HDAC8 regulates neural differentiation through embryoid body formation in P19 cells

https://doi.org/10.1016/j.bbrc.2018.02.195Get rights and content

Highlights

  • Selective HDAC8 inhibitor causes reduced embryoid body (EB) formation in P19 cells.

  • HDAC8-inhibited EBs show decreased neural differentiation activity.

  • HDAC8 inhibition leads to G2/M phase accumulation.

  • HDAC8 inhibition causes downregulation of cyclin A2 and cyclin B1 gene expression.

  • CRISPR-Cas9-mediated HDAC8 knockout cells show smaller EBs.

Abstract

Histone acetylation and deacetylation correlate with diverse biological phenomena through gene transcription. Histone deacetylases (HDACs) regulate deacetylation of histones and other proteins. However, as a member of the HDAC family, HDAC8 function during neurodevelopment is currently unknown. Therefore, we investigated HDAC8 function during neurodevelopment by examining embryoid body (EB) formation in P19 cells. HDAC8-selective inhibitor (NCC-149) (HDAC8i)-treated cells showed smaller EBs than non-treated cells, as well as reduced expression levels of the neuronal marker, NeuN. Additionally, HDAC8i treatment led to inhibition of cellular proliferation by G2/M phase accumulation and downregulated cyclin A2 and cyclin B1 gene expression. Furthermore, two independent HDAC8 knockout cell lines were established by CRISPR-Cas9, which resulted in smaller EBs, similar to HDAC8i-treated cells. These results suggest that HDAC8 regulates neural differentiation by exerting control of EB formation.

Introduction

Histone acetylation and deacetylation are epigenetic processes that regulate gene expression, and in turn, cellular function [1,2]. Histone acetyl transferases transfer acetyl groups to lysine residues on histones causing relaxation of the chromatin structure because of reduced interaction between DNA and histones. Histone deacetylases (HDACs) are a group of enzymes responsible for deacetylation of lysine residues on histones and non-histone proteins. Deacetylation of histones alters chromatin structure, thereby regulating downstream gene expression and subsequently many cellular processes. HDAC isoforms are classified into four groups: Class I (HDAC1, 2, 3, and 8), which are homologous to yeast RPD3; Class II (HDAC4–7, 9 and 10), which are homologous to yeast Hda1; Class III (Sirtuin 1–7); and Class IV (HDAC11) [3,4]. HDAC inhibition has extensively been shown to suppress proliferation and alter differentiation in tumor cells. Indeed, HDAC members are clinical target molecules for cancer management [5,6].

HDAC8 is a unique member of the Class I HDACs. HDAC8 shares only 43% sequence identity with other members of its class and has a shorter C terminal region [7]. Moreover, HDAC8 has a unique expression pattern and is abundantly expressed in the brain, kidney, and prostate [7,8]. Furthermore, HDAC8 is implicated in a number of diseases including neuroblastoma (with its high expression associated with poor prognosis) [9], and Cornelia de Lange syndrome (CdLS) [10,11].

However, little is known about the role of HDAC8 during development and differentiation of neurogenesis in vitro, which led us to hypothesize that HDAC8 inhibition might regulate these processes [9]. In this study, we investigated the involvement of HDAC8 inhibition on embryonic neurogenesis using retinoic acid (RA)-treated P19 cells as a model for neural progenitor cells, and a highly potent and selective HDAC8 inhibitor [10] that our group had previously discovery [12,13]. Moreover, using the CRISPR-Cas9 system, we constructed HDAC8 knockout (KO) cells and analyzed them. Here, we show that HDAC8 regulates neural differentiation through embryoid body (EB) formation.

Section snippets

Materials

Anti-NeuN antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-tubulin antibody was obtained from Wako Pure Chemical Industries (Osaka, Japan).

Cell culture and differentiation of P19 cells

P19 embryonic carcinoma cells were grown on Minimum Essential Medium Eagle, Alpha Modification (α-MEM; Wako) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA). Cells were passaged at least twice before differentiation. Cell differentiation was performed according to the procedure of

Selective HDAC8 inhibitor treatment causes reduced EB formation and neural differentiation in P19 cells

To clarify HDAC8 function in neural cells and during neural differentiation, we used the P19 embryonic carcinoma cell line. EB formation is essential for differentiation into neurons and glia cells [18]. Thus, we investigated the role of HDAC8 in EB formation in the presence of RA and a HDAC8 selective inhibitor, NCC-149 (i.e., HDAC8i) [12]. Accordingly, we found that EB size was reduced in a dose-dependent manner (Fig. 1A and B). To confirm that this phenotype was not due to downregulation of

Discussion

HDAC family proteins regulate gene expression through histone deacetylation. The HDAC family consists of 11 HDAC and seven sirtuin members [3,4]. Although there have been many HDAC studies, several HDAC8 functions are currently unclear. HDAC8 is likely associated with neural function and development. In fact, HDAC8 inhibition increases neurite outgrowth and expression in BE(2)-C neuroblastomas [24]. Thus, we further investigated HDAC8 function during neurodevelopment, and show that

Conflicts of interest

None to declare.

Acknowledgements

This work was supported in part by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2015–2019 to T.I. We thank Ms. Yuka Kimura and Ms. Kyoko Yakiyama for helping preliminary research. We thank Rachel James, Ph.D., from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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    We declare that this paper includes part of the doctoral dissertation of J. O. Makanga.

    1

    These authors contributed equally to this work.

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