- Email: [email protected]
[74] In Vivo footprinting of specific protein-DNA interactions
[74]
In Vivo FOOTPRINTING
735
[Editors' Note. In Chapter 73, methods were presented for determining sequence specific binding of proteins in vitro to cloned regulatory elements of DNA. Chapter 74 contains high resolution methods for mapping the location of proteins bound to DNA in vivo; the difference in sensitivity to DNase I of DNA in chromatin compared with naked DNA is used to predict specific protein-DNA interactions. A different version L2 of this technique involves digestion of chromatin with DNase I, isolation of DNA, and complete cleavage with a restriction endonuclease that cuts rarely, as in [74]. Then, the fragments are separated electrophoretically in gels [8], transferred to nitrocellulose [61] and hybridized to a 32p-labeled probe [43, 45] that abuts the particular restriction site chosen as the point of reference. The visualization step is called indirect end labeling because the end is generated by a restriction enzyme and "labeling" of all subfragments sharing that end is achieved by hybridization. Improvements in Chapter 74 include solution hybridization and the use of short, highly radioactive, highly purified probes.] S. A. Nedospasov and G. P. Georgiev, Biochem. Biophys. Res. Commun. 92, 532 (1980). 2 C. Wu, Nature (London) 286, 854 (1980).
[74] I n V i v o F o o t p r i n t i n g o f Specific P r o t e i n - D N A Interactions By
P. D A V I D JACKSON a n d GARY FELSENFELD
We describe in this chapter a method for mapping the pattern of cleavages within specific unique DNA sequences in a complex genome at near single base resolution. The method has been used to accurately map the sites of sequence-specific protein-nucleic acid interactions ("footprints"; Fig. 1A) upon regulatory sequences in various members of the a- and/3globin gene families within chicken cells and cell nuclei.t-3 We examined the pattern of cleavages introduced into the genomic copies of/3- and aglobin gene promoters by exogenously added nuclease, but any reagent (endogenous as well as exogenous) that cleaves DNA is a candidate for this analysis. The method can be applied to the study of any gene for which in vivo structural information is desirable. The method described here is an improved version of that previously published) The method (Fig. 1B) is related to that of Berk and Sharp for mapping mRNA cap sites 4 with modifications that increase sensitivity and specifici p. 2 B. 3 p. 4 A.
D. Jackson and G. Felsenfeld, Proc. Natl. Acad. Sei. U.S.A. 82, 2296 (1985). Kemper, P. D. Jackson, and G. Felsenfeld, Molec. Cell. Biol. 7, 2059 (1987). D. Jackson, B. E. Emerson, and G. Felsenfeld, unpublished observation. J. Berk and P. A. Sharp, Cell (Cambridge, Mass.) 12, 721 (1977).
METHODS IN ENZYMOLOGY,VOL. 152
736
IDENTIFICATION
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OF
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[74]
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ity. It (along with other mapping methods 5,6) also adapts the "indirect end-labeling" concept 7 to allow the high-resolution mapping of genomic cleavage sites with respect to an experimentally introduced reference point, the "indirect end label. ''8 Genomic DNA in cell nuclei or in whole cells is treated with nucleases or other DNA cutting agents to delineate the DNA contact points of sitespecific DNA binding proteins and other chromatin structures that protect DNA from attack (Fig. 1A). The partially digested genomic DNA (Fig. 1B, top) is then purified, cut with a restriction endonuclease to reduce the size of the DNA fragment containing the target sequence, and hybridized to a highly purified 5'-end-labeled single-strand DNA probe complementary to the site of interest (Fig. 1B, step 1). The probe is prepared by primed synthesis on a single-strand template (Fig. 2). The probe-genomic DNA hybrids are heat treated at low ionic strength to eliminate nonspecific hybridization and are then digested with a single-strand-specific nu5 C. Wu, Nature (London) 309, 229 (1984). 6 G. M. Church and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 81, 1991 (1984). 7 C. Wu, Nature (London) 286, 854 (1980). 8 "Indirect end label" concept as used here is diagrammed in Fig. 1B. Briefly, cleavages in the target genomic strand are mapped with respect to the "end label" on the probe strand hybridizing to the target strand. As the end label reference point is not covalently attached to the mapped strand, the label is referred to as "indirect." FIG. 1. (A) Footprint concept. Binding of proteins to DNA affects the accessibility of DNA sequences at the binding site to digestion with nucleases or other DNA cleavage reagents. Partial digestion of protein-DNA complexes and protein-free DNA reveals a site that is normally cleaved in protein-free DNA but is protected from cleavage by protein binding (site b) as well as sites that are unaffected by protein binding (sites a and c). The protection against cleavage at site b constitutes the footprint of the protein bound to its recognition site on the DNA molecule. (B) Genomic mapping. Information on the patterns of cleavage and protection along a genomic DNA sequence that can identify footprints of in vivo protein-DNA interactions is obtained by mapping the cleavage sites with respect to an experimentally introduced indirect end-label reference point. Genomic DNA from nucleic or from whole cells is partially digested with the DNA cleavage reagent of choice. Protein-free DNA from the same source is treated similarly. Both samples are subsequently purified and further digested (step 1) with restriction endonucleases (R in diagram) chosen to uncouple the sequence of interest from the extended adjacent lengths of genomic DNA that might otherwise interfere with hybridization. The restriction endonuclease-cleaved DNA is then hybridized (step i) to a single-strand 5'-end-labeled DNA probe complementary to the sequence to be mapped. After hybridization, the hybridization mixture is heated to denature nonspecific hybridization products and the single-strand tails trimmed from the hybrids and unhybridized probe and genomic strands degraded by digestion with a single-strand-specific nuclease (step 2). The resultant mixture is resolved by electrophoresis in nondenaturing polyacrylamide gels (step 3) and the indirect end-labeled hybrid bands (molecule 1) visualized by autoradiography.
