The long term objective of this proposal is to understand, at the molecular level, the mechanism of regulation of the alpha- fetoprotein-albumin gene pair during rat development. We have recently characterized the control region of the AFP gene of the rat and found it to contain three regulatory elements: a cell-type specific """"""""promoter"""""""", a cell-type specific enhancer(s), and a cell- type specific negative regulator. During the next period we will concentrate on the study of this region. We will describe in detail the elements of the system (DNA sequences and protein factors) in order to determine the mechanism of their action and their role during development. Emphasis will be on the study of the negative regulator. We will: I. Characterize the sequences required for AFP """"""""promoter"""""""" activity. II. Characterize the sequences required for AFP negative regulatory element activity. III. Characterize the sequences required for AFP enhancer activity. These studies will entail the use of transient expression assays of chimeric templates in hepatic and non hepatic cell lines. IV. Isolate and characterize the protein factors that interact with the sequences defined in I, II, and III. V. Develop and in vitro transcription system which, when supplied with DNA cntaining the negative regulatory element, is sensitive to the negative regulator and determine the mechanism of its action. These studies will entail the use of a number of biochemical procedures to detect factors and the development of in vitro systems dependent on the activity of the factors for use in their purification. VI. Investigate the role of the elements present in the AFP control region in the expression of AFP during development through the generation and analysis of transgenic mice. The knowledge acquired will allow us to understand the processes that lead to cell differentiation and to dedifferentiation during tumorigenesis.
