We have previously identified a C2H2 zinc-finger transcription factor [BTEB3 (basal transcription element-binding protein 3)/KLF13 (Krüppel-like factor 13)] that activates the minimal promoter for the smooth muscle-specific SM22α gene in other types of cell. We show that recombinant BTEB3 binds to three TGGG motifs in the minimal SM22α promoter. By mutation analysis, only one of these boxes is required for BTEB3-dependent promoter activation in P19 cells and BTEB3 activates or inhibits reporter gene expression depending on the TGGG box to which it binds. Transient transfection experiments show that BTEB3 also activates reporter gene expression from the SM22α promoter in VSMCs (vascular smooth muscle cells). Similar studies showed that BTEB3 did not activate expression from the promoter regions of the smooth muscle myosin heavy chain or smooth muscle α-actin promoters, which contain similar sequences, implying that promoter activation by BTEB3 is selective. The expression of BTEB3 is readily detectable in VSMCs in vitro and is modulated in response to injury in vivo .

Recent evidence has implicated CC(A/T richG )GG (CArG) boxes, binding sites for serum response factor (SRF), in the regulation of expression of a number of genes in response to changes in the actin cytoskeleton. In many cases, the activity of SRF at CArG boxes is modulated by transcription factors binding to overlapping (e.g. Yin Yang 1, YY1) or adjacent (e.g. ets) binding sites. However, the mechanisms by which SRF activity is regulated by the cytoskeleton have not been determined. To investigate these mechanisms, we screened for cells that did or did not increase the activity of a fragment of the promoter for a smooth-muscle (SM)-specific gene SM22α, in response to changes in actin cytoskeletal polymerization induced by LIM kinase. These experiments showed that vascular SM cells (VSMCs) and C2C12 cells increased the activity of promoters containing at least one of the SM22α CArG boxes (CArG near) in response to LIM kinase, whereas P19 cells did not. Bandshift assays using a probe to CArG near showed that P19 cells lacked detectable YY1 DNA binding to the CArG box in contrast with the other two cell types. Expression of YY1 in P19 cells inhibited SM22α promoter activity and conferred responsiveness to LIM kinase. Mutation of the CArG box to inhibit YY1 or SRF binding indicated that both factors were required for the LIM kinase response in VSMCs and C2C12 cells. The data indicate that changes in the actin cytoskeletal organization modify SRF activity at CArG boxes by modulating YY1-dependent inhibition.

Members of the three-zinc-finger family of transcription factors play an important role in determining basal transcription. We have cloned mouse BTEB3 (mBTEB3), a new member of the basic transcription element binding protein (BTEB) family, which is expressed in a wide variety of tissues. mBTEB3 activates transcription of the simian virus 40 early promoter (4-fold) and of the tissue-specific SM22α promoter (100-fold), suggesting that, like BTEB1 and Sp1, mBTEB3 is a basal transcription factor.

Serum response factor (SRF) is a key transcriptional activator of the c-fos gene and of muscle-specific gene expression. We have identified four forms of the SRF coding sequence, SRF-L (the previously identified form), SRF-M, SRF-S and SRF-I, that are produced by alternative splicing. The new forms of SRF lack regions of the C-terminal transactivation domain by splicing out of exon 5 (SRF-M), exons 4 and 5 (SRF-S) and exons 3, 4 and 5 (SRF-I). SRF-M is expressed at similar levels to SRF-L in differentiated vascular smooth-muscle cells and skeletal-muscle cells, whereas SRF-L is the predominant form in many other tissues. SRF-S expression is restricted to vascular smooth muscle and SRF-I expression is restricted to the embryo. Transfection of SRF-L and SRF-M into C 2 C 12 cells showed that both forms are transactivators of the promoter of the smooth-muscle-specific gene SM22α, whereas SRF-I acted as a dominant negative form of SRF.