The Bromodomain Mediates Transcriptional Intermediary Factor 1α-Nucleosome Interactions

Eumorphia Remboutsika,Ken Yamamoto,Matthias Harbers,Marc Schmutz

doi:10.1074/jbc.m203759200

Abstract

Nuclear histone acetyltransferases, DNA-dependent ATPases, and transcriptional intermediary factors (TIFs) all harbor a distinct structural module known as the bromodomain (BrD). Although the BrD can interact with histones H3 and H4 and their acetylated N-terminal tails in vitro, its function in a chromosomal environment remains elusive. We used the nuclear receptor coregulator TIF1alpha, a protein kinase that associates tightly with euchromatin, to analyze the properties of the BrD in a nucleosomal environment in vitro. Here, we report that TIF1alpha-chromatin association is direct and involves DNA and nucleosome interactions mediated by the BrD. Mutation of the BrD signature peptide, PMDL, abolishes DNA binding and disrupts BrD-nucleosome interactions. Based on our results, we propose that the BrD plays a critical role in vivo by directing transregulators to their cognate location on nucleosomal DNA.

Highlights

Nuclear histone acetyltransferases, DNA-dependent ATPases, and transcriptional intermediary factors (TIFs) all harbor a distinct structural module known as the bromodomain (BrD)
When chromatin generated with or without TIF1␣ was subjected to time-dependent microccocal nuclease (MNase) digestion [45], we found that TIF1␣ incorporation had no apparent effect on the regularity of nucleosomal spacing (Fig. 1E)
TIF1␣ Binds to DNA and Nucleosomes—We have examined the functional association of nuclear receptor coactivator TIF1␣ with chromatin templates

Summary

Introduction

DNA-dependent ATPases, and transcriptional intermediary factors (TIFs) all harbor a distinct structural module known as the bromodomain (BrD). We report that TIF1␣-chromatin association is direct and involves DNA and nucleosome interactions mediated by the BrD. Nucleosome Electrophoretic Mobility-shift Assay (EMSA)—Unless otherwise indicated, 1 ␮g (0.7 ␮M) of purified TIF1␣ protein was incubated either with 32P-labeled reconstituted nucleosomes or naked DNA in 10 mM Tris-HCl buffer, pH 7.5, 1 mM EDTA, 15 mM NaCl, and 5% glycerol for 15 min at room temperature.

Results

Conclusion