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)

Stimulation of quiescent AKR-2B mouse embryo cells with epidermal growth factor (EGF) results in a rapid and specific induction of actin mRNA sequences. These mRNAs include those coding for both P-and y-cytoskeletal, but not a-skeletal muscle, actin isotypes. Elongation of nascent RNA chains in isolated nuclei (run-off transcription) demonstrates that the mRNA accumulation is preceded by an increase in actin gene transcription. This increase is transient, however, and is followed by a rapid attenuation of transcriptional activity. An inhibitor of protein synthesis, cycloheximide, was also found to induce 18-and r-actin mRNA accumulation. Furthermore, the simultaneous addition of EGF and cycloheximide produced a synergistic effect on actin sequences in both steady-state nuclear and polysomal RNA. Run-off transcription experiments demonstrate that this synergistic effect results from an increase in the magnitude and duration of actin gene transcription. It is also specific in that a-tubulin gene transcription is not similarly affected. These data suggest the existence of a specific labile repressor of actin gene transcription.The binding of epidermal growth factor (EGF) to specific receptors in the membranes of quiescent cells initiates a variety of biochemical events that can culminate in the initiation of DNA synthesis and cell division (reviewed in ref. 1). Inhibitors of RNA synthesis can block this process, implying that the transcription of certain genes is required for a quiescent cell to reenter the cell cycle (2). The molecular mechanisms that regulate these genes are poorly understood but do not appear to involve the nuclear translocation of the hormone-receptor complex. Indeed, the complex itself is rapidly internalized and degraded several hours prior to the initiation of DNA synthesis (3,4). This observation has led to speculation that a second messenger(s) of hormone action must be responsible for initiating the expression of specific genes required for cell proliferation (1).A major constraint on any proposed mechanism of EGF or second messenger action is the requirement for a high degree of specificity. This consideration arises from several studies that have shown that peptide growth factors regulate a very limited domain of specific genes (5-8). This specificity could be achieved by a specific DNA binding protein acting either as a positive or negative regulator of gene transcription. Although theoretical constraints have been imposed on specific protein-DNA interactions in the context of a mammalian nucleus, these constraints are not absolute and a variety of compensating strategies are available to a eukaryotic cell (9). Therefore, it may be noteworthy that recent studies have shown that inhibitors of protein synthesis can potentiate the induction of specific mRNA sequences by platelet-derived growth factor (7, 10). Although this observation is consistent with a protein repressor of growth-factor-regulated genes, a direct effect on gene transcription rates was not shown.Prompted by a rep...

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Transcriptional activation or repression of RNA polymerase II-dependent genes is often mediated by sequence-specific DNA-binding proteins (i.e. transcription factors) that respond to extracellular signals by interacting with critical cis-acting regulatory elements. Activator or repressor proteins generally bind to double-stranded DNA recognition sites distal to the TATA box and influence promoter activity by interacting, either directly or through an adaptor protein, with factor(s) comprising the RNA polymerase II basal transcription complex (1, 2). Although most transcription factors demonstrate preferential binding to double-stranded DNA, a number of recent studies have associated sequence-specific, single-stranded DNA (ssDNA)

An asymmetric polypurine-polypyrimidine cis-element located in the 5 region of the mouse vascular smooth muscle ␣-actin gene serves as a binding site for multiple proteins with specific affinity for either singleor double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to transactivate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur␣ and Pur␤ with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur␣, Pur␤, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur␣, Pur␤, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur␣, Pur␤, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.Current models of transcriptional repression take into account the ability of negative-acting factors to function in multiple capacities with or without DNA binding specificity (1, 2). For example, evidence exists that certain activator proteins can simply be masked or sequestered by protein binding partners that do not interact with DNA directly, as in the case of retinoblastoma tumor suppressor protein-mediated repression of the E2F family of trans-activators (3). Alternatively, a repressor protein may bind and displace components of the basal transcription machinery as in the case of Drosophila evenskipped-mediated repression of the Adh proximal promoter (4). Recent work suggests that some repressors can participate, indirectly, in modifying chromatin structure by binding activator sites and recruiting specific histone deacetylases to silence genes through histone modification. This scenario is implicated in repression of E-box motifs through Mad/Max-mediated recruitment of histone deacetylases 1 and 2 (2). Still other models of repression propose that activator protein access to a DNA target sequence can be blocked by repressor protein binding to either the same site or an overlapping site. Such a mechanism appears to be operative in the competitive DNA binding of YY1 and serum response factor (SRF) 1 to serum response elements (SREs) of the c-fos promoter (5).An interesting variation on the theme of multiple proteins competing for overlapping binding sites arises from our attempts to elucidate the mechanism of vascular smooth muscle (VSM) ␣-actin pr...