During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin- modifying enzymes to muscle-specific loci 1,2 . The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway 3–5 targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38α/β repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38α-β in vitro, and forced activation of p38α/β in myoblasts by expression of a constitutively active MKK6 (refs. 5–7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements. Pharmacological blockade of p38α/β kinases by SB203580 inhibits the myogenic program 3–5 by repressing the transcription of early (myo- genin; Myog) and late (muscle-creatine kinase; Ckm) muscle genes in myoblasts induced to differentiate (Fig. 1a). We monitored by chro- matin immunoprecipitation (ChIP) the effect of p38 inhibition on the assembly of the myogenic transcriptosome and on chromatin modifi- cations at the Myog promoter and the Ckm enhancer during myoblast differentiation. The muscle-regulatory factors (MRFs) MyoD and MEF2C bound the Myog promoter within the first hours of incubation in differentiation medium (Supplementary Fig. 1 online) and throughout the differentiation process, regardless of the presence of SB203580 (Fig. 1b). At the Ckm enhancer, MRF binding was delayed (data not shown) and not influenced by SB203580 (Fig. 1b). p38α was selectively recruited to chromatin occupied by MRFs during muscle differentiation (Fig. 1b), as MRFs and p38 were not detected at these sequences in undifferentiated myoblasts (Fig. 1b) or in fibroblasts cul- tured in differentiation medium (data not shown). MRFs and p38 were also not found on the coding regions of Ckm (Supplementary Fig. 2 online) or on the Igh enhancer (Fig. 1b), which contains E-boxes occupied by E-proteins only in B cells 8 . An interaction between Mef2A-C and p38 was suggested by previous studies 4,5,9,10 , and MyoD could bind p38 in vitro (Supplementary Fig. 2 online), suggesting that MRFs might mediate p38 recruitment to muscle-regulatory elements. We next investigated the effect of p38 inhibition on chromatin mod- ifications produced by MRF-recruited enzymes, including p38, histone acetyltransferases (p300, PCAF) 1,2,11,12 and the chromatin-remodeling SWI-SNF complex 13 . In undifferentiated myoblasts, histones H4 and H3 within the regulatory elements of Myog and Ckm were hypoacety- lated and hypophosphorylated (Fig. 1b). We detected hyperacetylation of histones and phosphoacetylation of histone H3 at the Myog pro- moter and the Ckm enhancer after incubation in differentiation medium in cells treated with SB203580 and in untreated cells, which at times correlated with MRF binding (Fig. 1b and Supplementary Fig. 1 online). In contrast, treatment with SB203580 eliminated the increased phosphoacetylation of histone H3 at the Jun promoter caused by TNFα in myoblasts (Supplementary Fig. 3 online). These results suggest that p38 is involved in phosphoacetylation of histone H3 at promoters acti- vated by inflammatory cytokines 14 , but not muscle-specific promoters. Histone hyperacetylation reflects the activity of acetyltrans- ferases recruited by MRFs. Previous reports showed that p300-CBP and PCAF directly acetylate MyoD 15,16 . We evaluated the acetyla- tion status of MyoD at the regulatory elements of Myog and Ckm by using antibodies to acetylated MyoD 17 . Acetylated MyoD was detectable at the Myog promoter after a few hours in differentiation medium (Fig. 1b and Supplementary Fig. 1 online) and at the Ckm enhancer after 18–24 h in differentiation medium (Fig. 1b). The distinct kinetics of chromatin binding of acetylated MyoD at these p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci Cristiano Simone 1,2,4 , Sonia Vanina Forcales 1,4 , David A Hill 3 , Anthony N Imbalzano 3 , Lucia Latella 1 & Pier Lorenzo Puri 1,2 LETTERS 1 Laboratory of Gene Expression, Dulbecco Telethon Institute at Fondazione A. Cesalpino, Institute of Cell Biology and Tissue Engineering, San Raffaele Biomedical Science Park of Rome, Rome 00128, Italy. 2 The Salk Institute for Biological Studies, Peptide Biology Laboratory, La Jolla, California 92093, USA. 3 University of Massachusetts Medical School, Department of Cell Biology, Worcester, Massachusetts 01655, USA. 4 These authors equally contributed to this work. Correspondence should be addressed to P.L.P. (plpuri@dti.telethon.it). Published online 20 June 2004; doi:10.1038/ng1378 738 VOLUME 36 | NUMBER 7 | JULY 2004 NATURE GENETICS © 2004 Nature Publishing Group http://www.nature.com/naturegenetics