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rds > stem cells, epigenetics & programming > Flashcards

stem cells, epigenetics & programming Flashcards

1
Q

what is genomic imprinting

A

expression of specific genes from either only maternal or only paternal allele (not both)

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2
Q

levels of epigenetic regulation (3)

A
  1. DNA methylation: on/off switch (methylation → off)
  2. Histone modifications: modification of tail → tight/loose packaging → affects accessibility for DNA transcription
  3. miRNA regulation: after transcription, fine-tuning
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3
Q

levels of epigenetic regulation (3)

A
  1. DNA methylation: on/off switch (methylation → off)
  2. Histone modifications: modification of tail → tight/loose packaging → affects accessibility for DNA transcription
  3. miRNA regulation: after transcription, fine-tuning
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4
Q

DNA methylation:

  1. enzymes
  2. CpG island vs gene body methylation
A
  1. 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
  2. CpG island (in promoter) -> gene silencing
    Gene body -> gene expression
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5
Q

DNA methylation: mechanisms of gene silencing

A

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

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6
Q

example of DNA methylation and histone modification coupling

A

Methyl-CpG-binding domain proteins (MBD) may have SET domain (containing HMTs) which directly binds and methylates histone tails → gene repression

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7
Q

miRNA epigenetic regulation: mechanism

A

miRNA regulation occurs after transcription (vs histone/DNA methylation which occurs before)

  1. Pri-miRNA (primary form) expressed from genes
  2. Drosha cleaves pri-RNA into pre-miRNA
  3. Exportin 5 recognises overhang and exports from nucleus
  4. Dicer cleaves hairpin loop
  5. Strands unwind and dissociate from each other
  6. 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)
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8
Q

examples of epigenetic changes in cancer (2)

repeated essay question

A
  1. Inactivation of apoptotic pathways; DNA methylation of pro-apoptotic proteins e.g. DAPK (death associated protein kinase)
  2. Aberrant promoter CpG-island methylation (hypermethylation) → tumour suppressor silencing e.g. BRCA1 in breast cancer
  3. Loss of methylation (hypomethylation) → activation of oncogenes e.g. HOX11 proto-oncogene in leukemias
  4. UV light → ↑production of pyrimidine dimers → ↑p53 mutations (more common in methylated cytosine)
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9
Q

clinical uses of epigenetics (4)

A
  1. DNMT inhibitors: incorporated into DNA but cannot be methylated ∴ methylation marks not copied to new strands in DNA replication (leukaemia)
  2. Histone deacetylase (HDAC) inhibitors: binds to catalytic domain of HDAC, chelates zinc ion and inhibits deacetylation process (lymphoma)
  3. Biomarkers e.g. BRCA1 promoter methylation associated w better response to PARP (poly ADP ribose polymerase) inhibitors
  4. Epigenetic Editing using CRISPR: CRISPR allows targeting of modifications to specific genes
    challenges with drug delivery
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10
Q

The Dutch hunger winter

2017 SAQ

A

Adults born to mothers who were poorly nourished during early gestation:
↑early onset of coronary artery disease
↑prevalence of intra-abdominal obesity in men

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11
Q

programming: effects of culture

A

failure to activate embryonic genome in culture was overcome by modifying culture media
culture (mice) -> low birth weight

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12
Q

programming: fetal outcomes of ART

A
  1. increased imprinting disorders e.g. Angelman syndrome
  2. increased methylation in IVF babies vs natural conception (placenta and cord blood)
  3. low birth weight, congenital abnormalities
    however effects may be due to decreased fertility of ART pts
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13
Q

programming: effects of in utero environment on fetus (3)

A
  1. Stress → hypermethylation
  2. Sub-optimal in-utero environment → impaired β-cell function and T2DM in rats
  3. 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
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14
Q

fetal programming by glucocorticoids (5)

A 
Hyperglycaemia
Hypertension 
Obesity
Increased fear + anxiety
Compromised lung function
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15
Q

what prevents high levels of cortisol in fetus during 1st trimester (3)
importance of this?

