Testing the Effect of Histone Acetyltransferases on Local Chromatin Compaction.
Methods Mol Biol
; 2589: 361-376, 2023.
Article
em En
| MEDLINE
| ID: mdl-36255637
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Cromatina
/
Histona Acetiltransferases
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article