Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts Tomonobu M. Watanabe a,b , Sayaka Higuchi a , Keiko Kawauchi c , Yoshikazu Tsukasaki a , Taro Ichimura a , Hideaki Fujita a,⇑ a Laboratory for Comprehensive Bioimaging, Riken Qbic, Osaka 565-0874, Japan b World Premier Initiative, iFREC, Osaka University, Osaka 565-0871, Japan c Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore article info Article history: Received 17 January 2012 Available online 28 January 2012 Keywords: Histone Nucleus Imaging Epigenetics Myogenesis abstract Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, deter- mining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differen- tiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicat- ing that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics dur- ing differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on dif- ferentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myo- genesis using the state diagram developed with the parameters obtained in this study. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Skeletal muscle tissue is a unique tissue that can convert chem- ical energy into mechanical work at high efficiency, which is sup- ported by a highly ordered structure of thick and thin filaments [1,2]. Myogenesis requires the formation of such a structure, which has been well studied and documented in the last several decades. During skeletal muscle differentiation, myoblasts fuse to form huge elongated multi-nucleated myotubes having sarcomere structures inside [3]. Genetical studies revealed the presence of key transcription factors that govern myogenesis such as Myf5, Myf6, MRF4, MyoD and myogenin [4,5]. These transcription factors are serially expressed, resulting in the expression of muscle spe- cific proteins and terminal differentiation [6]. While gene expres- sion is altered upon differentiation, the epigenetic status is also altered, controlling the gene expression profile. Histone H3 lysine 27 methylation was shown to be important for the repression of muscle-specific genes in growing cells [7], whereas MyoD induces regional histone acetylation under differentiating conditions that modify the epigenetic landscape [8]. Recently, genome-wide changes in the epigenetic landscape during myogenesis were stud- ied using C2C12 myotubes, which show dynamic changes in his- tone modifications [9]. In these studies, epigenetic changes were compared between undifferentiated myoblasts and fully differenti- ated myotubes, but the detailed time course changes of the epige- netic landscape was not fully documented. Detailed time course studies of epigenetical changes are usually difficult because it is necessary to comprehensively investigate the epigenetic state, such as DNA methylation, at each time point, which usually in- volves disruption of the cells. To study the time course of the epigenetic changes of a cell, it is essential that observation is performed in a non-invasive manner; hence optical observation is suitable. One way to visualize the epi- genetic status in a live cell is to characterize the mobility of the chromatin structure. In early studies, the regions of the nucleus with tightly packed DNA were described as heterochromatin re- gions, and euchromatin regions having high gene concentration, 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.091 ⇑ Corresponding author. Address: Laboratory for Comprehensive Bioimaging, Riken Quantitative Biology Center, OLABB, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan. Fax: +81 6 6155 0112. E-mail address: hideaki.fujita@riken.jp (H. Fujita). Biochemical and Biophysical Research Communications 418 (2012) 742–747 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc