Negative Regulation of the Vascular Smooth Muscle α-Actin Gene in Fibroblasts and Myoblasts: Disruption of Enhancer Function by Sequence-Specific Single-Stranded-DNA-Binding Proteins

The Single-stranded DNA-binding Proteins, Purα, Purβ, and MSY1 Specifically Interact with an Exon 3-derived Mouse Vascular Smooth Muscle α-Actin Messenger RNA Sequence

“…Earlier analyses of mouse vascular smooth muscle ␣-actin gene transcription in fibroblasts and undifferentiated myoblasts led to the formulation of a molecular model in which the activity of an essential, transcription enhancer factor 1-dependent, MCAT enhancer element located within a polypurinepolypyrimidine tract (Ϫ165 to Ϫ195) was subject to negative regulation by sequence-specific, ssDNA-binding proteins (15,25). It was later discovered that the purine-rich strand binding activity, then termed vascular actin single-stranded DNA-binding factor 2 (VACssBF2), also interacted specifically with the sense strand of a protein-coding element (CE) sequence bearing similarity to the 5Ј promoter element (PE) sequence (16).…”

“…These proteins, recently identified as the mouse Y-box protein, MSY1 (13), and the purine-rich ssDNA-binding proteins, Pur␣ and Pur␤ (14), interact with opposite strands of a polypurine-polypyrimidine tract conserved in VSM ␣-actin gene promoters to negatively regulate transcription by a mechanism not yet fully understood (13,15). This tract, designated the promoter element (PE), resides between Ϫ165 and Ϫ195 relative to the start site of transcription, contains an inverted core MCAT enhancer element (AGGAATG), and exhibits a high degree of purine-pyrimidine asymmetry (15). Interestingly, an inspection of the complete VSM ␣-actin cDNA and genomic sequences revealed an exon 3-derived sequence with strong similarity to the 5Ј PE (16).…”

Transforming Growth Factor β1-mediated Activation of the Smooth Muscle α-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-α via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

“…Protein blots were blocked at 4°C in Tris-buffered saline (TBS) containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 3% (wt/vol) nonfat dry milk, and 0.5% bovine serum albumin and incubated with the indicated antibodies for 90 min at room temperature with gentle rocking. For each of the primary antibodies used in our analysis, preimmune serum or antibodies presaturated with blocking peptides (in the case of peptide epitope antibodies for YB-1) were used as negative controls and protein extracts prepared from mouse heart or AKR-2B mouse embryonic fibroblasts known to contain SM␣A gene repressors and activators Sun et al, 1995;Kelm et al, 1999) were used as positive controls and to monitor antibody binding specificity. Blots were washed four times at room temperature over a 20-min period in TBS containing Tween 20 (0.05% vol/vol) and then incubated for 45 min with a species-appropriate, horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom).…”

“…Synthetic oligonucleotide probes used in this report were derived from selected sequences present in the human SM␣A promoter Sun et al, 1995;Kelm et al, 1996;Subramanian et al, 2004;Zhang et al, 2005). Reaction mixtures containing protein extract (100 g of protein) and 3Ј-biotinylated oligonucleotides (100 pmol; Integrated DNA Technologies, Coralville, IA) were incubated in a buffer containing poly(dI-dC), 10 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 0.12 mM PMSF, and 4% glycerol.…”

“…Our laboratory has identified several cis-regulatory elements in SM␣A promoter that are highly conserved among vertebrate species and function as binding sites for multiple positive and negative trans-acting transcription factors (Foster et al, 1992;Cogan et al, 1995;Sun et al, 1995). Among these factors, Pur␣, Pur␤, and YB-1 show preferential binding to single-stranded DNA and function as transcriptional repressors (Kelm et al, 1999(Kelm et al, , 2003Carlini et al, 2002;Zhang et al, 2005;Knapp et al, 2006) whereas others such as transcription enhancer factor (TEF) 1; serum response factor (SRF); Smads 2, 3, and 4; and Sp1/Sp3 represent serum-or TGF␤1-responsive transcriptional activators (Kelm et al, 1999;Cogan et al, 2002;Subramanian et al, 2004).…”