The Histone Methyltransferase SETDB1 and the DNA Methyltransferase DNMT3A Interact Directly and Localize to Promoters Silenced in Cancer Cells

Hongwei Li,Tibor Rauch,Zhao-Xia Chen,Piroska E Szabó,Arthur D Riggs,Gerd P Pfeifer

doi:10.1074/jbc.m513249200

Abstract

DNA CpG methylation can cooperate with histone H3 lysine 9 (H3-K9) methylation in heterochromatin formation and gene silencing. Trimethylation of H3-K9 by the recently identified euchromatic histone methyltransferase SETDB1/ESET may be responsible for transcriptional repression of certain promoters. Here, we show that SETDB1 associates with endogenous DNA methyltransferase activity. SETDB1 interacts with the de novo DNA methyltransferases DNMT3A and DNMT3B but not with the maintenance methyltransferase DNMT1. The interaction of SETDB1 with DNMT3A was further characterized and confirmed by in vivo and in vitro interaction studies. A direct interaction of the two proteins occurs through the N terminus of SETDB1 and the plant homeodomain of DNMT3A. Co-expression of SETDB1 and DNMT3A was essential for repression of reporter gene expression in a Gal4-based tethering assay and resulted in their recruitment to the artificial promoter. We further demonstrate that the CpG-methylated promoters of the endogenous p53BP2 gene in HeLa cells and the RASSF1A gene in MDA-MB-231 cells are simultaneously occupied by both SETDB1 and DNMT3A proteins, which provides evidence for SETDB1 being at least partly responsible for H3-K9 trimethylation at the promoter of RASSF1A, a gene frequently silenced in human cancers. In summary, our data demonstrate the direct physical interaction and functional connection between the H3-K9 trimethylase SETDB1 and the DNA methyltransferase DNMT3A and thus contribute to a better understanding of the complexity of the self-reinforcing heterochromatin machinery operating at silenced promoters.

Highlights

DNA methyltransferases (DNMTs) have been demonstrated to silence gene expression, at least in part through recruitment of methyl-CpGbinding domain proteins (MBDs) to CpG-methylated DNA, which interact with components of histone deacetylase (HDAC) complexes and recruit them to methylated DNA regions, where a compacted chromatin structure is generated [1, 14, 15]
We find that SETDB1 associates with endogenous DNA methylase activity, which is most likely provided by DNMT3A, since SETDB1 interacts with DNMT3A in vitro and in vivo through its N-terminal domain but not with the maintenance methylase DNMT1
SETDB1 Associates with DNMT Activity—The H3-K9 trimethylase ESET/SETDB1 interacts with HDAC1 [17] and can repress euchromatic gene expression through methylated H3-K9 bound heterochromatin protein 1 (HP1) [40]

Summary

Introduction

DNMTs have been demonstrated to silence gene expression, at least in part through recruitment of methyl-CpGbinding domain proteins (MBDs) to CpG-methylated DNA, which interact with components of histone deacetylase (HDAC) complexes and recruit them to methylated DNA regions, where a compacted chromatin structure is generated [1, 14, 15]. HP1-␣ associates with DNMT3B, which suggests the recruitment of DNA methyltransferase upon histone H3-K9 methylation and thereby may explain the dependence observed in Suv39h knock-out ES cells [24]. We find that SETDB1 associates with endogenous DNA methylase activity, which is most likely provided by DNMT3A, since SETDB1 interacts with DNMT3A in vitro and in vivo through its N-terminal domain but not with the maintenance methylase DNMT1. Our results here provide a physiological and functional connection between DNA methylation and histone methylation and further support the concept of an interdependence of these two modifying enzyme systems in a self-reinforcing, circular pathway

Methods

Results

Conclusion