Interaksie van Geselekteerde Transkripsiefaktore met Reguleringsgebiede van die Mensgeen vir Pro-al(I)-Kollageen

Abstract

The fibrillar collagen Type I is abundant in a variety of connective tissue. Type I collagen is first assembled as two pro-a 1 (I) and one pro-a2(I) chains and the genes are under coordinated control. Preliminary studies in the elucidation of the transcriptional regulation of the collagen genes, showed that the human pro-al(I) collagen gene contains a strong promoter as well as an enhancer-like element in the first intron (Rossouw et al. , 1987). Publication of negative elements in the identified enhancer-like region, however, led to much controversy surrounding the existence of this enhancer-like element in the first intron of the human pro-a 1 (I) gene. Little knowledge about cis-acting sequences and their interaction with trans-acting elements in the human pro-al(I) gene is available in the literature. Therefore the aim of this study was to clarify the controversy surrounding the enhancer-like element in the first intron of the human pro-al(I) collagen gene and to further delineate the transcription regulatory regions in this gene between -442 and + 1697. The first part of this study deals with the functional data obtained from two different functional assays, the X. laevis oocyte system and the transfection system. Results from these experiments showed the existence of an enhancer-like region as well as negative elements in the first intron of the human pro-al(I) gene. The negative effect of these negative elements, however, is neutralized depending on the promoter region tested together with the intron region. In the second part of this study the nature and characteristics of the interaction between the cis-acting sequences and trans-acting elements between -442 and + 1697 of the human pro-al(I) gene was determined. DNase I footprinting with crude nuclear extracts from BJA-B and HeLa cells as well as S-300 fractions from BJA-B and HeLa cells, revealed long stretches of masked sequences. These long stretches of masked sequences indicate that there are regions where transcription factors overlap. To delineate 1 the interaction of specific factors, purified transcription factors are necessary. For this purpose three commercial available transcription factors: Spl , Apl and Ap2 were used to analyze the gene region between -442 and + 1697 systematically, employing DNase I footprinting. In a first step of delineation a computer search was done to determine all potential binding sites for the three selected transcription factors whereafter the DNase I footprinting experiments were performed. During these experiments sixteen Spl , one Apl and twentytwo Ap2 binding sites were identified in the human pro-o:1(1) gene between -442 and + 1697 and mapped. An indication of the binding affinities of each binding protein to its related binding site was obtained by the use of increasing protein concentrations with a constant DNA concentration during DNase I footprinting. Quantitative determination of each affinity binding constant was obtained by utilizing densitometry and liquid scintillation spectrometry as well as Marquard's method of non linear least square analysis. Detection of DNA-protein binding via transmission electron microscopy (TEM) supports the results obtained via DNase I footprinting . Analysis of DNA-protein binding via TEM also emphasized the ability of this technique to identify DNA-protein binding regions on long DNA-molecules at low resolution. A looping model was proposed during this study to elucidate the ranscriptional regulation of the human pro-o:1(1) collagen gene, using the results obtained in this study. The basis of this model is the binding of the homologuos pyrimidine-rich regions in the promoter and first intron of the gene with one another, bringing the promoter and enhancer in near proximity to one another while looping out the intervening DNA. The identified Spl and Ap2 binding sites in the enhancer can then bind to the Ap2 binding sites in the promoter region via protein-protein interaction thereby enhancing the transcription activity from the promoter. Protein-protein interaction between different proteins, as well as protein selfassociation, while looping out the intervening DNA, was observed during the electron microscopy study supporting the proposed model in this study. This study makes a real contribution to the elucidation of the transcriptional regulation of the human pro-a 1 (I) collagen gene. Part of the functional data was incorporated in the following paper: Rossouw, C.M.S., Vergeer, W.P., du Plooy, S.J., Bernard, M.P., Ramirez, F. & de Wet, W.J. (1987). "DNA sequences in the first intron of the human pro-al(I) collagen gene enhance transcription" Journal of Biological Chemistry 262, 15151-15157. The rest of the results emanating from this study are in preparation for publication. Various facets of this work have also been presented at the following meetings: de Wet, W.J., de Vries, W.N., Koedood, R. , Rossouw, C.M.S. & Vergeer, W.P. "Interaction of nuclear factors with the enhancer and promoter regions of the human pro-a 1 (I) collagen gene" Second International Conference on Molecular Biology and Pathology of Matrix. Philadelphia, USA (June 1988). Vergeer, W.P. , Rossouw, C.M.S. & de Wet, W.J. "Sequences in the first intron of the human pro-al(I) collagen gene enhance transcription in transfected fibroblasts" Ninth Congress of the South African Biochemical Society: Wilderness (27-31 March 1988). Vergeer, W.P., de Vries, W.N. & de Wet, W.J. "In vitro binding of Spl , Apl and Ap2 to regulatory regions of human pro-al(I) collagen gene" Eleventh Congress of the South African Biochemical Society: Sun City (28 June -1 July 1992).