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

• 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+)

Critical role of histone turnover in neuronal transcription and plasticity

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, H3.3is almost all H3 innucleosomes

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 H3proteins

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 enrichedPTMs for active expression

• Adult neurons lose theseactive 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