738
IDENTIFICATION
primer i~
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Single-Stranded End-Labeled Probe
FIG. 2. Preparation of highly purified single-strand 5'-end-labeled DNA probe. Singlestrand end-labeled probe is made by primed synthesis on a single-strand template. Singlestrand template is primed (I) by hybridization of a short oligonucleotide (primer) to vector sequences (solid semicircle) directly adjacent to the template sequence for the genomic probe to be synthesized (dashed semicircle). 5'-End-label is introduced (II) by extending the primer using large-fragment DNA polymerase I and limiting [a-32P]dNTP such that label is extended only into the first 10-30 nucleotides of the nascent probe sequence complementary to the genome. Probe synthesis is completed (III) by continuing the synthesis with a massive excess of unlabeled nucleotides on through the rest of the probe template sequence (dashed semicircle) and into the adjacent vector template sequences (solid semicircle). The probetemplate complex is digested (IV) with a restriction endonuclease (R) (HindlII in the example in the text) chosen to maximize the size difference between the subsequently denatured probe and template strands, and the probe strand purified away from the template by denaturing gel electrophoresis (V). The probe is further purified to remove the few remaining template strands (and template fragments) by sequential self-hybridization (VI) and hydroxyapatite (HAP) chromatography (VII) which separates the pure single-strand probe from contaminating template-probe duplex molecules. Intact probe molecules (VIII) are separated from the radiolytically degraded probe molecules that accumulate during probe purification by a second round of denaturing gel electrophoresis (VII) to yield single-strand end-labeled probe (VIII) for genomic mapping and footprinting (Fig. 1, A and B). clease (Fig. 1B, step 2). This e n z y m a t i c step d e g r a d e s u n h y b r i d i z e d p r o b e m o l e c u l e s and trims single-strand tails f r o m the h y b r i d s leaving a population o f r a d i o a c t i v e h y b r i d m o l e c u l e s that e x t e n d f r o m the p r o b e 5' end label to the p r o x i m a l cut in the c o m p l e m e n t a r y g e n o m i c strand, t h e r e b y allowing the m a p p i n g o f these g e n o m i c cuts with r e s p e c t to the end label r e f e r e n c e point (Fig. 1B, m o l e c u l e I). T h e p o p u l a t i o n o f labeled h y b r i d m o l e c u l e s thus g e n e r a t e d are sized by e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e gels u n d e r n o n d e n a t u r i n g c o n d i t i o n s (Fig. 1B, step 3) and the gels dried
[74]
In Vioo FOOTPRINTING
739
and autoradiographed to display the genomic cutting pattern comprising the footprint relic of in oioo DNA-protein interactions. Cleavage and Subsequent Preparation of Genomic DNA The choice of whether to perform the footprint analysis on whole cells or cell nucleic depends to some extent on the nature of the DNA cleavage reagent employed. Nuclei are permeable to most cleavage reagents whereas cells are more selective. Reagents such as dimethyl sulfate, 6 psoralen, 9 neocarcinostatin, 1°-~3 and related drugs can be used to cleave DNA in intact cells. We have used nucleases ~-3 as probes of chromatin structure in isolated nuclei and in concentrated lysed whole cells and we have used neocarcinostatin chromophore ~4,~5as a structural probe in intact cells. Nuclease digestion of isolated nuclei is technically the easiest experiment, but it is important to appreciate the lability of some chromatin structures in the design and interpretation of experiments on isolated nuclei, since structural components can be lost during isolation procedures. The exact procedure to be used in cell or nuclear preparation will vary with the tissue or cell type to be analyzed but in general the samples should be handled gently and quickly, and kept cold and protease free to stabilize nuclear structures. Aliquots of the preparation are treated with a series of concentrations of the cleavage reagent designed to produce one or two cuts per 500 bases within the