The studies described in this proposal are designed to provide a better understanding of the factors that control fibrinogen production by influencing the activity of the three linked genes for fibrinogen. Fibrinogen production in the liver is controlled by an indirect feedback mechanism in which the degradation products of fibrinogen interact with monocytes and Kupffer cells to induce a peptide, possibly IL-1, which acts directly on hepatocytes to stimulate fibrinogen production. Since previous work from our laboratory indicates tht fibrinogen production is controlled at the level of transcription, our studies will concentrate on the factors influencing and coordinating transcription of these three linked genes and will employ two model systems: the activation of fibrinogen gene transcription in the livers of intact animals during the acute phase reaction and the constitutive and induced production of fibrinogen in hepatocyte lines. We will complete the sequencing of the three fibrinogen genes in rats and humans, and search for sites of DNase sensitivity within the fibrinogen gene complex. In addition we will locate transcriptional enhancing elements within the fibrinogen gene complex and determine if one, two, or three transcriptional enhancing elements are necessary to activate three closely-linked genes. If an enhancing element is found neighboring each fibrinogen gene, as preliminary studies indicate, we will attempt to determine if the enhancing elements are using the same regulatory molecule by examining competition among the enhancing elements for cellular factors, after transfection into differentiated hepatoma cell lines. Since regional chromatin structure may be important in controlling the fibrinogen genes, we will test the hypothesis that transcriptional activation of the three linked fibrinogen genes occurs by a regional change in chromatin structure conducted along double-stranded DNA by superhelical torsional stress. In these studies we will analyze the function of the genes after gamma radiation of intact cells at doses calculated to make from 1 to 10 single-stranded nicks within the 70 kb gene complex and thereby relax torsional stress. We anticipate that our studies will provide specific information on the control of fibrinogen production by the liver as well as general information on the control of linked families of genes.
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