Genomic Sequencing by Template Purification: Principles and Mapping of Protein-Bound and Single-Stranded Sequences in Vivo

GENOMIC SEQUENCING BY TEMPLATE PURIFICATION: PRINCIPLES AND MAPPING OF PROTEIN-BOUND AND SINGLE-STRANDED SEQUENCES IN VIVO

Jovan Mirkovitch

Abstract ....................... . . . . . . . . . . . . . 26 I. Introduction .......................................... 26 omic Sequences ............................ 28 11. Purification of Specific A General Considerations. ................................. B. Isolation of Genomic DNA. . . . . . . C. Synthesis of Biotinylated RNA . . . . . . . . . . . . . . . . . . D. Hybridization and Purification of Genomic Sequenc E. High-Specific-Activity Primers . . . . . . . . . . . . . . . . . . . . . . . . . . 33 F. Primer Extension on Purified Ma . . . . . . . . . . . . 36 I11 Mapping Protein-DNA Interactions in Cells and Nuclei A. Liver-Specific Transcription. . . . B. Liver Transcription During Deve C. Induction of Transcription by Interferons .......................... 38

Advances in Molecular and Cell Biology Volume 21, pages 25-46. Copyright 0 1997 by JAI Press Inc. All rights of reproductionin any form reserved. ISBN 0-7623-0145-7

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IV. Mapping Melted Regions in Genomic DNA In Vivo. ..................... 39 A. General Considerations . . . . . . . . . . . . . . . . B. Transcription-DependentMelting in Cells and Nuclei . . . . . . . . . . . . . . . . . 40 C. Genomic Sequencing Detection of KMn04-Modified Residues V. Perspectives . . . . . . . . Acknowledgments . . . References ................................................ 44

ABSTRACT A new genomic sequencing procedure is described. This technique involves as a key step the purification of specific sequences from the bulk of the genomic DNA using a biotinylated RNA probe. The specific sequences purified from genomic material are then a suitable template for primer extension with a highly radiolabeled probe or for ligation-mediatedpolymerase chain reaction. This methodology allows the identification of stable DNA-protein complexes on genomic DNA as well as the mapping of genomic sequences presenting an altered DNA conformation. We present here details of the procedure and some particular aspects that permit the mapping of transcription-associated melted regions of DNA in whole cells and nuclei.

1.

INTRODUCTION

In recent years, our understanding of the mechanisms of gene expression control in eucaryotes has advanced dramatically. However, our present understanding of the mode of action of transcription factors derives from a limited number of techniques. With the exception of systems with simple genetics such as yeast, transcription cannot be studied on a gene in its natural location. Most eucaryotic systems rely on the technique of transient transfection for the mapping of DNA regulatory elements. Typically, DNA constructs are introduced into cultured cells and transcription is assayed 48 hours after transfection. More elaborate techniques exist for the stable introduction of DNA constructs into the genome either in cultured cells, or in whole organisms such as Drosophilu or the mouse. However, the use of homologous recombination to knock out a gene or to place an altered copy back in its natural context has been achieved only recently in mammals and is technically challenging. For example, liver-specific expression is probably driven by half a dozen factors, and a complete genetic analysis of the role of each factor would require an immense effort. In a now traditional analysis of the transcription of a gene, regulatory regions are first mapped by gene transfer techniques, then proteins that interact with these sequences are identified in vitro. Aprotein extract derived from whole cells or nuclei is incubated with a radiolabeled DNA fragment of specific sequence, and the DNA-protein complexes are detected

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by the techniques of gel-mobility shift assay, exonuclease I11 digestion, or footprinting. As can be predicted, these studies are subject to many pitfalls, as some proteins may need particular conditions to bind stably to DNA in vitro , or are simply not present in amounts large enough to be detected. This methodology has been the principal means of purifying and cloning proteins interacting with DNA. Although informative and necessary, the results obtained from such experiments must be confirmed by other methods that may not be easily practicable in higher eucaryotes. In the context of transcription control, the precise involvement of most described transcription factors has been difficult to ascertain. If a promoter contains a number of different DNA elements modulating transcription in transient assays, which of these are actually stably bound by proteins in vivo? Are all of these binding sites necessary for ongoing transcription? Are some of these sites necessary in a transient manner, either at each round of transcription or to induce a transcriptionally active state? Are other DNA sites binding proteins in vivo that could not be detected by transient assays or gel-shift? What is the role of chromatin structure in transcription? If a DNA sequence can bind different proteins present in the same cell, which one actually does bind in vivo and how is it discriminated from the other binding proteins? In vivo examination of the occupancy of DNA elements regulating transcription is therefore critical in revealing the mechanisms of action of different DNA-binding proteins during regulated gene expression. However, such an approach has been addressed only in very few cases, as the techniques were difficult to cany out. The availability of novel genomic sequencing methods opens new perspectives for the study of gene regulation and chromatin organization. Genomic sequencing consists in obtaining a sequence signal corresponding to a specific sequence using genomic DNA as the starting material. This permits the analysis of the structure of the genomic DNA and its occupancy by proteins using reagents specific for different DNA conformations. A number of genomic sequencing procedures for in vivo footprinting have been described in recent years (Ephrussi et al., 1985; Jackson and Felsenfeld, 1985; Selleck and Majors, 1987; Becker et al., 1989; Mueller and Wold, 1989; Saluz and Jost, 1989; Zhang and Gralla, 1989). However, clear mapping of genomic sequences interacting with proteins in vivo requires a simple and reliable procedure. This paper describes in detail a new genomic sequencing technique that involves the purification of the sequence of interest from the bulk of the genomic DNA (Mirkovitch and Darnell, 1991). This purification procedure permits the detection of a sequence ladder by primer extension with a highly radiolabeled nucleotide. Although the methodology is relatively simple, it necessitates rather large amounts of genomic DNA that may be difficult to obtain in some biological systems. However, purified genomic sequences appear to be adequate for the ligation-mediated polymerase chain reaction (LMPCR) technique, which can amplify sequences when one end is not defined (Mueller and Wold, 1989; Pfeifer et al., 1989).

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II.

