Technical Abstract: The genes encoding the caseins (CSN), major milk proteins, are located in an approximately 1MB mammalian specific domain on the human genome (~700kb in mouse genome). This domain harbors about 20 genes, is AT-rich (<37% GC), and has a repeat content slightly below average (39%) [1]. The genes in the casein domain encode secreted proteins expressed in tissues with an epithelial component (predominantly in mammary and salivary gland). Besides the nutritional function of the caseins many of these proteins have host defense properties as well as the capability to bind calcium. The CNS-region is characterized by its high evolutionary divergence. Comparative sequence analyses have shown that this domain has a conserved synteny and organization in human, chimpanzee, mouse, rat, rabbit, cow, dog and presumably most mammals. However, comparative analysis with the chicken, fugu and zebra fish genomes indicated the absence of this entire domain. By comparing the genomic sequence of human, mouse, rat and cow we identified 13 evolutionary conserved regions (ECR’s) at intergenic positions [1]. These might have a role in gene regulation based on their interspecies conservation. Preliminary analyses including more mammalian species have confirmed the conservation of a number of these ECRs. Chromatin remodeling is a hallmark of chromatin activity and gene expression. The acetylation, methylation and phosphorylation status of the tails of the core histones H3 and H4 are markers for the chromatin conformationand remodeling. To identify functional elements in the CSN-domain we have initiated the study of chromatin remodeling in the CSN-domain. We are using Chromatin Immuno-Precipitation (ChIP) assays, to identify histone modifications at the ECRs and promoters as a function of the developmental stage and tissue/cell type. Transfection assays in tissue culture have identified an enhancer activity for the human and bovine beta-casein enhancer (BCE) and potential chromatin remodeling involvement [2, 3]. We used the BCE as paradigm for the analysis of the other ECRs. Preliminary experiments in murine mammary epithelial cells (HC11) have shown a lactogenic hormone dependant acetylation of the beta-casein proximal promoter and BCE, and recruitment a transcription factors known to be important for milk protein gene expression, such as of Signal Transducer and Activator of Transcription (STAT) 5, correlating with the induction of beta-casein gene expression. We have started to investigate this region and others in lactating mouse mammary gland tissue in comparison to mouse liver. We show tissue specific histone acetylation on the casein gene promoters and some ECRs in mammary gland, correlating with gene expression. 1. Rijnkels, M., et al. Genomics, 2003. 82: p. 417-432. 2. Schmidhauser, C., et al. Mol. Biol. Cell., 1992. 3(6): p. 699-709. 3. Winklehner-Jennewein, P., et al. Gene, 1998. 217(1-2): p. 127-39.