An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

Abstract

Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

Fig. 1. A: Schematic representation of the immobilized template assay.

B: DNA reconstituted into a nucleosomal array was loaded on a 1.2% agarose gel and stained with ethidiumbromide afterwards. Naked DNA was loaded as a control. C: The purified LexA-Mad and LexA-TR(DE) proteins were loaded on an SDS-polyacrylamide (12%) gel and stained with Commassie brilliant blue.

Fig. 2. Determining the deacetylase capacity of the Sin3/HDAC complex and the amount of competitor nucleosome arrays needed to prevent non-targeted deacetylation.

A: Immobilized nucleosomal templates acetylated with the yeast SAGA complex were incubated with the purified Sin3/HDAC complex using various incubation times. The amount of HDAC2 binding to the templates and the amount of histone H3 acetylation was detemined by western blotting using an antibody against HDAC2 and an antibody that recognizes diacetylated histone H3 Lys 9, 14. B: Immobilized nucleosomal templates acetylated with the yeast SAGA complex were incubated for 1 hour at 37°C with the purified Sin3/HDAC complex in the presence of varying amounts of non-immobilized competitor nucleosomal arrays. The amount of histone H3 acetylation and HDAC2 association was subsequently determined as described in A.

Fig. 3. Targeted recruitment of the Sin3/HDAC to long nucleosomal templates pre-acetylated with the yeast NuA4 complex.

Immobilized nucleosomal templates acetylated with the yeast NuA4 complex were incubated with or wihout LexA-Mad and subsequently incubated with the Sin3/HDAC complex in the presence or absence of competitor nucleosomal arrays. The amount of histone H4 acetylation was determined with western blotting with an antibody that recognizes tetra-acetylated histone H4.