PURIFICATION OF SPECIFIC CENOMIC SEQUENCES A. General Considerations

A summary of the purified template genomic sequencing technique is presented in Figure 1. The procedure takes about a week from the treatment of cells to the detection of the sequence signal. In setting up the technique one should first define precisely the 200 to 400 bases that are to be studied. Although difficultiesmay appear at all steps, the most difficult task is faced at the very end of the protocol, when the purified sequences must be detected by a procedure that results in an accurate sequencing ladder. Therefore it is recommended to first obtain a dependable primer extension procedure with a radiolabeled primer, using cloned plasrnid DNA as a template. Be aware that for systems with genomes as large as those of mammals, one sample necessitatesabout 200 pg of DNA (about 3 x107 cells). This is the main limitation of the technique, and if you cannot afford it, try the ligation-mediated PCR technique (LMPCR) that uses only 1 pg of DNA per sample (Mueller and Wold, 1989; Pfeifer et al., 1989). We have successfully used the technique of Mueller and Wold on sequences after they were purified from total genomic DNA using our protocol, greatly increasing the sensitivity. Purified genomic sequences are a very good substrate for LMPCR, as three different sequences could be analyzed by the protocol of Mueller and Wold without concern for the primer sequences used (Mirkovitch and Darnell, unpublished results). The present technique has the principal advantage of being relatively simple to set up, although it may not be easy for novice molecular biologists. All of the sequences we tested were easily purified, and with slight variations, all purified genomic sequences could be primer extended and true sequence information could be obtained. The choice of primers and restriction enzymes should be made with the following criteria. Modified or cleaved genomic DNA should be digested with restriction enzymes so that the fragment to be purified is smaller than 1 kb (the largest we have tried was 800 bp). The presence of one or more repetitive elements in the fragment could lower the purification yield. If most fragments are large and contain two or more repetitive sequences, a gelatinous mass will form after hybridization and purification will be impossible. Thus it is necessary to use at least one 4-bp cutter enzyme that can be at the restriction fragment boundary. Alternatively, if the 4 bp cutter does not cut in the region under analysis, the boundaries can be subsequently defined by addition of other 6 bp cutter enzymes. Primers for extension can be placed anywhere along the purified template. However, we obtained better results when the primer was complementary to the end of the sequence, next to a restriction site. Primers annealing in the template can work, although the conditionsmay be more difficult to determine. Primers annealing next to a restriction site cut are usually easily extended by Tuq polymerase or Klenow, whereas primers annealing in the middle of the template seem to work better with T4 DNA polymerase.

Treat cells or nuclei with DNA modifying or cleaving reagent. -

Isolate genomic DNA. Digest with adequate restriction enzymes. Induce cleavage at modified residues (if necessary). L

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I I I I

I

Denature genomic DNA and hybridize with gene-specific biotinylated RNA probe.

1-

Immobilize hybrids on Streptavidin-agarose. Wash away bulk of genomic DNA.

Recover purified genomic sequences by alcali treatment. Hydrolysis of RNA probe.

L

Detect cleavage sites by primer extension or ligation-mediated PCR.

Figure 1. Outline of the purified template genomic sequencing procedure.

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Conditions for the isolation of genomic DNA, purification of a specific sequence, and detection of the purified sequences are described in detail below. The purification procedure is rather straightforward, the main cause for poor yields being the presence of free biotinylated ribonucleotides in the biotinylated RNA preparation. Free biotinylated ribonucleotides should be carefully removed by selective ethanol precipitation.

B.

Isolation of Genomic DNA

For in vivo footprinting,cells are treated with dimethyl sulfate (DMS) or nuclei are treated with DNase I as described (Mirkovitch and Darnell, 1991, 1992; Mirkovitch et al., 1992). Treated cells or nuclei are lysed by the addition of an equal volume of 2 x SK buffer (50 mM Tris-HCI, pH 8.0, 400 mM NaCI, 5 mM Na, EDTA, 0.4% sodium dodecyl sulfate, 0.1 mg/ml proteinase K). The lysate is incubated at 37°C for a few hours and then extracted twice at room temperature with PCI (phenol:CHC13:isoamylalcohol at 25:24:1, saturated with 10 mM TrisHCI, 1 mh4 Na,EDTA). The genomic DNA is precipitated by the addition of 2.5 volumes of ethanol. For DMS or KMn04 treatment of whole cells where the DNA is not cleaved yet and cellular RNA is still present, the precipitate is immediately centrifuged at room temperature for 10 min at about 2000 xg. This step results in the quantitativerecovery of genomic DNA, while most of the cellular RNAremains in the supernatant. For DNase I-treated or sheared genomic DNA, samples are incubated for 2 hours at -20°C after the addition of ethanol and centrifuged at 5000 x g for 30 min. All samples are dried under vacuum and then resuspended in TE (5 mM Tris-HC1, pH 8.0, 0.5 mM Na2EDTA). Resuspension is greatly facilitated if the DNA pellet has been well dried in a vacuum. However, if genomic DNA is contaminated by proteins, drying under avacuum is not recommended. At this step, genomic DNAcan be treated with RNase Aif necessary (20 pg/ml, 30 min at 37°C). Another option at this stage is to partially cleave with a restriction enzyme to facilitate further manipulations and use smaller volumes. After RNase A andfor partial restriction digestion, genomic DNA is extracted once with PCI and ethanol precipitated as described above. To prepare the genomic DNA for hybridization with a biotinylated probe, it must be digested with at least one 4-bp cutter restriction enzyme and other enzymes that will determine the extremities of the sequence to be analyzed. If the sequence is to be detected by primer extension with a radiolabeled probe, we use between 200 and 500 pg of genomic DNA for one sequencing lane. If LMPCR is used, 1-10 pg is adequate and provides enough material after the PCR step for multiple primer extensions. For large amounts of genomic DNA, samples are digested overnight with about one unit of each restriction enzyme for every 5 pg of genomic DNA. We like to use 4 bp cutters like HueIII, Hinfl, MspI, RsuI, or TuqI (in this last case for 4 hours at 65"C), which work well and are cheap. One of two inexpensive 6-bp cutters is also included, usually to define the ends of the sequence to be purified.

