Transcript Review (12/13/16)

12/13/16
Corbo
• Briefly describe the function and component
parts of a transcription factor.
What is a transcription factor?
A transcription factor is a protein with two parts:
(1) A sequence-specific DNA-binding domain
(2) An activation or repression domain
Activation
domain
Transcription
factor
DNA-binding
domain
DNA
CTAATCCC
• In a brief paragraph, describe how a neural
progenitor population can give rise to
different neuronal cell types at different time
points during development.
The competence model of retinal development
Competence model: progenitors pass through a
series of competence states, during each of
which the progenitors are competent to produce
a subset of retinal cell types.
Environmental factors:
(1) Small molecules
(2) Secreted proteins
(3) Cell surface molecules
Progenitor
Etc.
Competence
•
Time
Differentiated cell types
• Is neuronal cell type production probabilistic
or deterministic?
Cellular differentiation on an epigenetic landscape
Fertilized
egg
Differentiated cell types
• Describe the location within the ventricular
epithelium of a neural progenitor at the
various stages of the cell cycle. What is this
movement process called?
The cell cycle in the developing retina
Basal
Apical
Start near ventricle (apical)
Nuclei move towards basal surface
DNA replicates (s phase)
Nuclei move back towards ventricle
Reach ventricle and divide (m phase)
Repeat
Interkinetic nuclear migration
Taghert
• Papers
Kim
• Explain the distinction between the extrinsic
and intrinsic pathway of apoptosis.
• What are key differences in the morphology
and mechanism of apoptosis and necrosis?
• Apoptosis
– Regulated: enzymatic steps,
requires energy.
– Requires transcription and
protein synthesis
– No loss of membrane
integrity
– Cytoplasm shrinks
– Formation of apoptotic
bodies on membrane
(vesicles)
– Cell kind of
implodes/fragments into
smaller pieces
• Minimize damage to
neighboring cells
• Necrosis
– Unregulated no energy
requirement
– Does not require
transcription and protein
synthesis
– Loss of membrane integrity
– Cytoplasm swells
– No vesicle formation
– Complete lysis of cell.
• Can cause damage to
neighboring cells
• What is an explanation for the failure of
caspase inhibitors to protect injured neurons
in stroke or neurodegenerative diseases?
• What is an explanation for the failure of
caspase inhibitors to protect injured neurons
in stroke or neurodegenerative diseases?
– I think he said this last year: by the time the
caspase inhibitors are applied, the cell has already
committed to apoptotic death. Injury or stress has
yielded an irreversible death decision, on which
the inhibition of caspase has no effect.
– Necrosis rather than apoptosis?
• Co-receptor systems for neurotrophic factors
are ubiquitous. Name two examples
neurotrophic factors that use co-receptors
(name ligand and the receptors). What might
be a teleological reason for having a coreceptor system?
Basically anything + p75
P75 + Trk: NGF or NT are ligands.
P75 + NogoR/Lingo: Nogo is the
ligand
Why:
Efficient
Ensures death domain present
Campenot Chamber
Gabel
• What chemical modifications are added to
histones? What are the classes of enzymes
that write and erase each of these histone
modifications?
Histone modifications: Writers and Erasers
Histone acetyl transferases
Histone deacetylases (HDAC)
e.g. Creb binding protein CBP
e.g. HDAC1-6
Histone methyltransferases
e.g. MLL3
Histone demethylases
e.g. LSD1
Cota et al. Pluirpotent Stem Cells 2013
• What chemical modifications are added to
histones? What are the classes of enzymes that
write and erase each of these histone
modifications?
– Epigenetic writers:
• histone acetyltransferases (HATs)
• histone methyltransferases (HMTs)
• kinases
– Epigenetic erasers:
• histone deacetylases (HDACs)
• lysine demethylases (KDMs)
• phosphatases
• Name two different consequences that
histone modifications can have when present
on a nucleosome.
– Do the different chemical modifications on
histones have the same effect regardless of which
amino acid they are added to on the histone?
– What histone modifications are associated with
active or repressed gene-regulatory elements?
• Name two different consequences that
histone modifications can have when present
on a nucleosome.
– facilitate or repress transcription
• Do the different chemical modifications on
histones have the same effect regardless of
which amino acid they are added to on the
histone?
– No.
• H3K4me2/3 is associated with transcriptional activity.
• Methylation of H3K9me2/3 is associated with
repression
• Name two different consequences that
histone modifications can have when present
on a nucleosome.
– Do the different chemical modifications on
histones have the same effect regardless of which
amino acid they are added to on the histone?
– What histone modifications are associated with
active or repressed gene-regulatory elements?
• What histone modifications are associated with
active or repressed gene-regulatory elements?
– Acetylation typically activates (HATs).
– De-acetylation represses (HDACs)
– Histone ubiquitination also represses
• What proteins and DNA modifications are
uniquely enriched in neuronal chromatin?
