The HeLa transcription factor LSF has been purified by heparin-agarose and DNA affinity chromatography, and its DNA binding and transcription properties have been characterized. LSF is a 63-kD polypeptide that binds to two distinct bipartite sites within the SV40 promoter region. One binding site consists of GC motifs 2 and 3 within the 21-bp repeats (LSF-GC site), and the other consists of sequences centered 44 bp upstream of the major late initiation site, L325 (LSF-280 site). Four guanine residues within the LSF-GC site, when methylated, strongly interfere with LSF binding. Alteration of the spacing, but not the sequence, between the two directly repeated GC motifs dramatically reduces the binding affinity of LSF for the site. Thus, LSF appears to recognize directly repeated GC motifs, when their center-to-center distance is 10 bp. The LSF-GC and LSF-280 sites share limited sequence homology. Only half of the LSF-280 site contains a short GC-rich sequence homologous to the GC motif. However, the binding affinity of LSF to the two sites is similar. LSF activates transcription from the SV40 late promoter in vitro from initiation site L325, via its binding to the template DNA. SV40 offers an excellent model system for studying transcriptional regulatory mechanisms in mammalian cells: The virus uses the host cell transcription machinery for expression of its genes, and both biochemical and genetic means can readily be applied in investigating its transcription. During lytic infection, transcription of SV40 is precisely regulated. Before the onset of viral DNA replication, the SV40 early genes (encoding the large-T and small-t antigens) are primarily transcribed; after the onset of viral DNA replication, the SV40 late promoter is activated, and the late genes (encoding the viral capsid proteins) are predominantly expressed (Tooze 1981).The DNA sequences in the SV40 genome that regulate early and late transcription are located within a region of -400 bp (see Fig. 8, below). The SV40 late promoter appears to be very complex and may actually be a combination of multiple overlapping promoters capable of stimulating transcription from the same initiation sites. Many DNA sequence elements have been implicated in promoting late transcription in vivo and/or in vitro. These sequences include the origin of DNA replication (Brady and Khoury 1985;Keller and Alwine 1985), the three 21-bp tandem repeats containing six directly repeated GC motifs (GGGCGG) (Fromm and Berg 1982;Hansen and Sharp 1983;Brady et al. 1984;Hartzell et al. 1984a;Rio and Tjian 1984;Vigneron et al. 1984), the 72-bp direct repeats containing many enhancer elements (Fromm and Berg 1982; Hartzell et al. 1984b;Keller and Alwine 1985; Emoult-Lange et al. 1987), and the region from the 72-bp repeats up to and beyond the major in vivo initiation site, L325 (Brady et al. 1982;Keller and Alwine 1985; Emoult-Lange et al. 1987;Ayer and Dynan 1988). The major late initiation site L325 is not preceded by a typical TATA box (Brady et al. 1982). Environmental factors may...

Regulation of the human multidrug resistance gene (hMDR1) was studied by mapping DNA elements in the proximal promoter necessary for efficient transcription. Transient transfection analysis in tumor cell lines (HCT116, HepG2, and Saos2) of promoter deletions identified several regulatory domains. These cell lines expressed hMDR1 mRNA. Removal of an element between +25 and +158 reduced promoter activity by 2-3-fold, whereas deletion of sequences from approximately -5000 to -138 base pairs gave a approximately 2-fold increase. The activity of the hMDR1 promoter (-137 to +25) was comparable in activity to the SV40 early promoter and enhancer combination. Deletion of the hMDR1 promoter between -86 and -44 reduced activity by 5-10-fold, identifying an important regulatory region. This minimal region (-88 to -37) activated transcription when inserted upstream of a synthetic promoter, suggesting that it acts independently of other regulatory sequences. Two DNA elements within 85 base pairs of the transcriptional start site were required to confer efficient gene expression. A double-point mutation in the Y box (inverted CCAAT box) between -70 and -80 reduced activity of the promoter by 5-10-fold, and a single-point mutation at -52 within a GC-rich element reduced activity by 3-fold. Thus, both the Y-box and GC elements must each remain intact for optimal promoter activity. DNA-binding analyses suggest that the transcription factor NF-Y, but not YB-1 or c/EBP, is most likely responsible for controlling the activity of the Y-box element in these tumor cell lines. DNA-binding analyses also suggest that Sp1, alone or in combination with other nuclear factors, likely controls the activity of the GC element.

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Spl. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Spl versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of a-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an a-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10-to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.Within eukaryotic cells, gene expression is regulated in the context of chromatin, a complex of genomic DNA, histones, and nonhistone proteins. Localized biochemical and structural changes of the chromatin have been correlated with the ability of individual genes to be actively transcribed. Such changes include higher concentrations of high-mobility-group proteins, modified histones, methylated cytosine residues (for reviews, see references 53 and 73), and the appearance of DNase I-hypersensitive sites, both 3' and 5' to the gene (for reviews, see references 20 and 29). Hypersensitivity to DNase I is believed to be due to the binding of specific transcription factors to promoter elements and to the displacement or the specific positioning of nucleosomes. Therefore, it is argued that chromatin plays a role in the regulation of gene expression.To investigate directly the role of chromatin in regulating transcription, in vitro assays have been developed to study the initiation and elongation of transcription on chromatin templates. Such in vitro transcription studies require substantial quantities of a defined DNA complexed into nucleosomes. Several previous studies have used as templates chromatin assembled in vitro by incubating DNA with either * Corresponding author.t Present address:

Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein.

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