Molecular Interactions between Single-stranded DNA-binding Proteins Associated with an Essential MCAT Element in the Mouse Smooth Muscle α-Actin Promoter

Abstract

Eukaryotic gene transcription requires the coordinated assembly of upstream cis-element binding proteins, intermediary cofactors, and components of the basal transcription machinery into a multicomponent complex competent to initiate transcription. During this process, sequence-specific DNA-binding transcriptional activators and/or repressors play a pivotal role in modulating the cell-type specific expression of genes. While most such proteins bind to double-stranded DNA target sequences, a small but intriguing subclass has been identified that show enhanced affinity and specificity for either the sense or antisense strands of certain cis-regulatory elements required for promoter-specific activation (1-4) or repression (5-9). We have recently cloned and identified two single-stranded DNA (ssDNA)

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

“…YB-1 has well documented duplex-DNA unwinding activity (Gaudreault et al, 2004), and we previously presented evidence showing that YB-1 and Smads compete for binding to the region of the SM␣A promoter harboring the THR element (Subramanian et al, 2004). YB-1 binds with high-affinity to the reverse strand of the MCAT/THR motif and may maintain this segment of DNA in an unwound state with assistance from the Pur␣ and Pur␤ corepressor proteins that bind the forward strand of this same region (Strauch et al, 1997;Kelm et al, 1999;Carlini et al, 2002). We are examining duplex MCAT/THR melting in the presence and absence of recombinant YB-1 and Smads to validate various aspects of the working hypothesis presented in Figure 9.…”

“…Commercial antibodies used in this study were specific for Smad2/3 (rabbit polyclonal antibody, 1:1000; Cell Signaling Technology), Egr-1 (clone 44D5 rabbit monoclonal antibody [mAb], 1:1000; Cell Signaling Technology; or C-19 rabbit polyclonal antibody, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), Smad7 (goat polyclonal antibody, 1:250; Abcam, Cambridge, MA), phosphorylated Smad2 (Ser 465/467-specific, rabbit polyclonal antibody, 1:1000; Cell Signaling Technology), SM␣A (EPOS anti-human smooth muscle ␣-actin/ HRP, clone 1A4, 1:200; Dako North America, Carpiteria, CA), Erk1/2 (anti-MAP kinase p44/p42, rabbit polyclonal antibody, 1:1000; Cell Signaling Technology), phosphorylated Erk1/2 (anti-MAP kinase phospho-p44/p42, T202/ Y204-specific, clone E10 mouse mAb, 1:1000; Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (anti-GAPDH/HRP, 1:200; Santa Cruz Biotechnology). Rabbit polyclonal antibodies specific for YB-1 (anti-YB1 M85-110 and anti-YB1 M276-302, 1:2000) were described in our previous reports (Kelm et al, 1999). The two Egr-1-specific antibodies gave slightly different results on immunoblots owing to Egr-1 size variation, possibly because of protein phosphorylation.…”

“…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). A DNA element referred to as SPUR recently was identified in the mouse as a key component in both basal and TGF␤1-dependent transcriptional activation of the SM␣A gene through its ability to bind heteromeric complexes of the Sp1/Sp3 activators and Pur␣/Pur␤ protein repressors (Subramanian et al, 2004;Knapp et al, 2006).…”

“…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) et al, 1999). The two Egr-1-specific antibodies gave slightly different results on immunoblots owing to Egr-1 size variation, possibly because of protein phosphorylation.…”

Deletions of PURA, at 5q31, and PURB, at 7p13, in myelodysplastic syndrome and progression to acute myelogenous leukemia

“…27 In contrast, binding of the Pur␣/Pur␤ heterodimeric complex to the mouse vascular smooth muscle ␣-actin gene enhancer results in transcriptional repression. 30 The kinetics of dimerization of these two proteins is very likely sensitive to any change in the intracellular levels of each. We report here that the human gene that encodes the Pur␤ protein is mapped to the short arms of chromosome 7, band region 7p13.…”