tetraploid DNA cannot lead to proper development
challenges of clinical ESC use + how they can be overcome (3)
- tumorigenesis
clone toxic gene in promoter of tumour cells (difficult to find promoters only present in tumour cells) - immune rejection
immunosuppressants (increased infection risk), transplant of haematopoietic stem cells before ESC transplant shown to decrease rejection - ethics: use and destruction of human embryos
methods of reprogramming cells to pluripotency (3)
- cloning / somatic cell nuclear transfer (SCNT)
Whole donor cell (any somatic cell) injected into enucleated oocyte, activation by electric shock → totipotent cell → culture; either
a. transfer to surrogate mother
b. isolate ES cells for differentiation to other cell types - fusion of embryonic stem cell w somatic cell -> tetraploid cell, not suitable for regenerative medicine
- iPSC formation by reprogramming with defined factors:
Oct4, Sox2 -> Nanog complex -> expression of Nanog -> ESC genes
c-Myc, Klf4
sources of cardiac stem cells (6)
advs and disadvs
- Skeletal myoblasts: thought that may act like cardiac myocytes upon transplantation
Do not form true cardiomyocytes (different electrical control systems)
Arrhythmic complications - Bone marrow-derived stem cells
Immune matching
Bone marrow cells from heart disease pts are less active (may be contributing to disease or same risk factors cause reduced bone marrow stem cell activity and heart disease)
Rarely form cardiomyocytes
Beneficial effects due to growth factors and angiogenesis - Mesenchymal stem cells (e.g. bone marrow stromal cells)
Ongoing debate regarding whether true cardiomyocytes formed
Immune matching - Cardiac stem cells
Self-renewing and multipotent
Clinical safety unknown - Embryonic stem cells
Unlimited supply; large scale proliferation
Form true cardiomyocytes
Ethical problems
Immune rejection
Tumorigenesis
Immature cardiomyocytes → arrhythmias - iPSCs/induced cardiomyocytes
Same as ESCs except no ethical issues or immune rejection
