REGULATION OF CD4 GENE EXPRESSION

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DESCRIPTION: (Adapted from Applicant's Abstract) This application is directed at understanding the transcriptional regulation of the gene encoding CD4. The CD4 cell surface molecule serves as an essential receptor component responsible for the T lymphocyte recognition of foreign antigens, and mediates an intracellular signal for T-cell activation. Its cell surface expression is regulated in a stage-specific manner during the ontogeny of T lymphocytes, and, along with CD8, it is used to define the lineage relationships of developing thymocytes. In addition, it serves as the primary receptor for the binding of the human immunodeficiency virus (HIV) to CD4+ T lymphocytes and monocytes. The CD4 cell surface molecule is, thus, most important in the normal physiological immune response, and its abnormal interactions in the presence of HIV infections may be responsible, in part, for the immunodeficiency portion of acquired immunodeficiency syndrome (AIDS). An understanding of the regulation of expression of the CD4 molecule will be informative with respect to the T lymphocytes development, and may be useful in designing therapies for immunodeficiency diseases. A 75 kilobase chromosomal segment containing the CD4 gene has been mapped for coding exons and a transcriptional start site, and this proposal is directed at understanding the structure and activity of the cis-acting DNA sequences and trans-acting factors that determine the cell-type and developmental control of CD4 gene expression. Various segments of DNA upstream of the transcriptional start site will be ligated to a reporter gene (luciferase), and transcriptional activity will be measured in transfected CD4+ T-cells by measuring luciferase activity. Each of the difference constructs showing activity will be further analyzed by primer extension to insure that transcription is correctly initiated. All transfections will be reproduced in CD4+/- T-cells and in B-cells. Gene segments that appear to be important for positive or negative control of transcription will be examined by linker-scanning mutational analysis, and these gene segments will be further analyzed for the presence of nuclear factors that may specifically bind. A second set of experiments will be directed at locating more distant cis-acting regions that serve as enhancers and nuclear matrix association regions (MARS). Large regions of the CD4 gene and its surrounding sequence will be ligated into enhancerless promoter constructs, and assayed by transfection for transcription enhancement. The actual enhancer elements will be tested in transgenic mice to determine whether the isolated sequences are sufficient to direct appropriate tissue and development control. Differential regulation of CD4 and CD8 expression during thymic differentiation will be analyzed by recombining the regulatory and structural elements of the two genes.