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Critical role of histone turnover in
neuronal transcription and plasticity
Quick Background
• Histones are DNA “packaging” proteins that
are a base unit of an 8-protein macromolecule
known as a “nucleosome.”
• Important for cell division / DNA replication
• They also influence DNA transcription by
altering availability to DNA and binding affinity
for transcription factors
Histone Families
• Multiple histones (H1, H2a, H2b, H3, H4)
• Multiple different “types” of H3
– H3.1,H3.2 are replication dependent
– H3.3 is replication independent
• Multiple different genes for each type of H3
Critical role of histone turnover in
neuronal transcription and plasticity
• Some theories of epigenetics rely on
nucleosome stability
• If histones are recycled/replaced, then this
doesn’t hold true
• So: is there histone turnover?
– Note: they used FACS to sort cells to only look at
neurons (NeuN+)
Increase in H3.3
• Across lifespan, H3.3 increases relative to
other H3 variants using liquid chromatography
- mass spec (LC-MS)
• Note that weeks
3-7 are relatively
stable
• After 2 years, H3.3
is almost all H3 in
nucleosomes
Turnover of H3.3
• Rats fed chow with heavy labeled lysines
– Non-radioactive lysine isotope, shows up in new
protein in MS
• Ratio increased over
the 2-4 weeks, even
though overall
numbers are stable
• Taken from human post-mortem brains
• Like in mice, H3.3 almost completely replaces
the other H3
proteins
Four things can influence H3.3 levels
• Neurogensis – new neurons could be made with
H3.3 (no turnover)
• Neurodegeneration – neurons with H3.1/2 could
die off (no turnover)
• Histone
synthesis /
degradation
would suggest
turnover
Bomb Pulse Test
• C14 put into air by nukes
• Can measure the C14/C12 ratio and if the H3 protein at
time of death was made before or after the bomb pulse
• This shows that early neurodevelopment during the bomb
trials results in more C14 in H3.3 at death
Pre-bomb
• Looking at just pre-bomb births, a “no turnover”
doesn’t fit the predictive data.
• Adding a slow turnover does fit, however.
Post-bomb
• Slow turnover not enough to explain C14 ratio in
post-bomb people
• Slow turnover AND fast turnover does
Switching to ESC
• Genes expressed in cultured neurons correlate with
gene expression in embryonic neurons (RNA-Seq)
Chip-Seq
• H3.3 bound to DNA in almost all the same places
in embryonic neurons and cultured neurons.
• H3.3 increases rapidly, then levels out for a
while
• Heavy labeling of cultured neurons shows similar incorporation in new
H3.3 neurons
• Eviction shows that turnover keeps happening even during “stable” times
• Matches different turnover rates seen in the human bomb data
H3.3 and gene expression
• H3.1/2 shown to be associated with intragenic
regions
• H3.3 shown to be associated with genes and
promoters, and associated with gene
expression
• Association with gene expression not as
strong in adult neurons
• Embryonic has enriched
PTMs for active expression
• Adult neurons lose these
active PTMs
RT-qPCR
• H3f3b mRNA expression goes up in response
to many stimuli
• Also went up similarly with optogenetically
controlled depolarization
H3 gene translation (western)
• H3/H3.3 levels unchanged by stimulation by
KCl
• More H3f3b translated
Stimulation drives
turnover
• Turnover goes up with
stimulation
• Shown by increasing heavy
ratio in presence of heavy K,
and eviction in absence of
heavy K
Environmental Enrichment
• EE also drives increased expression of H3f3b
mRNA in the hippocampus, similar to direct
neuronal stimulation
EE on H3.3 translation
• As expected, the new mRNA is translated into
new protein in the hippocampus
H3.3 and Chaperone Proteins
• These three proteins are known to be altered
by neuronal stimulation (Atrx, Daxx, Hira)
• No difference in expression, but increased
binding of H3.3 to Hira
shRNA knockdown
• Knocking out Daxx does nothing, but knocking
out Hira decreases the amount of new H3.3,
especially after depolarization
Breakdown of H3.3
• Ubiquitination is a
tag on proteins that
marks them for
degradation
• Ubiquitinated H3.3
goes up in response
to depolarization
Degradation mechanisms
• MG132 is a proteasome inhibitor
• MG132 inhibits production of new H3.3
• MG132 also blocks some eviction (H3.3 levels
stay higher)
Mini-summary
• H3.3 is associated with active genes
• H3.3 turnover is increased in response to
stimulation
• H3.3 turnover is dependent on Hira chaperone
protein for incorporation
• Turnover is dependent on degrading old H3.3
protein
• Inhibiting degradation results in less turnover