sequence of interest. For DNase I digestion of protein-free DNA or nuclease hypersensitive sites in isolated nuclei these concentrations are in the range 5-200 ng enzyme/mg DNA for 30 min at 20° in 50 mM NaCI, 20 mM HEPES (pH 7.5), 5 mM MgCl2, and 1 mM CaCI2 with protease inhibitors when necessary. Digestions are terminated by the addition of NaEEDTA (pH 8.0), to 10 mM, NaC1 to 0.5 M, and protease K to 100 /zg/ml. After 30 min at 37° sodium dodecyl sulfate is added to 0.25% and protease K to 200 /zg/ml and digestion allowed to proceed for 24 hr at 37°. Cutting frequency can be assayed by Southern blotting ~6to follow the appearance of cutting within a hypersen-
9 M. M. Becker and J. C. Wang, Nature (London) 309, 682 (1984). l0 L. S. Kappen, I. H. Goldberg, and T. S. A. Samy, Biochemistry 18, 5123 (1979). 11 T. Hatayama, I. H. Goldberg, M. Takeshita, and A. P. Grollman, Proc. Natl. Acad. Sci. U.S.A. 75, 3603 (1978). 12 p. D. Jackson and G. Felsenfeld, unpublished observations. t3 L. S. Kappen, M. A. Napier, I. H. Goldberg, and T. S. A. Samy, Biochemistry 19, 4780 (1980). 14 L. S. Kappen, M. A. Napier, and I. H. Goldberg, Proc. Natl. Acad. Sci. U.S.A. 77, 1970 (1980). 15 L. S. Kappen and I. H. Goldberg, Biochemistry 19, 4786 (1980). ~6E. M. Southern, this series, Vol. 68, p. 152.
740
IDENTIFICATION AND CHARACTERIZATION OF CLONES
[74]
sitive site 7,17 of interest or to follow the disappearance of a short (-500 bp)
restriction fragment containing the site of interest. The indirect end-label Southern blotting method for low-resolution mapping of cutting within hypersensitive sites is well documented. 7,16,~7 Briefly, purified genomic DNA froM, e.g., nuclease-digested nuclei is digested with restriction endonucleases that will cut at least 500 and not more than 4000-6000 base pairs away from possible nuclease-hypersensitive sites near the gene of interest. The DNA is resolved by electrophoresis in agarose gels (see this volume [8]), transferred to a solid support [61], and hybridized to a radioactive probe [45]. TM The 300- to 600-bp probe is chosen such that it hybridizes specifically with the first 300-600 bases adjacent to the restriction endonuclease cut at one end of the genomic restriction fragment and such that it does not overlap the restriction site. The combination of restriction endonuclease digestion of genomic DNA and hybridization to short probes specific for one end of the genomic restriction fragment allows the mapping of genomic cleavage sites (e.g., hypersensitive sites) with respect to the apparent end label (indirect end label) provided by the short probe. Deproteinized, unrestricted genomic DNA is used to prepare controls displaying the cleavage pattern of the cleavage reagent on protein-free DNA and to make up a mixture of various restriction endonuclease-digested samples to act as size standards (Fig. 5, lanes 1-2 and lanes marked STD, respectively). We have found that genomic DNA serves quite well as substrate for the preparation of protein-free DNA controls, obviating the need to use cloned sequences for this purpose as was previously described. Genomic DNA is prepared for high-resolution mapping by deproteinization following by restriction endonuclease digestion and treatment with ribonuclease A to eliminate endogenous RNA that would otherwise compete for radioactive probe during hybridization. The deproteinized genomic DNA is treated with 1/xg of heated ribonuclease A (100 ° for 15 min, then cooled to room temperature) per 20/xg of genomic DNA in 10 mM Tris-HC1 (pH 8.0), 1 mM Na2EDTA (TE buffer) at 37° for 30 min and followed directly by restriction endonuclease digestion, phenol-chloroform extractions to remove protein (this volume [4]), alcohol precipitation (this volume [5]), and resuspension of the DNA to 2 g/liter in TE. The restriction endonuclease digestion is chosen to isolate the genomic DNA sequence of interest on a small (300-1000 bp) fragment within the genomic digest. In the example of the mapping method presented here, digestion with PvulI serves to center the -200-bp 5' nucle17 j. D. McGhee, W. I. Wood, M. Dolan, J. D. Engel, and G. Felsenfeld, Cell (Cambridge, Mass.) 27, 45 (1981).
