Regulation of mammalian histone deacetylases by phosphorylation

Nuclear acetylation of nucleosomes by histone acetyltransferases (HATs) generally stimulates transcription, while deacetylation of these substrates by histone deacetylases (HDACs) is correlated with transcriptional repression and silencing. In addition to acetylation, histones may also be modified by methylation, phosphorylation, poly-ADP ribosylation, and ubiquitination. The contribution of these modifications to chromatin structure and metabolism is largely unknown, and the cellular regulation of the enzymes responsible for these events remains to be determined. We utilized mass spectrometry to characterize how the proportion and combinations of these modifications exist on single histone proteins within the cell. We identified the core histones and variants present in human K562 cells, and have determined the modification profiles from asynchronously growing cells. To further understand how these modifications were regulated, we utilized the phosphatase inhibitor, okadaic acid, to increase phosphorylation events within the cell, and compared the modification patterns on histones between untreated and okadaic acid treated cells. One of the most dramatic changes was the loss of acetylation on H2A and H4 after treatment with 1000 nM okadaic acid. We also observed that okadaic acid also partially blocks histone hyperacetylation caused by trichostatin-A, a deacetylase inhibitor, implicating regulation of both HAT and HDAC activities by phosphorylation. Fractionation of HAT and HDAC activities demonstrates that elution profiles for both activities are different after okadaic acid treatment, and that both activities increase. HDAC1 and 2 are detected in multiple fractions containing deacetylase activity and are shown to be phosphoproteins that are hyperphosphorylated after one hour treatment with okadaic acid. Hyperphosphorylation of either HDAC results in a gel migration shift that is sensitive to calf intestinal phosphatase, lambda-phosphatase, and protein phosphatase-1, but not protein phosphatase-2A. This gel migration shift occurs in the absence of protein synthesis and is lost with removal of okadaic acid, suggesting it is a reversible event. Hyperphosphorylation of HDAC2 is also seen in cells blocked in mitosis, but not in cells blocked in S phase. HDAC containing fractions as well as immunoprecipitation of okadaic acid treated HDAC activities show little change after phosphatase treatment. Okadaic acid treatment disrupts co-precipitation of HDAC1 and 2, as well as each HDAC interaction with the co-repressors Sin3A and YY1. In contrast, the interactions between HDAC1 or 2, and RbAp46/48 are not disrupted by treatment with okadaic acid. Under the conditions used, both Sin3A and YY1 are also phosphorylated in an okadaic acid dependent manner. While these phosphorylations may contribute to the disruption of interactions, the removal of phosphorylation from HDAC1 and 2 is sufficient to increase interactions with mSin3A. This establishes the importance of HDAC phosphorylation in controlling protein interactions and provides a mechanism to remove deacetylase mediated transcriptional repression immediately downstream of phosphorylation signaling events. These results suggest that targeted transcriptional repression may be controlled by disruption of HDAC and repressor complexes through phosphorylation.