Recruitment of serum response factor and hyperacetylation of histones at smooth muscle-specific regulatory regions during differentiation of a novel P19-derived in vitro smooth muscle differentiation system

Little is known regarding transcriptional regulatory mechanisms that contro l the sequential and coordinate expression of genes during smooth muscle ce ll (SMC) differentiation. To facilitate mechanistic studies of SMC differen tiation, we established a novel P19-derived clonal cell line (designated A4 04) harboring a smooth muscle (SM) cr-actin promoter/intron-driven puromyci n resistance gene. Retinoic acid plus puromycin treatment stimulated rapid differentiation of multipotential A404 cells into SMCs that expressed multi ple SMC differentiation marker genes, including the definitive SM-lineage m arker SM myosin heavy chain. Using this system, we demonstrated that variou s transcription factors were upregulated coincidentally with the expression of SMC differentiation marker genes. Of interest, the expression of serum response factor (SRF), whose function is critical for SMC-specific transcri ption, was high in undifferentiated A404 cells, and it did not increase ove r the course of differentiation. However, chromatin immunoprecipitation ana lyses showed that SRF did not bind the target sites of endogenous SMC marke r genes in chromatin in undifferentiated cells, but it did in differentiate d A404 cells, and it was associated with hyperacetylation of histones H3 an d H4. The present studies define a novel cell system for studies of transcr iptional regulation during the early stages of SMC differentiation, and usi ng this system, we obtained evidence for the involvement of chromatin remod eling and selective recruitment of SRF to CArG elements in the induction of cell-selective marker genes during SMC differentiation.