Role for tyrosine phosphorylation of SUV39H1 histone methyltransferase in enhanced trimethylation of histone H3K9 via neuregulin-1/ErbB4 nuclear signaling

https://doi.org/10.1016/j.bbrc.2019.02.130Get rights and content

Highlights

  • 4ICD phosphorylates SUV39H1 upon NRG-1 stimulation.

  • Major phosphorylation sites are Tyr-297, -303, and −308.

  • Tyrosine phosphorylation of SUV39H1 enhances H3K9me3 levels in NRG-1/ErbB4 signaling.

Abstract

Protein-tyrosine kinases transmit signals by phosphorylating their substrates in diverse cellular events. The receptor-type tyrosine kinase ErbB4, a member of the epidermal growth factor receptor subfamily, is activated and proteolytically cleaved upon ligand stimulation, and the cleaved ErbB4 intracellular domain (4ICD) is released into the cytoplasm and the nucleus. We previously showed that generation of nuclear 4ICD by neuregulin-1 (NRG-1) stimulation enhances the levels of trimethylation of histone H3 at lysine 9 (H3K9me3). However, it remains unclear how nuclear 4ICD enhances H3K9me3 levels. Here we show that the histone H3K9 methyltransferase SUV39H1 associates with NRG-1/ErbB4-mediated H3K9me3. Knockdown of SUV39H1 blocked NRG-1-mediated enhancement of the levels of H3K9me3. Nuclear 4ICD was found to phosphorylate SUV39H1 primarily at Tyr-297, -303, and −308 that are conserved among humans, mice, and flies. Furthermore, knockdown-rescue experiments showed that the unphosphorylatable SUV39H1 mutant (3 YF) was incapable of enhancing the levels of H3K9me3 upon NRG-1 stimulation. These results suggest that nuclear ErbB4 enhances H3K9me3 levels through tyrosine phosphorylation of SUV39H1 in NRG-1/ErbB4 signal-mediated chromatin remodeling.

Introduction

Protein-tyrosine phosphorylation plays key roles in a variety of intracellular signaling pathways involved in cell proliferation, differentiation, gene expression, cell adhesion, and metabolic changes [1]. While receptor-type tyrosine kinases localize to the plasma membrane, most of non-receptor-type tyrosine kinases are present in the cytoplasm but some are at various intracellular locations. We have shown that tyrosine kinases residing in the nucleus are capable of phosphorylating their nuclear substrates to regulate chromatin structural changes and gene expression [[2], [3], [4], [5], [6], [7], [8], [9]].

The receptor-type tyrosine kinase ErbB4 is a member of the epidermal growth factor receptor subfamily and is essential for development or maintenance of the heart, the mammary glands, and the nervous system [[10], [11], [12]]. Notably, ErbB4 has a unique characteristic that ligand stimulation generates the soluble ErbB4 intracellular kinase domain. Stimulation with neuregulin-1 (NRG-1) sequentially cleaves the ErbB4 extracellular domain (4ECD) by the tumor necrosis factor-α-converting enzyme (TACE) and the ErbB4 intracellular domain (4ICD) by γ-secretase, resulting in the release of 4ICD into the cytoplasm [13,14]. 4ICD acts as a non-receptor-type tyrosine kinase having one functional nuclear localization signal (NLS) and three putative nuclear export signals and can shuttle between the cytoplasm and the nucleus, suggesting the possibility of ErbB4's nuclear functions [14,15]. In fact, 4ICD was shown to act as a transcriptional regulator in the nucleus through protein-protein interactions [16,17]. Importantly, we showed that nuclear 4ICD enhances the levels of trimethylation of histone H3 at lysine 9 (H3K9me3), which is crucial for heterochromatin formation and epigenetic gene silencing [17]. These functions of nuclear ErbB4 depend on its kinase activity, suggesting the involvement of tyrosine phosphorylation of its unidentified substrate(s).

In this study, we showed the histone methyltransferase SUV39H1 as a nuclear substrate of 4ICD and the importance of tyrosine phosphorylation of SUV39H1 for NRG-1/ErbB4-induced H3K9me3 in chromatin dynamics.

Section snippets

Cells and transfection

COS-1 and HeLa S3 cells (JCRB) were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 1% fetal bovine serum (FBS) and 4% bovine serum. T47D cells (provided by M. Tagawa, Chiba Cancer Center Research Institute) were cultured in IMDM supplemented with 5% FBS. Cells were transfected with plasmid DNA using polyethylenimine [18]. Stimulation with NRG-1 was performed as described previously [17]. The ErbB-family inhibitor AG1478 (20 μM) was added 30 min before NGR-1

Requirement of SUV39H1 for nuclear ErbB4 kinase-dependent H3K9me3

We showed that nuclear 4ICD enhances the levels of H3K9me3, which is critical for heterochromatin formation and epigenetic gene silencing [17]. To explore how nuclear ErbB4 enhances H3K9me3 levels, we examined the role of the histone methyltransferase SUV39H1 [21] that selectively trimethylates H3K9 in nuclear ErbB4-dependent H3K9me3. We established a stable SUV39H1 knockdown HeLa S3 cell line using shRNA against SUV39H1 (HeLa S3/shSUV39H1) (Fig. 1B). Consistent with previous studies [22,23],

Discussion

Our previous study demonstrated for the first time that NRG-1/ErbB4 nuclear signaling enhances the levels of H3K9me3 in a manner dependent on 4ICD's tyrosine kinase activity [17]. Now, this study shows that NRG-1/ErbB4 nuclear signaling enhances H3K9me3 levels through SUV39H1 phosphorylation at Tyr-297, -303, and -308. Our previous study also showed that the enhanced levels of H3K9me3 by NRG-1/ErbB4 nuclear signaling further lead to transcriptional repression of the human telomerase reverse

Conflicts of interest

The authors declare that there is no conflict of interest.

Acknowledgements

We thank Dr. H. Miyoshi and Dr. M. Tagawa for the invaluable plasmid and cells. This work was supported in part by grants-in-aid for scientific research from the MEXT, Japan (15K07922) and the scholarship donation from the Daiichi Sankyo Co., Ltd.

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