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Digestions are stopped by the addition of EDTA, and the digested DNA samples are extracted with PCI, precipitated with ethanol at -2O”C, dried in vacuum, and resuspended in RNase-free HE (20 mM Hepes-NaOH, pH 7.4, 1 mM Na2EDTA) at a final concentration of 2 to 5 mg/ml of DNA for samples of a few hundred micrograms (usually 200 pl), and samples for LMPCR (1 to 10 micrograms) are resuspended in 20 p1 of HE. From this step on, it is important to carry out all manipulations with silanized piasticware in an RNase-free condition. C. Synthesis of Biotinylated RNA The biotinylated ribonucleotide is synthesized by conventional procedures using sP6 and T7 RNA polymerases (Krieg and Melton, 1987) in the presence of biotin-ll-UTP. Biotin-11-UTP can be obtained from Enzo and can also be purchased from Sigma. We use a 3: l ratio of UTP:biotinUTP, as it is quite expensive and a single biotin molecule per nucleic acid is sufficient to immobilize it on streptavidin agarose. Other enzymes such as T3 RNA polymerase have not been tested but should work as well. We have not tried photobiotinylation after synthesis with regular nucleotides. The RNA product does not need to be full-length; actually, “smeary” preparations work as well as RNA preparations that show a clean band. The biotinylated RNAdoes not need to be longer than 50 nt, but it should encompass the sequence that will anneal to the primer. Although the preparation of biotinylated RNA uses standard procedures, two aspects are critical for satisfactory purification of genomic sequences. The RNA preparation should be freed as much as possible of the template DNA. Indeed, any template left will later hybridize with the RNA, copurify with genomic sequences, and produce artifactual bands in the detection procedure. Therefore the RNA is extensively digested with DNase I. If convenient, an aliquot of the RNApreparation is used in a primer extension or LMPCR assay to check that no signal is produced. Another step critical for successful purification is the elimination of unincorporated biotinylated UTP from the RNA preparation. This is done by selective ethanol precipitation in the presence of 0.75 M NH40Ac with 2.5 volumes of ethanol at room temperature. A complete protocol that yields large amounts of biotinylated RNA is presented below. Smaller amounts may be synthesized if LMPCR will be the main procedure to detect purified sequences. The reaction mix is prepared in the following order at room temperature: 20 pg of a plasrnid digested by a restriction enzyme, purified by PCI and ethanol precipitation (as for all SP6 and T7 protocols do not use enzymes that produce 3‘ overhangs, or eliminate overhangs by T4 DNA polymerase treatment); H,O to make a final reaction volume of 100 ml, 2 p1 of 1 M dithiothreitol (DIT); 10 p1 of ATP, CTP, and GTP at 10 mM, 7.5 p1 of UTP at 10 mM; 1.25 pl of biotin-1 1-UTP at 20 mM, 20 pl of 5 x buffer (1 x is 20 mM Tris-HC1, pH 7.4, 10 mM NaCl, 6 mM MgCl,, 1 rnM spermidine, and it works well for both SP6 and T7), 10 ml of RNasin at 40 units/pl (Promega); and 150 units of SP6 or 300 units of T7 RNApolymerase.

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The reaction is left to proceed at 40°C (SP6) or 37°C (Ti')for 1 hour. After that time, another aliquot of polymerase and RNasin is added and incubation is continued for another hour. Two hundred units of DNase I ( B E or Boehringer Mannheim, RNase-free) is then added with 200 units of RNasin (5 pl), and the samples are incubated at 37°C for 1-2 hours. One hundred seventy microliters of HE, 30 ml of 7.5 M NH40Ac, and 0.8 ml of PCI are then added. The samples are vortexed, and spun for 1 min in a microfuge, and the phenol layer is removed when the interphaseis left with the aqueous phase. This is important, as biotinylated RNA may partition at the interphaseat this point. The interphase may dissolve when 0.75 ml of ethanol is added. Samples are left for 10 min at room temperature (they should appear a little cloudy), after which they are spun in a microfuge at room temperature for another 10 min. Ethanol is carefully removed, and the pellets are left to dry on the bench for a few minutes and then resuspended in 170 p1 HE. MgCl, is added to a final concentration of 10 mM, NaCl to 50 mM, and 5 pl of 1 M DlT,10 p1 of RNasin (40 U/pl), and 200 units of DNase I are also added. Digestion is left to proceed for 1-2 hours at 37"C, after which 20 pl of 7.5 M NH40Ac and 0.8 ml of PCI are added. The aqueous phase and interphase are recovered and added to another tube containing 500 pl of ethanol, and the RNA is precipitated again at room temperature. The pellet is resuspended in 200 p1 of HE, and 20 p1 of 7.5 M NH40Ac and 0.5 ml of ethanol are added and the RNA is again precipitated at room temperature. The biotinylated RNA is resuspended in a final volume of 100 p1 of HE, and 2 pI are used for UV quantification and another 2 p1 for analysis on a small denaturing acrylamide gel (optional but informative, biotinylated RNA runs about 20% slower than regular RNA). This protocol yields on average about 50 pg of clean biotinylated RNA. D. Hybridization and Purification of Genomic Sequences

The following protocol is devised for samples of 400 pg to 1 mg of DNA for primer extension with a highly labeled probe. Samples for LMPCR are processed in 10 times smaller volumes during hybridization. To genomic DNA in 200 p1 of HE is added 100 p1 of 0.6 M NaOH, and the samples heated at 65OC for 5 min. The samples are cooled to room temperature, and 30 pl of 1 M HEPES-NaOH (pH 7.4) and 100 p1 of 0.6 M HCl are added in this order and immediately vortexed. seventy microliters of 5 x HB (5 - is 100 mM HEPES-NaOH, pH 7.4, 3 M NaCI, 5 mM Na2EDTA,0.5 mg/ml yeast tRNA and 0.25% Tween 20) containingthe biotinylated RNA freshly added to it is mixed with the denatured genomic samples. The final concentration of biotinylated probe is 0.1-0.3 ml/ml. Tubes are placed in NEN lead containers that are submerged in a 65°C water bath so that no condensation forms. Hybridization is left to proceed overnight. Hybrids are immobilized on streptavidin-agarose(Sigma). To prevent nonspecific adsorption on beads, the Sigma preparation is diluted with 5 volumes of 1 x HB and washed three or four times before addition to the hybridization mix.

