Using an in vitro chromatin remodeling system, we developed new technology to study protein/DNA interactions in vitro in real time. We established a laser-based, UV crosslinking system that allows us to efficiency detect receptor/DNA and remodeling complex/DNA interactions with short, 5 nanosec. UV pulses. Using this system we showed that receptor template interactions during chromatin remodeling are transient and periodic, and that the receptor is actively ejected from the template during the remodeling reaction. This unexpected finding has led to a paradigm shift in the field, and provides a new mechanistic basis for the transient interactions of receptors with promoters we first reported in living cellsWe have discovered that histone deacetylase 1 (HDAC1) is acetylated during hormone induction by GR. Acetylated HDAC1 is found enriched in a GR complex, and the acetylation pattern of the protein corresponds closely to the induction profile of the MMTV promoter. We found by ChIP analysis and immunofluorescence that HDAC1 is present on MMTV chromatin at all times. The general coregulator p300 is also present in the GR bound HDAC1 complex, and increases in concentration in parallel with HDAC1 acetylation. An in vitro acetylation assay demonstrates that HDAC1 can be acetylated by p300 and PCAF. Our findings document the first evidence for acetylation of the HDAC family, and provides new insight into the mechanism of coactivator and corepressor function during gene activation and repression.
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