, 2011). Two of them, the transcription factor Egr2 and the serine/threonine/tyrosine phosphatase Dusp6, were enriched in DAXX immunoprecipitates ( Figure S2H). Overall, DAXX association with c-Fos, Egr2, and Dusp6 was not affected by KCl treatment

( Figures 2B and S2H). We next investigated whether the DAXX-interacting protein ATRX displays similar selectivity for IEG regulatory regions. Indeed, ChIP analysis showed that ATRX interacts with the Bdnf and c-Fos regulatory elements, but it failed to bind the Npas4 gene ( Figure S2I). We confirmed that DAXX and ATRX could interact in isolated cortical neurons ( Figure S2J). KCl treatment did not affect this selleck chemical interaction or ATRX association buy PD173074 with Bdnf and c-Fos regulatory regions ( Figures S2I and S2J). Thus, DAXX and ATRX interact in neurons and display similar binding selectivity for IEG regulatory elements. DAXX has been recently implicated in loading of the histone variant H3.3 as part of a chaperone complex containing ATRX (Elsaesser and Allis,

2010). In view of the presence of both proteins at regulatory regions of selected IEGs, we speculated that DAXX could promote H3.3 loading at these loci. No data was available on induction of H3.3 deposition upon neuronal activation and the potential chaperones involved. To test this hypothesis, we first studied whether DAXX and H3.3 interact in neurons. Coimmunoprecipitation experiments showed that yellow fluorescent protein (YFP)-H3.3 pulled down endogenous DAXX (Figure 3A). Based on these data, we analyzed H3.3 association with regulatory regions of activity-regulated genes

by using an H3.3-specific antibody (Figures S3A and S3B). DAXXFlox/WT or DAXXFlox/Flox neurons infected with CRE particles were depolarized with KCl for 3 hr. We found that neuronal activation clearly induced CYTH4 H3.3 deposition at regulatory regions of all genes included in this study (Bdnf Exon IV, c-Fos, Npas4, Zif 268, Nurr1, Ier2, Gadd45g, Egr2, Dusp6, and Arc; Figures 3B–3D and  S3C). This was not due to increased nucleosome density, because anti-H4 ChIP failed to show increased H4 binding at regulatory regions of Bdnf Exon IV and c-Fos upon membrane depolarization ( Figure S3D). DAXX depletion led to clear impairment in KCl-triggered loading of H3.3 at the regulatory elements of Bdnf Exon IV ( Figure 3B; see regions 2 and 3 in Figure 2A), c-Fos ( Figure 3C; see regions 1–3 in Figure 2B), Egr2, and Dusp6 ( Figure S3C). DAXX depletion did not interfere with nucleosome density at these genes ( Figure S3D). Deposition of H3.3 at the c-Fos transcribed region was DAXX-independent, indicating that DAXX is not required for loading at this region ( Figure 3C). This is in agreement with the HIRA-dependent deposition of H3.3 at actively transcribed genes ( Goldberg et al., 2010).