Histone chaperones function in histone transfer and regulate the nucleosome occupancy and the activity of genes. HIRA is a replication-independent (RI) histone chaperone that is linked to transcription and various developmental processes. Here, we show that HIRA interacts with Mef2 and contributes to the activation of Mef2-target genes during muscle differentiation. Asf1 cooperated with HIRA and was indispensable for Mef2-dependent transcription. The HIRA R460A mutant, which is defective in Asf1 binding, lost the transcriptional co-activation. In addition, the role of Cabin1, previously reported as a Mef2 repressor and as one of the components of the HIRA-containing complex, was delineated in Mef2/HIRA-mediated transcription. Cabin1 associated with the C-terminus of HIRA via its N-terminal domain and suppressed Mef2/HIRA-mediated transcription. Expression of Cabin1 was dramatically reduced upon myoblast differentiation, which may allow Mef2 and HIRA/Asf1 to resume their transcriptional activity. HIRA led to more permeable chromatin structure marked by active histone modifications around the myogenin promoter. Our results suggest that histone chaperone complex components contribute to the regulation of Mef2 target genes for muscle differentiation. (C) 2011 Elsevier Inc. All rights reserved.