At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

Abstract

We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI-and EFH-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.Enhancers are a class of cis-acting regulatory DNA sequences that can activate transcription from both homologous and heterologous eucaryotic promoters in a relatively orientation-, position-, and distance-independent manner.Enhancers have been identified in many viral genomes (for reviews, see references 23, 43, 55) and near or within a variety of cellular genes (1, 13, 22, 24, 26, 41, 44, 60 64), where they often potentiate transcription in a cell typespecific, and sometimes inducible, fashion. Different enhancer elements bear no extensive sequence homologies, although several short, degenerate consensus sequences occur in certain subsets of enhancers (27, 38, 68, 71, and references therein). Despite the lack of sequence homology, enhancers do seem to share an organizational consensus. Extensive site-specific mutagenesis and deletion analyses have established that the simian virus 40 (SV40) and polyomavirus enhancers (28, 29, 71), as well as several other well-characterized enhancers (4,14,32,46,67), are mosaics of multiple different and often redundant sequence motifs. Each relevant sequence motif appears to be contained within a separable functional domain that possesses little enhancer activity unless combined with another domain. The functional domains of an enhancer, although nonhomologous, can compensate for each other in satisfying this multiplicity requirement (29,30,32,63,65,71).The mechanism(s) by which this modular arrangement of multiple short sequence motifs synergistically leads to the long-range activation of transcription from a promotor is not known for any enhancer. However, evidence from both functional (36,50,52,54,69) and structural (6, 16) studies has been rapidly accumulating that the action of viral and cellular enhancers involves their interaction with specific protein factors. Recently, the interaction of such transacting factors in nuclear extracts with specific domains of the SV40 (70), polyomavirus (3,18,45), and insulin gene (40) enhancers has been directly demonstrated in vitro by DNase * Corresponding author.

Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein.

“…The EFIII, EFIIIB (48-mer), and 100 mM NaCl, and 10% glycerol (buffer I). Samples were c-fos SRE oligonucleotides were first ligated, then treated analyzed on polyacrylamide gels as previously described with DNA polymerase I to generate blunt ends, and finally (44). Similar analyses of SRE BP binding activity were inserted into pe-CAT linearized with AccI (at unique site performed as described above except that 500 ng of poly(dI--235 relative to the start site of transcription).…”

“…Nuclear extracts were prepared from 14-day-old chicken embryos (SPAFAS Inc., Preston, Conn.), chicken embryo fibroblasts (CEF), and BK3a and NIH 3T3 cells as previously described (44) except that lysis of the cells was not followed by a wash of the nuclei in buffer A but rather was followed by a single extraction with buffer B containing 0.53 M NaCl. The phosphatase inhibitors 3-glycerolphosphate (10 mM), sodium vanadate (100 ,um), and sodium molybdate (10 mM) were also added to all solutions used during and after cellular lysis.…”

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

“…To define the factor-binding sites more precisely, preparative mobility shift gels were combined with DNase I footprinting (17,50). In this method, a brief DNase treatment was used to nick the probe subsequent to binding and before nondenaturing electrophoresis.…”

Transcriptional Regulation of the Chicken Caldesmon Gene

“…CArG1, CArG2, CArG3, and CArG1M were prepared by annealing respective sense and antisense synthesized oligonucleotides to form duplex DNA. Nuclear extracts from SMCs and CEFs were prepared according to the procedures described elsewhere (31). For characterization of DNA-protein binding, samples of nuclear extracts were mixed with 0.1-0.2 ng of 32 P-labeled probe and 3.5 g of heatdenatured herring sperm DNA in the presence or absence of nonradiolabeled competitor at room temperature for 30 min in 20 l containing 5 mM HEPES, pH 7.8, 5 mM ␤-mercaptoethanol, 1 mM EDTA, 60 mM NaCl, 5 mM spermidine, and 10% glycerol.…”