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Magnetic beads were not tested but should work as well: however, we would recommend washing them with 1 x HB before use. Washed streptavidin-agarose diluted 10 times in 1 x HB is added to the hybridization mix (100 yI for large samples, 20 pI for LMPCR samples), and the hybrids are left to bind for 30-60 min at 37°C with shaking. Beads are sedimented by spinning for 10 sec in a microfuge, and the supernatant is recovered if the other strand has to be purified. The beads are washed twice with 400 y1 of HE. Care should be taken not to pipet out the bead pellet, since it is rather small and may be difficult to see with some brands of microtubes. Genomic DNA is recovered from the beads by alkali treatment. One hundred eighty microliters of 0.2 M NaOH (90 pl for LMPCR samples) is added to the beads, and the tubes are vortexed and left for 2 rnin (or longer if convenient) on the bench. The tubes are spun for 10 sec, and the supernatant is recovered in a silanized microtube using a silanized tip. It is important to work with silanized plasticware, as the amount of purified DNA is very small. The tubes are placed in a 7OoC water bath for 20 rnin to hydrolyze the RNA. Tubes are chilled on ice and 30 pl(15 for LMPCR) of 1 M Tris-HCI (pH 7.4) containing 100 pg/ml yeast tRNA is added, immediately followed by 90 pl (45 for LMPCR) of 0.4 M HCI, and the samples are immediately vortexed. PCI (0.5 ml) is added (0.3 ml for LMPCR); the samples are heated for 2 rnin at 37°C vortexed, and microfuged for 2 min and the aqueous phase is placed in a clean, silanized microtube containing 0.9 ml (0.45 ml for LMPCR) of ethanol. The tubes are left overnight at -20°C or chilled twice on dry ice before centrifugationfor 15 rnin in a microfuge. The visible small pellet is then dried on the bench (all of the ethanol should be easy to pipette out from silanized tubes) and is resuspended in 3 or 6 y1 of HE. E.

High Specific Activity Primers

Purified sequences can be detected either by primer extension with a highly labeled probe or by LMPCR. In both cases though, a similar primer extension is performed on the purified material, with either a labeled or a cold primer. We will describe here in detail the synthesis of a highly radioactive primer and the conditions affecting primer extension so as to obtain accurate sequencing ladders. A 25-mer primer that contains 8 to 16 radioactive nucleotides, allowing a specific activity of about 30,000 Ci/mmol to be obtained, is prepared mainly according to the method of Saluz and Jost (1989). Two hundred picomoles of a 29-mer template is annealed to 1000 pmol of a 9-mer primer in a final volume of 50 yl in 8 x MS buffer (from Boehringer Mannheim, 1 x is 10 mM Tris-HC1, pH 7.4, 10 mM MgCI,, 50 mM NaCI, 1 mM dithioerythritol), which yields 5 pmol template/yl in 8 x MS buffer. Both oligos are purified on polyacrylamide before annealing. The 9-mer anneals to the 29-mer so as to produce a 4-bp 3' overhang. Fill-in of 16 nucleotides produces a 25-mer labeled primer. Synthesis of the 25-mer

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radiolabeled primer is carried out in a final volume of 4 pl. Hot nucleotides (10 microliter of each one at 3000 Ci/mmol, 33 pmol) and different cold nucleotides (250 pmol) are dried in a silanized tube. Nucleotides are resuspended in 3 p1 of H,O, and then 0.5 p1 of annealed oligos (2.5 pmol of template) and 0.5 p1 of Klenow enzyme at 2 units/pl are added. The number of hot nucleotides is not important. The reaction works if only one, or all four, of the nucleotides are radioactive. For economic reasons it is usually convenient to use only one or two hot nucleotides that will introduce 10 or more radioactive residues in the primer. The extension reaction proceeds for 10 min at room temperature and is stopped by the addition of 4 p1 of gel loading formamide buffer. The mixture is electrophoresed on a small 12% acrylamide-ureagel. The position of the 25 mer is detected by autoradiography (a 1-sec exposure is usually sufficient), and the corresponding region of the gel is excised. The gel piece is crushed with a small pestle, and 400 p1 of elution buffer is then added (25 mM Tris-HC1, pH 8.0,400 mM NH40Ac, 2 mM Na2EDTA, 0.1% SDS). The hot primer is left to diffuse out for 1 hour with mixing, after which the gel pieces are sedimented, and the supernatant is recovered, corresponding to about 80% of the probe as estimated by monitor. Two micrograms of yeast tRNA are added as a carrier, and the mixture is extracted once with PCI and precipitated with 4 volumes of ethanol overnight at -2OOC or by chilling twice on dry ice. After precipitation, the probe is resuspended in 20 pl of HE for a final concentration of about 0.1 pmol/pl. The primer is then used in the following days. Primers older than about 5 days tend to produce high backgrounds.

F.

Primer Extension on Purified Material

Primer extension is done with identical protocols for direct detection with a highly radiolabeled primer, or for LMPCR with an unlabeled primer. However, reactions are stopped and processed differently if they have to be directly run on sequencing gels or amplified by LMPCR. Different parameters may be tested to obtain a true sequencing ladder. These include annealing temperature and time, as well as primer amount, but in our hands the nature of the DNA polymerase is the most critical parameter. Tests are most easily carried out on plasmid DNA that has been cleaved by a Maxam-Gilbert sequencing reaction (for example, a G-ladder produced by DMS modification and piperidine cleavage). For highly labeled primers, 1 ng of cleaved plasmid (about 3 kb) is equivalent to sequences purified from about 1 mg of human or rodent genomic DNA. Conditions are varied until a Gladder corresponding to the sequence is obtained. Conditions may be tested using a primer labeled by kinase reaction, but higher amounts of plasmid then have to be used. In this case, it is important to use short annealing times, as the two strands of the plasmid DNA may renature and inhibit primer extension. The purified template is resuspended in 3 p1 of HE in a silanized tube to which 1 p1 of 0.2 M NaOH and 1 pl of primer are added (2 pl of a freshly prepared mix

