Calcium-Dependent Dephosphorylation of the Histone Chaperone DAXX Regulates H3.3 Loading and Transcription upon Neuronal Activation

Summary Activity-dependent modifications of chromatin are believed to contribute to dramatic changes in neuronal circuitry. The mechanisms underlying these modifications are not fully understood. The histone variant H3.3 is incorporated in a replication-independent manner into different regions of the genome, including gene regulatory elements. It is presently unknown whether H3.3 deposition is involved in neuronal activity-dependent events. Here, we analyze the role of the histone chaperone DAXX in the regulation of H3.3 incorporation at activity-dependent gene loci. DAXX is found to be associated with regulatory regions of selected activity-regulated genes, where it promotes H3.3 loading upon membrane depolarization. DAXX loss not only affects H3.3 deposition but also impairs transcriptional induction of these genes. Calcineurin-mediated dephosphorylation of DAXX is a key molecular switch controlling its function upon neuronal activation. Overall, these findings implicate the H3.3 chaperone DAXX in the regulation of activity-dependent events, thus revealing a new mechanism underlying epigenetic modifications in neurons.

Total RNA was prepared using RNeasy kit (Qiagen). For reverse transcription, High Capacity RNA-to-cDNA Master Mix (ABI) was used and quantitative real-time PCR was performed in an Eppendorf Realplex Mastercycler using Maxima™ SYBR Green qPCR Master Mix (Fermentas).
Relative abundance of the specific mRNAs was normalized to GAPDH mRNA. Primer sequences are in Supplemental table1.

Virus Preparation and Infection
Lentiviral supernatants were prepared as described previously (Salmon and Trono, 2006).
Briefly, 293T cells plated in 15cm petri dish were transfected using calcium phosphate. Culture supernatants were collected at 48 h after transfection and lentiviral particles were concentrated using PEG (System Bioscience). Virus titrations were performed as described previously on 293T cells by qPCR (Salmon and Trono, 2006). Cortical neurons were infected with lentiviral particles 8 hours after plating and medium changed the day after.

Immunofluorescence
Cortical neurons were grown on glass coverslips coated with poly D-lysine (Sigma-Aldrich).