However, this reduction in B NHEJ efficiency cannot be reversed by HDAC2 knockdown, despite the efficient pro tein down regulation achieved. We inquired whether the efficient silencing of HDAC2 modifies the acetylation status of chromatin in Lig4 MEFs. Figure 2D shows that despite nearly 90% deple tion of http://www.selleckchem.com/products/Calcitriol-(Rocaltrol).html HDAC2, chromatin acetylation remains low both in exponentially growing as well as in serum deprived cells. We conclude that multiple HDACs contribute to histone deacetylation in Lig4 MEFs, and that as a con sequence inhibition of HDAC2 alone fails to generate detectable effects on chromatin acetylation. Effect of TSA on chromatin acetylation and B NHEJ The lack of histone hyperacetylation following HDAC2 knockdown suggested that inhibition of multiple HDACs is required for global changes in chromatin acetylation.

Therefore, we tested TSA, a non specific inhibitor of class I and II HDACs. Treatment of Lig4 MEFs with 0. 5 uM TSA causes strong hyperacety lation of H3K9Ac, detectable already 2 h after drug ad ministration that is maintained for up to 24 h. This effect is observed both in exponentially growing, as well as in serum deprived cells, although hyperacetylation occurs faster in growing cells. TSA mediated H3K9Ac is reversible within about 2 h of drug removal, irrespectively of treatment duration between 2 8 h, in exponentially growing and serum deprived Lig4 MEFs, as well as in the human tumor cell line, M059K. The cell cycle distribution of TSA treated exponentially growing Lig4 MEFs shows accumulation in S phase and the formation of a sub G1 peak indicative of apoptotic cell death after prolonged incubation with the drug.

Cell cycle effects and toxicity, evidenced as sub G1 peak, are not detectable in serum deprived cells. We conclude that TSA causes fast and rever sible global changes in chromatin acetylation, irrespectively of growth state, within 2 4 h without overly affecting cellu lar integrity or the distribution of cells throughout the cell cycle. To study the effect of global chromatin hyperacetylation on B NHEJ, exponentially growing Lig4 MEFs were trea ted with 0. 5 uM TSA for 4 h and subsequently exposed to 20 Gy X rays. After IR, one set of dishes was incubated with TSA for repair, whereas a second group of dishes was transferred to TSA free growth medium for repair.

Figure 5A shows the level of hyperacetylation achieved and the kinetics of loss of this hyperacetylation upon TSA re moval. There are only minor changes observed in cell cycle distribution in cells treated with Brefeldin_A TSA. Despite the strong hyperacetylation observed and the presumed chromatin decondensation, induction of DSBs by IR remains unchanged. Notably, extensive chro matin hyperacetylation leaves unchanged the kinetics of DSB rejoining by B NHEJ. TSA treatment as described above but for serum deprived Lig4 MEFs shows a prolonged per sistence of hyperacetylated chromatin with out significant shifts in cell cycle distribution.