Accordingly, we hypothesized HDACi induced increases in b catenin stabilization and nuclear localization accounted for Olig2 suppres sion in our cell fate assays. However Western dilution calculator blot ana lysis failed to detect significant differences in nuclear protein levels of b catenin in adult mouse NSCs trea ted with HDACi under proliferation culture conditions. We speculate HDACi effects on other targets offset this competitive interaction. One possible candidate is Hdac6, a class IIb HDAC. Hdac6 deacetylates b cate nin at lysine 49 to reduce b catenin phosphor ylation and promote b catenin nuclear localization and c myc induction. Thus inhibition of Hdac1 2 and Hdac6 activity has the capacity to promote opposing effects on b catenin stability and nuclear localization by increasing stability via inhibition of Hdac1 2 as well as decreasing stability and nuclear localization as a result of increased Lys49 acetylation and phosphorylation.

Indeed differential sensitivities of Hdac1 2 and Hdac6 to SAHA and NaB inhibition might underlie the different fold changes in b catenin nuclear localization compared to vehicle controls. The fact that the effects of HDACi are con sistent with anti proliferative responses to pharmacolo gical and genetic interventions targeting the canonical Wnt b catenin signaling pathway in adult NSCs suggests the net effect of these molecules is to inhibit rather than activate this signaling pathway. Conclusion In summary, the broad class I and class II HDAC inhi bitors SAHA and NaB blocked G1 to S phase progres sion in proliferating adult NSCs in vitro.

Gene expression changes induced by SAHA and NaB treat ment in adult NSCs vary in fold change but not direc tionality, consistent with the comparable treatment outcomes of G1 arrest. In addition, the direction of gene changes induced by SAHA and NaB treatment is consistent with G1 arrest accompanied by a reduction of stem progenitor state and activation of neuronal lineage commitment programs. SAHA and NaB treat ment induces increases in the transcription of Cdk inhibitors p21 and p27 in adult NSCs which was asso ciated with elevated H3K9 acetylation levels at proxi mal promoter regions. This association is consistent with direct SAHA and NaB effects on cell cycle arrest genes in adult NSCs, in common with widely reported HDACi induced growth arrest in normal and trans formed cells.

Finally, we show HDACi treat ment under proliferation culture conditions leads to long term changes in cell fate in adult NSCs induced to differentiate in vitro. Methods Animals 8 10 week old male and female C57BL 6J mice were purchased from The Jackson Laboratory and housed in the Uniformed Services Universitys Cen ter for Laboratory Animal Medicine prior to experimen tal use. Batimastat Animals were handled in accordance with procedures approved by the Uniformed Services Univer sity of the Health Sciences Institutional Animal Care and Use Committee.