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introduced on the side of the tube and left to fall to the bottom results in adequate mixing). Samples are incubated for 5 min at 65°C and then chilled on ice. The amount of primer necessary is variable, depending of the sequence. In our hands, 0.01 pmol of radioactive primer (0.1 pl of a highly labeled primer preparation, 5 about 10 cpm) produces nice results. Higher amounts may increase the specific signal but sometimes also the background. To the samples chilled on ice, 2.5 p1 of a freshly prepared mix of 1 pl of 0.2 M HCl and 1.5 microliter of 10 x extension buffer are added along the side of the tube and left to fall into the sample (1 x extension buffer is 10 mM Tris-HC1, pH 8.5,at room temperature, 25 mM KCl, and 3.5 mM MgCl,). Annealing is typically done for 10 rnin at 6OoC, but the temperature may vary between 55 and 65OC, depending on the sequence of the 25-mers used as primers. For longer annealing times, usually not necessary, samples are put into submerged NEN lead containers for up to 40 min. After annealing, samples are chilled on ice and 12.5 pl of nucleotides and enzyme mix is added, the samples are rapidly vortexed and briefly centrifuged to bring up all the liquid at the bottom, and extension is carried out for 10 min at 7OoC for Tug polymerase, or 50°C for Klenow. The freshly prepared 12.5 ml mix consists of 0.5 p1 of 10 x buffer, 0.8 pl of dNTPs (each at 10 mM in this stock), and polymerase for a final concentration of 10 unitdm1 (Tuqor Amplitaq) or 25 unitdm1 (Klenow) in the final extension reaction (20 pl). It is important to use low amounts of polymerase, as primer extension in the presence of higher concentrations does not result in readable sequencing ladders. It is also important to incubate samples with Tug or Amplitaq for up to 10 min since these enzymes have a terminal deoxynucleotidyl transferase activity that is variable, and this will ensure that every extended product has an extra protruding residue. Other enzymes such as Vent or T4 DNA polymerase can work as well. Vent seemed to work as Tuq and Klenow, whereas T4 DNA polymerase seems to be adequate for substrates where the primer anneals in the middle of the molecule (Mirkovitch, unpublished results). An advantage of T4 DNA polymerase is that it degrades most of the nonextended primer, which results in lower background when radioactive primers are used. However, T4 works efficiently only at 37°C and seems to be more susceptible in theseconditions to secondary structure than Tag or Klenow, which are used at higher temperatures (Mirkovitch, unpublished results). Vent is used with the buffer supplied by New England Biolabs, and T4 DNA polymerase is used in a 1 x buffer consisting of 50 mM Tris-HC1 (pH 8.5), 15 mM NH40Ac, 25 mM NaCl, 7 mM MgCl,. Bovine serum albumin and 2-mercaptoethanol are added to the 12.5yl nucleotide-enzyme mixture to obtain final concentrations of 0.1 mg/ml and 0.1%, respectively. Vent and T4 DNA polymerase are both used at a final concentration of 25 unitdml. Primer extensions are stopped differently, depending on whether they are to be detected by LMPCR or directly run on a sequencing gel. Unlabeled primer extension reactions for LMPCR are stopped by the addition of 80 pI of 10 mM Tris-HC1 (pH 8.0),2 mM Na2EDTA,200 mM NaCI, 25 pl/ml yeast tRNA, extracted

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once with PCI and precipitated with 4 volumes of ethanol in dry ice. LMPCR is then performed exactly as described (Mueller and Wold, 1989),except that the PCR products are purified by PCI extraction and ethanol precipitation, and various amounts of amplified products are primer extended with a kinased primer as described above. Samples extended with labeled primers are stopped by the addition of 170 p1 of 25 mM Tris-HC1 (pH 7.4), 1 mM Na2EDTA, 0.2% sodium dodecyl sulfate, and 5 pl/ml yeast tRNA or denatured salmon sperm DNA. Tubes are then briefly vortexed, microfuged, and incubatid at 80°C for 20-30 min. This step seems to decrease the background quite significantly. Samples are cooled at room temperature, and 10 pl of 5 M NaCl and 300 p1 of PCI are added. After vortexing and centrifugation, the aqueous layer is placed in a new silanized tube containing 0.9 ml of ethanol and put twice on dry ice until solid. After a 15-min spin, the supernatant is carefully removed and a small pellet should be visible. The sample is resuspended in 3 p1 of 90% formamide, 0.5 x TBE containing 0.2% bromophenol blue and 0.2% xylene cyanol. Tubes are vortexed to resuspend the samples and heated at 80°C for 5 min prior to electrophoresis on sequencing gels that have been poured the day before. Unincorporated primer is run out of the gel, which is then fixed in 10% methanol, 10% acetic acid. Gels are dried on cellophane sheets and exposed between two intensifying screens for 12 hours to 3 days.

111.

MAPPING PROTEIN-DNA INTERACTIONS IN CELLS AND NUCLEI A.

Liver-SpecificTranscription

The template purification genomic sequencing technique was used to footprint the promoter of the transthyretin gene (TTR, also called prealbumin) in liver and spleen nuclei using DNase I or copper-phenanthroline as cleaving agents (Mirkovitch and Darnell, 1991). Many regions were protected in liver nuclei, a tissue where the gene is highly expressed, but not in spleen nuclei, where the gene is not expressed. The TATA box region and two binding sites for the liver factors HNF3 were well protected. The sequences binding HNF3 were previously shown to be required for TTR expression by transient assay, and the importance of HNF3 in vivo was demonstrated by these footprinting experiments. In addition, a site just upstream of the TATA box was also protected in liver nuclei, and gel-shift experiments showed that it was bound by an NF-1 like protein. Surprisingly,the sequences in the TTR promoter corresponding to binding sites for the liver-enriched proteins CEBPand HNF4 were not protected from cleavage in either liver or spleen nuclei. Further experiments showed that these CEBP and HNF4 unoccupied promoter sites do not influence transcription in the presence of a functional enhancer (Costa and Grayson, 1991). However, both CEBP binding sites in the TTR enhancer were strongly protected. Indeed, the integrity of these sites was previously found to be

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necessary for enhancer function in transfection assays. Had the footprinting on nuclei been done to start with, a lot of time and energy could have been spared in the analysis of the relevant sequences in the promoter and enhancer. These observations demonstrate the importance of determining the occupancy of binding sites in vivo, to assess the role of the putative binding sites identified in vitro.

