THE HUMAN CLOTTING FACTOR-VIII CDNA CONTAINS AN AUTONOMOUSLY REPLICATING SEQUENCE CONSENSUS-LIKE AND MATRIX ATTACHMENT REGION-LIKE SEQUENCETHAT BINDS A NUCLEAR FACTOR, REPRESSES HETEROLOGOUS GENE-EXPRESSION, AND MEDIATES THE TRANSCRIPTIONAL EFFECTS OF SODIUM-BUTYRATE

Expression of the human blood-clotting factor VIII (FVIII) cDNA is ham pered by the presence of sequences located in the coding region that r epress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R. C. Hoeben , F. J. Fallaux, S. J. Cramer, D. J. M. van den Wollenberg, H. van Orm ondt, E. Briet, and A. J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicatin g sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffol d-attached regions. One of these elements, consisting of nucleotides 1 569 to 1600 of the FVIII cDNA (nucleotide numbering is according to th e system of Wood et al. (W. I. Wood, D. J. Capon, C. C. Simonsen, D. L . Eaten, J. Gitschier, B. Keyt, P. H. Seeburg, D. H. Smith, P. Holling shead, K. L. Wion, et al., Nature [London] 312:330337, 1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can rep ress the expression of a chloramphenicol acetyltransferase (CAT) repor ter gene and also loses this capacity upon mutation. Furthermore, we d emonstrate that repression by FVIII sequences can be relieved by sodiu m butyrate. We demonstrate that the synthetic heptamer (FVIII nucleoti des 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studie d long terminal repeat promoters. Our data provide a functional charac terization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between transcriptional repression by FVIII cDNA elements and the stimulatory effect of butyrate on FVIII cD NA expression.