Experiments determining the chromatin association of histone acetylases (HATs) and deacetylases (HDACs) at the genome-wide level provide precise maps of locus occupancy, but do not allow conclusions on the functional consequences of this locus-specific enrichment. Here we describe a protocol that allows tethering of HATs or HDACs to specific genomic loci upon fusion with a fluorescent protein and a DNA-binding protein such as the E. coli Lac repressor (LacI), which binds to genomically inserted lac operon sequences (lacO) via DNA/protein interactions. Integration of these lacO sequences into a genomic region of interest allows to monitor the functional consequences of HAT/HDAC targeting on chromatin (de)compaction, histone modification, and interaction with other proteins by quantitative light microscopy, as described here. As DNA-binding of LacI can be tightly controlled by the addition of galactose-derivatives, this method also allows to monitor the effects of locus-specific recruitment in a time-resolved manner.
- Pfisterer, M.
- Schmitz, M. L.
Keywords
- *Histone Acetyltransferases/genetics/metabolism
- *Chromatin/genetics
- Lac Repressors/genetics
- Histones/metabolism
- Escherichia coli/genetics/metabolism
- Galactose
- Histone Deacetylases/metabolism
- DNA/genetics/metabolism
- Acetylation
- Acetyltransferases/metabolism
- Chromatin
- Histone acetyltransferases
- Histone modification
- Immunofluorescence
- LacI/lacO
