Identification of a CArG-independent region of the cysteine-rich protein 2 promoter that directs expression in the developing vasculature

Abstract

Cysteine-rich protein (CRP)2 is a member of the LIM-only CRP family that is expressed in vascular smooth muscle cells (VSMC). To gain insight into the transcription of CSRP2 (gene name for CRP2) in VSMC, we analyzed the 5′-flanking sequence of the CSRP2 gene. We showed previously that 4,855 bp of the 5′-flanking sequence of the CSRP2 gene directed lacZ reporter gene expression, primarily in the VSMC of transgenic mice. To further define the regulatory sequences important for CSRP2 expression in VSMC, a series of promoter constructs containing deletions of the 5′-flanking sequence upstream of a nuclear-localized lacZ reporter gene were generated and analyzed. Similar to that observed in the –4855CSRP2-lacZ mice, β-galactosidase reporter activity was detected in the developing great vessels, aorta, intersegmental arteries, umbilical vessels, endocardial cushions, and neural tube in the –3513-, –2663-, –795-, and –664CSRP2-lacZ lines. However, an internal deletion of bp –573 to –550 abolished the vascular, but not the neural tube, staining. Interestingly, no CArG box [CC(A/T)6GG] was present in the –795-bp fragment. Cotransfection experiments showed that dominant-negative serum response factor (SRF) did not repress CSRP2 promoter activity, which was different from the repressive effect of dominant-negative SRF on the SM22α promoter. Our data suggest the presence of a VSMC-specific element(s) within bp –573 to –550 of the CSRP2 5′-flanking sequence; however, in contrast to many other smooth muscle genes, transcriptional regulation of the CSRP2 gene is not dependent on SRF.