Chromatin organization regulated by EZH2-mediated H3K27me3 is required for OPN-induced migration of bone marrow-derived mesenchymal stem cells

Highlights

• OPN-induced BMSC migration increases chromatin compaction and the levels of heterochromatin-specific epigenetic markers.

• OPN-induced BMSC migration increases the levels of EZH2 histone methyltransferase.

• Pharmacological inhibition or depletion of EZH2 suppresses BMSC migration.

• Chromatin structure may affect the mechanical properties of the nucleus and BMSC migration.

• OPN increases BMSC chromatin condensation via the ERK1/2 signaling pathway.

Abstract

Osteopontin (OPN) is a chemokine-like extracellular matrix-associated protein involved in the migration of bone marrow-derived mesenchymal stem cells (BMSCs). An increasing number of studies have found that chromatin organization may affect cellular migration. However, whether OPN regulates chromatin organization is not understood, nor are the underlying molecular mechanisms. In this study, we investigated the link between chromatin organization and BMSC migration and demonstrated that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3) through the methyltransferase EZH2. The expression of EZH2 reorganizes the chromatin structure of BMSCs. Pharmacological inhibition or depletion of EZH2 blocks BMSC migration. Moreover, using an atomic force microscope (AFM), we found that chromatin decondensation alters the mechanical properties of the nucleus. In addition, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signals represses OPN-promoted chromatin condensation and cell migration. Thus, our results identify a mechanism by which ERK1/2 signalling drives specific chromatin modifications in BMSCs, which alters chromatin organization and thereby enables OPN-mediated BMSC migration.

Introduction

Osteopontin (OPN), also known as secreted phosphorylated glycoprotein, is a soluble protein present in most bodily fluids. OPN acts intracellularly as a regulator of gene expression. Extracellular OPN functions by binding to multiple cell surface receptors, including CD44 and various integrins (such as α4β1 and α9β1) (Stemberger et al., 2014). The binding of OPN to these receptors regulates a diverse range of biological processes (Kahles et al., 2014), such as cell activity, adhesion, migration, and survival, in many cell types (Hirano et al., 2015).

Bone marrow-derived mesenchymal stem cells (BMSCs) are capable of transmigrating into other tissues far from their niche and may have roles in tissue regeneration, repair and reconstruction (Pittenger and Martin, 2004). Tissue injuries are often accompanied by various factors that may provide cues to mobilize BMSCs to the site of tissue damage. By directly differentiating into multiple cell lineages and/or by releasing growth factors to aid in tissue repair, BMSCs play beneficial roles in tissue repair (Smart and Riley, 2008). It has also been found that OPN is significantly upregulated in response to inflammation and injury in many tissues (Kahles et al., 2014). Our previous study indicated that the binding of OPN to integrin β1 promotes BMSC migration (Zou et al., 2013). However, little is known about the downstream molecules involved in the regulation of OPN-promoted BMSC migration.

Recent findings have suggested that migrating cells have high deformability, and this property may be necessary for them to migrate through small spaces (McGregor et al., 2016; Harada et al., 2014; Swaminathan et al., 2016). Indeed, it has been implied that for proper cell migration, the nucleus, which is the largest and most rigid organelle in the cell, must change its morphology (Friedl et al., 2011). Dynamic reshaping of the nucleus during cell migration has been observed in various cell types (Lammermann et al., 2008; Gerlitz and Bustin, 2011; Lautscham et al., 2015). Despite these findings, very little research has been done to address the nature of the structural changes occurring within the nucleus during cell migration. Inside the nucleus, the chromatin fibre continuously changes in response to a variety of internal and external signals.

During interphase, chromatin can be configured into actively transcribed euchromatin domains, which are relatively decondensed, or as non-transcribed, condensed heterochromatin (Gerlitz and Bustin, 2011). Important structural changes in the nucleus occur through epigenetic mechanisms (Zhang et al., 2016). The organization and level of chromatin condensation are determined by DNA methylation, posttranslational modifications of histone proteins and architectural proteins (Crider et al., 2012). Given that the chromatin fibre occupies a large portion of the total nuclear volume (Gerlitz and Bustin, 2010), a relationship between chromatin organization and cell migration should be expected. Indeed, increases in the level of the heterochromatin marker trimethyl Lys27 in histone H3 (H3K27me3) in response to migration cues have been previously observed (Gerlitz et al., 2007). H3K27me3 is mediated by Enhancer of zeste homologue 2 (EZH2), a member of the polycomb group family of methyltransferases. EZH2 is the catalytic component of polycomb repressive complex 2 (PRC2) (Maleszewska et al., 2016), which binds to target gene promoters, leading to epigenetic repression via trimethylation of histone H3 at the residue lysine 27 (Lv et al., 2015). Multiple recent results have suggested that EZH2 is involved in the regulation of cell migration (Lv et al., 2015; Liu et al., 2016; Rao et al., 2010). However, the mechanisms connecting these changes in the nucleus with BMSC migration are unclear.

Here, we show that proper cell migration is not only associated with but also contingent on chromatin condensation. Next, we demonstrate that induction of BMSC migration leads to an increase in the levels of additional epigenetic H3K27me3 markers associated with facultative heterochromatin. We identify EZH2 as the enzyme responsible for these epigenetic changes and show how it affects BMSC migration. Thus, we show that changes in chromatin structure affect cellular migration. Together, our results reveal that condensed chromatin has a structural role in supporting nuclear movement, which is required for efficient BMSC migration.