Abstract
Changes in the environment of a cell precipitate extracellular signals and sequential cascades of protein modification and elicit nuclear transcriptional responses. However, the functional links between intracellular signaling-dependent gene regulation and epigenetic regulation by chromatin-modifying proteins within the nucleus are largely unknown. Here, we describe novel epigenetic regulation by MAPK cascades that modulate formation of an ATP-dependent chromatin remodeling complex, WINAC (WSTF Including Nucleosome Assembly Complex), an SWI/SNF-type complex containing Williams syndrome transcription factor (WSTF). WSTF, a specific component of two chromatin remodeling complexes (SWI/SNF-type WINAC and ISWI-type WICH), was phosphorylated by the stimulation of MAPK cascades in vitro and in vivo. Ser-158 residue in the WAC (WSTF/Acf1/cbpq46) domain, located close to the N terminus of WSTF, was identified as a major phosphorylation target. Using biochemical analysis of a WSTF mutant (WSTF-S158A) stably expressing cell line, the phosphorylation of this residue (Ser-158) was found to be essential for maintaining the association between WSTF and core BAF complex components, thereby maintaining the ATPase activity of WINAC. WINAC-dependent transcriptional regulation of vitamin D receptor was consequently impaired by this WSTF mutation, but the recovery from DNA damage mediated by WICH was not impaired. Our results suggest that WSTF serves as a nuclear sensor of the extracellular signals to fine-tune the chromatin remodeling activity of WINAC. WINAC mediates a previously unknown MAPK-dependent step in epigenetic regulation, and this MAPK-dependent switching mechanism between the two functionally distinct WSTF-containing complexes might underlie the diverse functions of WSTF in various nuclear events.
Highlights
Intracellular signaling impacts gene regulation and thereby modulates cell proliferation, differentiation, migration, and apoptosis
Vitamin D receptor (VDR)3 is a member of the steroid/thyroid hormone nuclear receptor superfamily regulating bone metabolism, calcium homeostasis, and cell differentiation by binding with 1␣,25-dihydroxyvitamin D3 (D3), a physiologi
WICH serves as an ISWI-type chromatin remodeling complex, which is responsible for recovery from DNA damage at various steps [3, 24]
Summary
Plasmids—The expression vector for the WSTF-S158A and -S158E mutants was constructed with a site-directed mutagenesis kit (Stratagene, La Jolla, CA) using a pcDNA3-FLAG-WSTF plasmid [1] as a template vector. The pGEX-WAC-S158A mutant was constructed with the site-directed mutagenesis kit using pGEX-4T-WAC (amino acids 1–162) with the same primers as described above. The expression vector for activated transforming growth factor- receptor (TR-I(TD)) was kindly provided by Dr Kohei Miyazono as described in our previous paper [25]. Kinase Inhibitors—ERK inhibitor (U0126), JNK inhibitor (SP600125), and Akt inhibitor (Akt inhibitor X; 124020) were purchased from Calbiochem. P38 inhibitor (SB203580) was purchased from Sigma Kinase Inhibitors—ERK inhibitor (U0126), JNK inhibitor (SP600125), and Akt inhibitor (Akt inhibitor X; 124020) were purchased from Calbiochem. p38 inhibitor (SB203580) was purchased from Sigma
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