B. Liver Transcription During Development Genomic sequencing has been difficult to carry out on higher organisms. Although the new technique described here facilitates genomic sequencing, it requires a fair amount of genomic DNA, typically 200 pg or more for one sample, and this may not be practical for some biological systems. A technique for amplifying the specific sequences would therefore be of great value. In this respect, the introduction of the PCR methodology has revolutionized the study of nucleic acids. This procedure for amplifying DNA sequences usually necessitates that the two extremities of the target molecule be known to allow a match to specific primers. Unfortunately, in sequencing usually one end of the molecule is known and can be labeled, and the other end is variable and has to be identified. However, a protocol has recently been described (LMPCR) for the PCR amplification of a population of molecules in which one end is not defined (Mueller and Wold, 1989). Although difficulties with LMPCR can originate at many steps, we found that isolation of a specific sequence from the bulk of genomic DNAprior to the LMPCR greatly facilitates the procedure (Mirkovitch and Darnell, unpublished results). In fact, each strand of three different sequences was obtained at the first try without a particular strategy in the creation of the primers. Therefore, some difficultiesthat may be encountered with LMPCR may be attributed to the presence of a large excess of irrelevant DNA during the first enzymatic reactions. With a method that permits the analysis of as little as 1 pg of genomic DNA, it was possible to look at the developmental pattern of proteins binding to the 'ITR promoter and enhancer in mouse embryos. From fertilization to birth, mouse embryological development lasts 20 days. By day 9, a liver bud can be detected differentiatingfrom the developing foregut, where it is induced by the presence of the neighboring precardiac mesenchyme. By day 11 a liver can be detected by eye as a diffuse reddish spot. At this stage the liver is the recipient of hematopoietic cells that migrate from the yolk sac and is the major hematopoietic tissue. At day 13hepatocyteshave clearly appeared among the hematopoietic cells. The transcription of some typical liver genes such as a-fetoprotein, albumin, and TTR can be detected at this 13-day stage, although it takes place at a much lower rate than later in development. Interestingly, we could clearly detect a protection over the TATA box of the 'ITR promoter in nuclei derived from 13-dayembryo liver that correlates with the initial but low expression of TTR during development. Expression of l T R remains at low levels during the rest of gestation, and only a protection over the TATAregioncan be seen during that period. However, in the first 2 weeks following

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birth, expression of l T R is increased to reach its highest levels in the young adult. At this stage, clear protection over the HNF3 sites appeared after birth and correlated with increased expression of ‘ITR. Protection of the enhancer CEBP sites appeared only in adult hepatocytes where transcription reaches its highest levels. Therefore ‘ITR transcription seems to be switched on at the first appearance of hepatocytes at day 13, where it is directed mainly by the TATA binding factor. After birth, increased levels of TIT transcription result from the binding of the HNF3 protein at the promoter, and by C/EBP in the adult. These results presented the first analysis at the nucleotide level of proteins binding to the genome during developmental regulation. The signals that induce the TATA protection from day 13 and HNF3 binding after birth are not known. TATA binding factors are present in all cells during the whole development, and HNF3 proteins appear at day 9, before the formation of a liver and long before the induction of high levels of transcription of l T R . It may well turn out that the mechanisms that trigger liver-specific transcription are not the “liver-specific” transcription factors of the HNFl, CEBP, HNF3, and HNF4 families. However, these factors could be the tools used by other signals to regulate proper transcription at different developmental stages (Mirkovitch, unpublished observations). C.

Induction of Transcription by Interferons

The regulation of genes induced by type I and type I1 interferons has been extensively studied in Dr. Darnell’s laboratory (Levy and Darnell, 1990). The chromatin of the a-interferon induced gene ISG54 presented a footprint on the ISRE, the upstream regulatory sequence necessary and sufficient to confer a-interferon inducibility on a basal promoter. The TATA box was also well protected. Again, footprinting on nuclei identified the relevant control element. Occupancy of the ISRE DNA element was also demonstrated by protection against DMS. Since DMS can penetrate cells easily to methylate purines on the genomic DNA and does not require the prior isolation of nuclei, this kind of analysis was performed on whole cells (Mirkovitch et al., 1992). We have also investigated the chromatin structure of the GBP promoter, a gene with a complex pattern of regulation that can be induced by both type I and type I1 interferons (Mirkovitch et al., 1992). These studies again demonstrated the constitutive occupancy of the ISRE, but another DNA element, adjacent to and overlapping the ISRE, was also found to be occupied by proteins. This latter region, called GAS, was previously shown to be necessary for optimal y-interferon induction, but no specific protein binding to it could be detected by gel mobility shift assay. However, another in vitro assay for DNA-protein interactions demonstrated the existence of a low-affinity binding activity only in induced cells (Decker et al., 1991). This binding activity had the remarkable property of being translocated from the cytoplasm to the nucleus when cells were treated with y-interferon (Decker et al., 1991). In vivo footprinting results, using both DMS treatment of whole cells or DNase I digestion of isolated nuclei, showed

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a strong protection of the GAS binding site upon induction of GBP transcription. Two observations concerning this GAS footprint are important. First, in vivo footprinting clearly demonstrated the binding of a y-interferon induced protein to a region to which no DNA-binding protein could be detected by conventional gel-shift analysis. A second important observation is that this binding was transient and at a maximum just after y-interferon treatment, but disappeared later when transcription of the GBP gene reaches its maximum level. An altered DMS sensitivity on the GAS has been observed to correlate with transcription,confirming the importance of the GAS in GBP transcription control. Interestingly, different additional footprints were observed in cell lines that present different kinetics of GBP induction. The constitutive presence of these still unidentified factors in some cell lines seemed to put the chromatin in a conformation that allows a very rapid induction of the gene (Mirkovitch et al., 1992). These experiments on two very different systems, TTR, a developmentally regulated gene, and interferon induced genes, have shown the necessity of analyzing at the nucleotide level the chromatin of active genes for understanding transcription regulation. In both cases, these studies have allowed some of the most complete chromatin structure analysis of gene control elements.

IV.

MAPPING MELTED REGIONS IN GENOMIC DNA IN VlVO A.

General Considerations

If genomic sequencing can identify sequences bound in vivo by proteins, it should also detect altered DNA structures as long as these can be recognized by a specific modifying reagent. One important altered structure of genomic DNA is the single-stranded conformation. Single-stranded DNA can be found in different processes such as replication, recombination or transcription. Synthesis of RNA results in the creation of a melted transcription bubble during initiation and elongation. The last step in the assembly of the transcription preinitiation complex consists of the formation of a so-called open complex, where the DNA at the initiation site is melted so that the polymerase can engage in RNA synthesis upon the addition in ribonucleotides. An open preinitiation complex consists of an RNA polymerase structure, associated with initiation factors, that has melted part of the DNA template so that it can read the template DNA sequence to synthesize the complementary RNA. Open complexes have been described in vivo in bacterial systems (Chamberlin, 1976;Von Hippel et al., 1984). More recently, in vitro studies on eucaryotic transcription initiation have described open complexes for RNA polymerase I (Bateman and Paule, 1988), RNA polymerase I11 (Kassavetis et al., 1990), and RNA polymerase I1 (Wang et al., 1992). The presence of a melted region around the start site is indicative of a complete preinitiation structure, the addition

