A retinoic acid/cAMP-responsive enhancer containing a cAMP responsive element is required for the activation of the mouse thrombomodulin-encoding gene in differentiating F9 cells.

Activation of thrombomodulin-encoding gene (TM) transcription in murine teratocarcinoma F9 cells occurs during differentiation in response to retinoic acid (RA) and dibutyryl cAMP (dbcAMP). To define regulatory elements involved in TM activation, reporter plasmids containing various lengths of the 5' flanking region of the mouse TM promoter fused to the cat gene were examined in transfected F9 cells. Stable transfectants showed no CAT activity in undifferentiated or RA-treated cells, and only those plasmids containing a 431-bp region approximately 700 bp upstream of the transcription start point showed significant CAT activity on subsequent treatment with RA/dbcAMP. Transfer of this region to an enhancerless SV40 promoter conferred position-independent responsiveness to RA/dbcAMP. Within this region is a 46-bp domain that contains potential transcription factor binding sites, including a cAMP responsive element (CRE) and a direct repeat sequence (DR). CAT assays using reporters lacking these sites showed that both contribute to TM expression in differentiating F9 cells. Gel retardation experiments demonstrated protein binding to both the CRE and DR sequences, and suggested that the factor interacting with the DR influences binding of a factor to the CRE site. Binding of both factors disappeared in differentiated cells, suggesting they are required for induction but not continued expression of TM. The CpG methylation pattern of the TM promoter was identical in undifferentiated and differentiated F9 cells. DNase I hypersensitivity studies showed increased DNase I resistance and the loss of a hypersensitive site in differentiated F9 cells. We conclude that an enhancer-like region containing a DR sequence and CRE is necessary for the induction of TM expression in differentiating F9 cells, that methylation does not play a role in the regulation of TM, and that the RA/cAMP response is associated with specific alterations to chromatin structure.