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, H3.3 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