Molecular mechanisms involved in the regulation of Il12b

The transcriptional regulation of inducible mammalian genes is complex. Mammalian genes are controlled by a promoter and typically at least one enhancer found at a distance either up or downstream from the transcription start site. The majority of mammalian genes contain highly conserved control regions and are often more conserved at the nucleotide level in the control regions than in the coding exons. Furthermore, the control regions for many mammalian genes are wrapped in chromatin, which must be unwound prior to the initiation of transcription. We believe that the current techniques of analyzing the functions of control regions by the introduction of mutations in promoter-reporter gene transfection assays and by using transcription factor knock-out mice are not capable of determining the precise molecular mechanisms involved in gene regulation. These assays have provided a lot of knowledge but for full understanding of regulation, mutations must be analyzed in the context of distant enhancers and native chromatin.;We have used the gene Il12b as a model to study inducible gene induction in an endogenous context. Il12b is a highly inducible gene in macrophages and is turned on in response to bacterial products through the activation of Toll like receptors. We have set up two systems to construct Il12b regulatory element mutations: (1) introduction of mutations into large 1112b BACs by homologous recombination in E. coli and then transfecting the BACs into mouse ES cells and (2) introduction of mutations into the endogenous locus by homologous recombination in mouse ES cells. For both strategies, we then differentiated the ES cells into macrophages for analysis.;We show that ES cells differentiated into macrophages are an ideal system for the study of gene regulation. We next find that even large 191 kb BACs randomly integrated do not recapitulate the native chromatin environment and only a few integrated copies are active. It is, however an attractive system to assay the requirements of elements for transcription in a more endogenous chromatin context. We then set up a system to modify control elements at the Il12b endogenous locus, which does recapitulate the endogenous chromatin. A preliminary analysis suggests that NF-kappaB is required for chromatin remodeling at the Il12b promoter, while AP-1 and NFAT are only required for transcription regulation downstream of chromatin remodeling.