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of ribonucleotides to which results in RNApolymerization. A melted bubble is also associated with the elongation complex, as the polymerase has to read the DNA sequence and the nascent RNA chain is transiently paired with the DNA template. Open complexes are most easily observed using the single-stranded cleaving reagent KMnO,, which has a high specificity for single-stranded DNA. Whereas double-stranded DNA is not very reactive, thymine residues in single-stranded DNA are strongly modified and are oxidized by KMn0, to a glycol form (for a review on chemical modification of DNA, see Nielsen, 1990). The modified residues can be cleaved by piperidine treatment so that aT-ladder is obtained (Rubin and Schmid, 1980). These modified residues can also be detected by primer extension, as various DNApolymerases cannot proceed easily through the modified residues (Ide et al., 1985; Rouet and Essigmann, 1985; Hayes and LeClerc, 1986; Borowiec et al., 1987). A strong advantage of KMn04 is that it penetrates cells (probably by creating gaps in the cell membrane) and can rapidly modify genomic DNA in tissue culture cells. Indeed, KMnO, has now been used in a number of studies to identify single-stranded regions in vivo. KMn0, sensitivity was first used to detect open complexes in vivo in bacteria (Sasse-Dwight and Gralla, 1988,1989,1990; O’Halloran et al., 1989).In mammals, KMn04-sensitive regions were detected at the SV40 control elements in infected CV-1 cells (Zhang and Gralla, 1989, 1990). More recently, transcription-associated melting was detected in genomic DNA by using template purification genomic sequencing (Mirkovitch and Darnell, 1992) or LMPCR (Giardina et al., 1992; Krumm et al., 1992). Although these studies did not identify open complexes at the initiation site, they showed the presence of elongating polymerases along active genes (Mirkovitch and Darnell, 1992) or a paused polymerase complex about 25 nucleotides downstream of the RNA start site in various genes (Giardina et al., 1992; Krumm et al., 1992; Mirkovitch and Darnell, 1992). The possibility of mapping at the nucleotide level the position and relative density of RNA polymerases should provide important information in the future regarding the control of transcription initiation and elongation. We describe below how to map KMn0,sensitive residues by purified template genomic sequencing, a method that appears particularly appropriate for that particular purpose. B.

Transcription-Dependent Melting in Cells and Nuclei

We have recently described the localization of RNA polymerase I1 along the promoter and transcribed region of two genes by using the purified template genomic sequencing procedure and KMn0, sensitivity (Mirkovitch and Darnell, 1992). Analysis of the interferon-induced ISG54 gene showed that the transcribed region became hypersensitive to KMn0, modification when transcription was induced. This sensitivity was proportional to the transcriptional level of the gene and was homogeneously distributed along the first 300 bp of the transcribed region that was analyzed. This sensitivity was retained when nuclei were isolated from

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cells actively transcribing ISG54. Run on conditions that allow engaged polymerases to extend initiated products in nuclei resulted in the loss of this sensitivity, as the two DNA strands reanneal after the polymerases move along the template. However, the presence of a-amanitin during the run on prevented this loss of sensitivity, demonstrating that it was due to the presence of engaged RNA polymerases. Interestingly, no KMn04 sensitivity was seen around the TATA box upstream of the cap site, where transcription complexes assemble and result in the described in vitro open complexes. This absence could have two possible causes. First, the transcription complex might protect the DNAfrom KMn04 modification. We think this is unlikely, as transcription complexes assembled in vitro with a number of polymerases showed that KMn0, can modify single-stranded DNA in open complexes (Bateman and Paule, 1988; Kassavetis et al., 1990; Wang et al., 1992). However, as discussed elsewhere (Sasse-Dwight and Gralla, 1989), if there is no rate limiting step after formation of the open complex at the promoter, it could be very short lived and therefore difficult to detect. Another interesting observation was that no KMn0, sensitivity was seen in the uninduced state on the ISG54 gene, showing that unlike some other rapidly induced genes, there was no polymerase complex engaged and paused just downstream of the RNA start site. Such promoter-proximal paused RNA polymerases have been observed on a number of Drosophilu genes (Rougvie and Lis, 1988, 1990; Giardina et al., 1992), as well as on the human c-myc gene downstream of the P2 promoter (Krumm et al., 1992) and the mouse TTR gene (Mirkovitch and Darnell, 1992).

C . Genomic Sequencing Detection of KMn04-Modified Residues The treatment of cells or nuclei with KMnO, has already been described in detail (Mirkovitch and Darnell, 1992). We will describe here some aspects of KMn04 modification of DNA that make the template purification genomic sequencing procedure particularly suitable for the detection of single-stranded conformation in vivo. In v i m , thymine residues in single-stranded DNA are susceptible to modification by KMn0, at submillimolar concentrations, and further treatment with piperidine results in efficient cleavage specifically at T residues (Rubin and Schmid, 1980). However, experiments involving whole cells, nuclei, or samples with large amounts of proteins make it necessary to use KMn04 concentrationsof 10 to 30 mM. Under these conditions, KMnO, introduces additional modifications in both single-strandedand double-stranded DNA that result in strand scission after piperidine treatment. This is most inconvenient, as most of the cleavage appears at sites other than modified thymines, which results in increased background and low sensitivity of detection of single-stranded regions. However, it is possible to identify modified thymines by primer extension, as thymine glycol residues stop the elongation of various DNA polymerases (Ide et al., 1985; Rouet and Essigmann, 1985; Hayes and LeClerc, 1986; Borowiec et al.,

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1987). Under these conditions, we have observed that primer extension on a template treated with high KMn04 concentrations resulted in a T-ladder only if the template was not subsequently treated with piperidine (Mirkovitch, unpublished results). As a result, the template purification genomic sequencing procedure is optimal for detecting modified residues in vivo. The genomic DNA is not treated with piperidine, and modified T residues are detected by primer extension with a high-specific activity primer, as described (Mirkovitch and Darnell, 1992). Figure 2 compares two genomic sequencing procedures on the same template. Nuclei isolated from HeLa cells actively transcribing the ISG54 gene were treated with KMn04 before (lanes 1 of each panel) or after run on (Figure 2A, lanes 2; Figure 2B, lane 3). The template purification procedure results in a low background on which almost only T residues are visible (stars next to sequence). However, LMPCR on the same samples after treatment with piperidine results in the appearance of many cleavages at non-T residues, and some T residues do not appear. LMPCR requires cleavage with piperidine, so that blunt ends can be obtained for ligation of the linker used for PCR. We are currently testing procedures to obtain blunt ends on samples that have not been treated with piperidine, so as to eliminate the non-specific background and increase the sensitivity when LMPCR is used to identify KMn04-modified residues.

