Technical Abstract: Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent system in which to perform kinetic studies of chromatin remodeling and transcriptional activation. Using HC11 cells as a model, we have investigated the effects of prolactin and glucocorticoids both singly and in combination at different time points following hormone treatment. Using chromatin immunoprecipitation (ChIP) analysis we have determined the dynamics of assembly and disassembly of Stat5, GR, C/EBP beta, and YY-1 at the hormonally activated beta-casein proximal promoter, as well as the distal mouse beta-casein enhancer located approximately -6 kb upstream of the transcription start site. Prolactin alone resulted in a rapid recruitment of both Stat5 and HDAC1 to the beta-casein promoter and enhancer, and reciprocally the dissociation of YY1 from the proximal promoter. In addition, we have examined the recruitment of co-activator p300 and determined chromatin acetylation status as a function of hormonal treatment. Finally, we have established the time course of RNA polymerase II and phospho-RNA polymerase II accumulation at the beta-casein promoter and enhancer following stimulation with hydrocortisone and prolactin. While glucocorticoids alone led to a rapid increase in histone H3 acetylation, treatment with both hormones was required for stable association of p300 and phospho-RNA polymerase II at both the promoter and enhancer. Collectively, these data suggest a model for the assembly of a multi-protein complex that helps to define how the signaling pathways controlled by these lactogenic hormones are integrated to regulate beta-casein gene expression.