Actin, Alpha-Cardiac

Transcriptional regulatory regions

• The mouse cardiac alpha-actin distal enhancer increased transcription of the proximal homologous promoter (100-fold) and is capable of activating heterologous tk and pox promoters in C2,7 myotubes. The enhancer was inactive in C2,7 myoblasts [Biben et al. 1994]*.

• The sequences between -416 and -217 of the frog cardiac alpha-actin gene is necessary for tissue-specific expression in early frog embryos. A segment of cardiac alpha-actin between -217 and +1 showed very low levels of CAT activity in embryos compared to the segment extending upstream to -416 bp [Mohun et al. 1986]*.

• A segment of human cardiac alpha-actin including 485 bp upstream, the first exon, and 24 bp of the first intron conferred high levels of transient CAT expression in differentiating C2C12 cells. The expression level of this construct in C2C12 cells was 20- to 40-fold higher than observed in transfected rat PC-G2 pheochromocytoma cells or mouse L fibroblasts. Expression in C2C12 myotubes decreased by 30% with deletion of -443 to -395 and decreased further, to 2%, with deletion of the segment between -177 and -47; expression levels in L cells was not affected by these deletions. Sequences further upstream and that between -395 and -177 had little affect on transcriptional activity [Minty & Kedes 1986]*.

• The proximal regulatory region of human cardiac alpha-actin between -177 and -65 is crucial for promoter activity and is distance- and orientation-dependent. The transcriptional activity of the promoter decreased in constructs in which the proximal regulatory region (-177 to -65) was separated from the basal promoter (-65 to +68); a separation of 105 bp resulted in 65% decrease, separation of 207 showed 77% decrease and separation of 272 bp showed a 83% decrease in promoter activity [Miwa and Kedes 1987]*.

• The chicken cardiac alpha-actin sequence from -315 to +15 is able to promote high levels of activity in chicken breast muscle cells. Deletion of the segment between -200 and -100 (containing CArG2-4) reduced activity to 50% and lowered tissue-specific expression 3-fold. Conversely, the segment between -315 and -100 (without CArG1) is able to confer tissue-specific gene expression when joined to the alpha- or beta-actin TATA region [Quitschke et al. 1989]*.

• A segment of human cardiac alpha-actin (-485 to +68) transfected into CV-1 and HeLa cells was transactivated by cotransfected MyoD; a shorter segment (-117 to +68) was also transactivated. No transactivation occurred when the segment of cardiac alpha-actin was shortened to -47 [Sartorelli et al. 1990]*.

• A region of frog cardiac alpha-actin between -348 and -282 (designated the "M region" by Taylor et al. [1991]*) is necessary for expression of this gene in frog embryos. Deletion of nucleotides between -104 and -224 did not reduce expression of the test fusion gene; deletion of the region between -348 and -282 (the "M region") dramatically decreased expression of the fusion gene. The frog analog to mouse MyoD binds specifically to the M region [Taylor et al. 1991]*.

• Developmental stage- and tissue-specific regulation of transcription is conferred by a region of human cardiac alpha-actin between -440 and +6 in P19 cells. Promoter activity was abolished when nucleotides between -237 and -158 were deleted [Pari et al. 1991]*.

• A segment of human cardiac alpha-actin between -485 and +68 shows a high level of CAT activity (used as 100% reference for succeeding constructs). Truncation to -263 reduced expression to 50%, and truncation to -177 reduced expression to 30% of wild-type levels [Sartorelli et al. 1992]*.

• 315-bp of the chicken cardiac alpha-actin promoter (-315 to +1) was transactivated 600-fold by MyoD, 400-fold by myogenin, and 100-fold by myf5 in 10T1/2 cells. MRF4 failed to transactivate the promoter segment. Gel mobility shift assays with 100-bp promoter fragment showed that specific binding of bHLH myogenic proteins to the chicken cardiac alpha-actin promoter occurs only in the presence of added E12. MRF4, unless as a MRF4/E12 heterodimer, does not bind alone to this promoter E-box [Moss et al. 1994]*.

• A transgene containing human cardiac alpha-actin between -600 and +7350 is down-regulated ~104-fold during skeletal muscle maturation. Between birth and adult mRNA levels decrease ~6-fold in the heart. Deletion of the nucleotides between +6200 and +7350 resulted in 4.5-fold decrease of maturation-based expression in heart and 70% of transfected cells showed very little decrease in mRNA levels between birth and adult. In developing striated muscle, expression of a transgene containing the sequence between -800 and +6200 varied an average of 9-fold, but transgenes containing either a sequence between -5700 and +7350 or -800 and +7350 varied greatly (110-fold and 104-fold respectively) during striated muscle development [Dunwoodie et al. 1994]*.