V.

PERSPECTIVES

Among the scientists trying to understand the molecular mechanisms of life, those working with yeast may be the most fortunate. The availability of simple homologous recombination permits the easy replacement of a gene by an altered allele. Even though it is now possible to execute gene targeting in higher organisms, it is still a very challenging technique. However, the study of biological processes involving DNA can take advantage of various genomic sequencing techniques. As described in different chapters of this book, it is possible to identify those sequences of the genome occupied by proteins in the cell. If, in the past, genomic sequencing was a very challenging technique, the particular methods that have been described here make it much simpler. This in vivo approach describes the gene in its natural milieu, the genome of the cell. The examples described here have shown that in vivo examination of the occupancy of DNA elements regulating transcription is critical in revealing the mechanisms of action of different DNA binding proteins during regulated gene expression. The genomic sequencing technique, is not only very useful for the detection of transcription factors and regulatory elements; it also appears to open new perspectives for other problems. Determining the position and density of RNApolymerases in active genes provides an important approach for studying the molecular events associated with transcription. The results discussed here show that the mapping of KMn04-induced cleavage sites in whole cells or isolated nuclei can be used to

Figure 2. Comparison of purified template and LMPCR procedures for identifying KMn04-induced cleavages. ISG54 transcription was induced to high levels in HeLa cells by interferon treatment. Nuclei were isolated and treated with KMn04 before (lane 1 ) or after (lanes 2 and 3) being incubated in run-on conditions KMn04-modified residues were mapped either by the purified template procedure using a high specific activity primer (A) or by LMPCR (B). Transcription starts at the arrow and proceeds downward (modification on the upper strand is presented). Stars represent the positionsof expectedthymine residues downstream of the RNA start site. For details, see Mirkovitch and Darnell (1992). 43

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determine the position of engaged polymerases at the nucleotide level. In addition, the magnitude of the KMnO, cleavage is proportional to the transcribing activity and therefore measures the relative density of polymerase complexes. The possibility of precisely mapping the distribution and density of active RNA polymerases on specific sequences in intact cells or isolated nuclei should help promote our understanding of the complex mechanisms involved in transcription initiation, elongation, and termination. The ability to study the requirements for reinitiation in isolated nuclei should provide ameans to determine which components are stably associated with chromatin and which are necessary only transiently at each round of transcription initiation. Experiments that are today conducted with cloned DNA and protein extracts in vitro could use cell nuclei or chromatin to ask the same questions on a material that has been assembled in vivo. The template purification procedure described here opens some interesting opportunities in areas not directly related to genomic sequencing. For example, purification of specific sequences from the bulk of genomic DNA can help in the detection of a tiny minority of mutant template. In the process of tumor progression, a number of mutational events take place in different genes. Most mutations can be scored only after a phenotypic alteration has occurred, by identifying the phenotypically different cell population from the rest of the population. It would therefore be very useful to have a tool to detect mutations that occurred in a very minor subset of cells before they develop into phenotypically different cells. For example, a specific mutation could destroy the recognition site of a restriction enzyme. The presence of mutant alleles can then be detected by the presence of fragments undigested by the enzyme by a PCR reaction with primers on each side of the restriction site. A regular PCR analysis would typically use not more than 10 pg of genomic DNA. But after the purification of specific sequences by biotinylated RNA, the equivalent of a few milligrams of genomic DNA could be used in a single PCR reaction. This would make possible the detection of a small mutant minority in a large population of wild-type cells. Very early stages of tumor progression could be studied by this methodology.

ACKNOWLEDGMENTS I am indebted to Dr. James E. Darnell, Jr., in whose laboratory most of the discussed experiments have been performed, and to Susan Gasser for reading the manuscript. Since the submission of the manuscript, a number of protocols have been improved and investigators are encouraged to contact the author. These studies have been supported by grants from the NIH, American Cancer Society, Swiss National Foundation and the Swiss League against Cancer.

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Rougvie, A. E., & Lis, J. T. (1990). Postinitiation transcriptional control in D.rnelanogasfer.Mol. Cell. Biol. 10,6041-6045. Rubin, C. M., & Schmid, C. W. (1980). Pyrimidine-specific chemical reactions useful for DNA sequencing. Nucleic Acids Res. 8,4613-4618. Saluz, H., & Jost, J.-P. (1989). A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level on higher eucaryotes. Proc. Natl. Acad. Sci. USA 86,2602-2606. Sasse-Dwight, S., & Gralla, J. D. (1988). Probing the E. coli glnALG upstream activation mechanism in vivo. Proc. Natl. Acad. Sci. USA 85, 8934-8938. Sasse-Dwight, S., & Gralla, J. D. (1989). M n 0 4 as a probe for lac promoter DNA melting and mechanism in vivo. J. Biol. Chem. 264, 80748081. Sasse-Dwight, S . , & Gralla, J. D. (1990). Role of eucaryotic-type functional domains found in the prokaryotic enhancer receptor factor sigma54. Cell 62,945-954. Selleck, S., &and Majors, J. E. (1987). In vivo DNA-binding properties of a yeast transcription activator protein. Mol. Cell. Biol. 7,3260-3267. Von Hippel, P. H., Bear, D. G., Morgan, W. D., & McSwiggen, J. A. (1984). Protein-nucleic acid interactions in transcription: A molecular analysis. Annu. Rev. Biochem. 53, 389-446. Wang, W., Carey, M., & Gralla, J. D. (1992). Polymerase I1 promoter activation: closed complex formation and ATP-driven start site opening. Science 2 5 5 , 4 5 0 4 3 . Zhang, L., & Gralla, J. D. (1989). In sifu nucleoprotein structure at the SV40 major late promoter: melted and wrapped DNA flank the start site. Genes Dev. 3, 1814-1822. Zhang, L., & Gralla, J. D. (1990). In sifu nucleoprotein structure involving origin-proximal SV40 DNA control elements. Nucleic Acids Res. 18, 1797-1803.