A
  1. cortisol-binding globulin (maternal circulation)
  2. placental 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2): deactivates cortisol to cortisone
  3. high GR levels in fetus
    allows growth (vs differentiation w high levels of cortisol)
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16
Q

how do changes in cortisol occur in the 3rd trimester?

important of this?

A

fetal GR is downregulated -> decreased -ve feedback -> increased cortisol
allows tissue differentiation e.g. alveolarisation in lungs -> surfactant production (prevent respiratory distress syndrome)

17
Q

define:

  1. totipotent
  2. pluripotent
  3. multipotent
A
  1. Totipotent: capacity for form an entire organism e.g. zygote
  2. Pluripotent: able to form all body’s cell lineages, including germ cells (but not placenta) e.g. hESCs
  3. Multipotent: can form multiple cell types that constitute an entire tissue or tissues e.g. haematopoietic stem cells
18
Q

how are embryonic stem cells derived

A

complement anti-human serum Ab destroys trophoectoderm cells → isolation of ICM cells

19
Q

types of stem cells (3)

A
  1. somatic stem cells: multipotent e.g. haematopoeitic stem cells
  2. embryonic stem cells: pluripotent
  3. iPSCs: pluripotent
20
Q

advs and disadvs of somatic stem cells

A 
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
21
Q

methods for assessing pluripotency of stem cells (2)

A
  1. in vitro: stain embryoid body for markers of 3 germ cell layers
  2. 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
22
Q

challenges of clinical ESC use + how they can be overcome (3)

A
  1. tumorigenesis
    clone toxic gene in promoter of tumour cells (difficult to find promoters only present in tumour cells)
  2. immune rejection
    immunosuppressants (increased infection risk), transplant of haematopoietic stem cells before ESC transplant shown to decrease rejection
  3. ethics: use and destruction of human embryos
23
Q

methods of reprogramming cells to pluripotency (3)

A
  1. 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
  2. fusion of embryonic stem cell w somatic cell -> tetraploid cell, not suitable for regenerative medicine
  3. iPSC formation by reprogramming with defined factors:
    Oct4, Sox2 -> Nanog complex -> expression of Nanog -> ESC genes
    c-Myc, Klf4
24
Q

sources of cardiac stem cells (6)

advs and disadvs

A
  1. Skeletal myoblasts: thought that may act like cardiac myocytes upon transplantation
    Do not form true cardiomyocytes (different electrical control systems)
    Arrhythmic complications
  2. 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
  3. Mesenchymal stem cells (e.g. bone marrow stromal cells)
    Ongoing debate regarding whether true cardiomyocytes formed
    Immune matching
  4. Cardiac stem cells
    Self-renewing and multipotent
    Clinical safety unknown
  5. Embryonic stem cells
    Unlimited supply; large scale proliferation
    Form true cardiomyocytes
    Ethical problems
    Immune rejection
    Tumorigenesis
    Immature cardiomyocytes → arrhythmias
  6. iPSCs/induced cardiomyocytes
    Same as ESCs except no ethical issues or immune rejection
25
Q

how do polycomb repressive complexes work

A
  1. ESCs : key development genes primed for activation and held in check by polycomb repressive complexes (PRC) and RNA polymerase II (RNAPII)
    These genes have bivalent chromatin structure
    a. Contains both activating and repressing epigenetic modifications in the same area; repressing modification are dominant → inactivation of genes
    b. Once repression removed, transcription machinery recruited to activating modifications → gene expression
    c. PRC2-mediated repression via H3K27me3
    d. PRC1-mediated regulation of RNAPII activity
    Primed chromatin associated w pluripotency is best defined by PRC1 and RNAPII occupancy at silent developmental genes
  2. Trophoectoderm stem cells: loss of gene priming for transcription at bivalent genes
    a. PRC1 and poised RNAP are not recruited to genes that retain bivalent signatures in TE-derived stem cells
26
Q

active vs repressive histone modifications

A
  1. Euchromatin (open) → transcriptionally active
    Histone 3: Acetylated K9 and K14, methylated K4
  2. Heterochromatin (closed) → transcriptionally repressive
    Histone 3: deacetylated K9 and K14, methylated K9 and K27

rds (13 decks)

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