Unique forms of DNA methylation are enriched in the neuronal
genome
Tahiliani et al. Science, 2009
5’
3’
m
CG
GCm
3’
5’
5’
3’
m
CA
GT
Xie et al. Cell 2012
All cell types
3’
5’
5’
3’
hm
CG
GCm
Kriaucionis et al. Science, 2009
Enriched in neurons
3’
5’
• What are two examples of disrupted
epigenetic mechanisms that lead to
neurodevelopmental disease?
Cytosine methylation at CpGs is associated with gene repression
5’
3’
m
CG
GCm
3’
5’
Xie et al. Cell 2012
Aberrant DNA methylation underlies Fragile X-syndrome
DNA methylation typically represses transcription
-
~25% of males with the disorder have autism.
-
Prominent cause of syndromic autism, accounts
for 1-2% of all autism.
-
Gene identified by Verkerk et al. Cell 1991
Robertson & Wolfe Nature reviews genetics 2000
Fragile X: methylation of the CGG repeat expansion and FMR1 promoter, leading to the
silencing of the FMR1 gene and a lack of its product
Mutations in the Methyl-cytosine binding protein2 (MeCP2)
lead to Rett syndrome
-Mapped to X chromosome and cloned using rare familial
cases, confirmed by sequencing sporadic cases
-Because of random X-inactivation Females with Rett are
MECP2 -/+ and are a
mosaic of MeCP2-expressing and MeCP2-null cells. Boys that
are MECP2 -/y die just after birth with severe encephalopathy.
-MeCP2 is expressed in all cells to some extent, but is highly
enriched in neurons, building up postnatally.
NCoR
me
Ac
Ac
me
Ac
me
Ac
Ac
MeCP2 MeCP2
me
Ac
Ac
Ac
me
MeCP2
me
DNA methylation & genomic imprinting contribute to Angelman
Syndrome
-Angelman syndrome is characterized by little or no verbal communication, Intellectual disability,
ataxia, seizures, happy demeanor.
-It is caused by mutation of Ube3a. Because this gene is imprinted in the brain
(expressed only from one allele), heterozygous disruption causes complete loss of ube3a.
Wild type
Expression of maternal Ube3a allele
Ube3a m-/p+
No Ube3a expression
Adapted from Li & Bartolomei Cell 2013
• What are three major criteria for judging the
validity of a disease model?
MeCP2 knockout mice: a model model
Evaluating a disease model:
Construct Validity
How well does the underlying cause
of dysfunction in the model replicate
the human cause.
Normal MeCP2 KO
Face Validity
How well does the model reflect
disease phenotypes and pathologies.
Predictive Validity
How well do new findings, effects of
trial therapeutics predict the effects
in humans with the disorder.
Kazdoba et al. Curr. Topic. Behav. Neurosci. 2016
Guy et al. Nature genetics 2001
Layer II/III Pyramidal cells
MeCP2 KO
Normal
Kishi & Macklis Mol Cell Neurosci. 2004
Breathing abnormalities
MeCP2 Mutant
Normal
Kron et al. Disease Mod. & Mech. 2014
Rubin
• Here are two very open-ended study
questions meant to get students thinking very
broadly about the topic.
– What can brain tumors teach you about normal
brain development and function, and how do they
do it?
– In what ways is brain tumor biology constrained
by normal brain biology?
3. Growth regulation: Brain region
Adult
P53, PTEN, CDKN2A, EGFR, NF1, IDH1,
Pediatric
H3G34V, p53
H3K27M,p53, ATVR1, PDGFR, BRAF
SHH, INI1, BRAF,p53, ATVR1, MYC
Glial tumors with many mutations
Glial, neuronal, ependymal, choroid
plexus tumors with few mutations
4. Brain tumor cells of origin: medulloblastoma
Subependymal
Germinal
Matrices
Fink (2006) J Neurosci
Miller & Kotzbauer
• Name the two proteins/peptides that
pathologically accumulate in Alzheimer’s
disease
• Longer CAG repeat length in Huntington’s
disease increases/decreases/does not affect
age of disease onset.
• Longer CAG repeat length in Huntington’s
disease increases/decreases/does not affect
age of disease onset.
• You have discovered mutations in the noseein
gene responsible for a neurodegenerative disease
in a family that develops progressive loss of
vision. Your genetic studies suggest autosomal
recessive inheritance. Which would be the best
approach to generate a mouse model for this
disorder.
A. Transgenic expression of the WT human noseein gene
B. Transgenic expression of mutant human noseein gene
C. Knockout of the mouse noseein gene
• You have discovered mutations in the noseein
gene responsible for a neurodegenerative disease
in a family that develops progressive loss of
vision. Your genetic studies suggest autosomal
recessive inheritance. Which would be the best
approach to generate a mouse model for this
disorder.
A. Transgenic expression of the WT human noseein gene
B. Transgenic expression of mutant human noseein gene
C. Knockout of the mouse noseein gene