[74]
In Vivo FOOTPRINTING
741
ase-hypersensitive region of the chicken erythrocyte/3 A-globin gene on a 580-bp fragment. Having the target sequence on such a small genomic DNA fragment limits the ability of otherwise long genomic DNA to compete kinetically with single-strand probe for hybridization to the complementary genomic strand, TM and similarly lowers the initiation rate of strand displacement reactions which would favor the longer, more stable genomic renaturation products over the shorter probe-genomic DNA hybrids at thermodynamic equilibrium. The loss of signal due to these phenomena is further limited by driving the hybridization reaction with a -30-fold molar excess of probe over genomic complement. The preferential stability of genomic renaturation products and the resulting negative effects of strand displacement on signal intensity can be completely eliminated by choosing the 5' end of the genomic restriction fragment to coincide with that of the probe. Probe Preparation and Purification Construction o f Probe Template
5'-End-labeled single-strand probe is prepared by primed synthesis on a single-strand template followed by isolation and purification of the newly synthesized probe strand. The basic procedure is as previously described I (Fig. 2) with technical modifications that increase probe yield. The template consists of ->300 bp of the genomic sequence of interest cloned into a vector which yields filamentous phage particles containing single-strand DNA copies during infectious growth in bacterial cultures. We have used the M13 phage vectors developed by Messing 1,3A9as well as pTZ plasmid vectors (P-L Biochemicals) bearing filamentous phage origins of replication 2,2°-22with equal success for the preparation of template. Recombinant phage are prepared from infected bacterial cultures ~9-2~(see this volume [13]) and the single-strand DNA template purified from the phage by phenol-chloroform extraction and alcohol precipitation. The choice of genomic fragment to be cloned and how to orient it within the vector sequence is made on the basis of two criteria: (1) each template will produce a probe that is strand specific (i.e., two templates will be required to examine completely the cutting pattern on both strands ~8j. G. Wetmurand N. Davidson,J. Mol. Biol. 31, 349 (1968). t9j. Messing, this series, Vol. 101, p. 20. 20L. Dente, G. Cesareni, and R. Cortese, Nucleic Acids Res. 11, 1645(1983). 21B. P. H. Peeters, J. G. G. Schoenmakers,and R. N. H. Konings,Gene 46, 269 (1986). 22D. A. Mead and B. Kemper, in "Vectors: A Survey of MolecularCloningVectors and Their Uses" (D. T. Denhardtand R. L. Rodriguez,eds.). Butterworth,Boston, in press.
742
I D E N T I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF CLONES
[74]
within a given genomic sequence), and (2) the mapping technique has best resolution 60-220 bases from the 5'-end-labeled region of the probe complementary to the genome (Fig. 3). As with any mapping technique, it is desirable to choose the end-labeled region such that it will avoid intense cutting sites in the genome. In the worst case, in which 100% of the genomic DNA is cut within the region complementary to the 5'-endlabeled stretch of the probe, it is very difficult to map the other genomic cut sites properly. Placement of the 5'-end label so that it is complementary to areas less heavily cut just adjacent to hypersensitive regions in chromatin eliminates this problem. The ml9XB probe used here (Figs. 35) is about 300 bases long and extends at each end about 50 bases beyond the sequence of the 200-bp 5'-nuclease-hypersensitive region of the flA_ globin gene. We have used synthetic primers complementary either to vector sequences directly adjacent to the cloned genomic sequences (e.g., 17-base "universal sequencing primer" used as primer for the ml9XB probe), or to regions within the cloned genomic sequences. The amount of [a32p]dNTPs necessary to extend label into probe sequences complementary to genomic DNA is directly proportional to the distance between the 3' end of the primer and the 5' end of genomic complement in the probe sequence. It is a consideration of economy to minimize this distance when designing a cloning strategy. A related point is that shorter labeled stretches will suffer less radiolytic damage and thereby increase probe yield, as will become obvious in descriptions of probe purification below.
Probe Synthesis Priming of Single-Strand Template (Fig. 2, step I). Approximately 0.75/xg of M13 phage DNA or 0.35 ~g of single-strand pTZ DNA with a 300-base insert is required for each 250/.Lg of genomic DNA analyzed. This starting amount represents a 1000-fold molar excess of the cloned template sequence over its chicken genomic complement and compensates for radiolytic and other losses incurred during probe purification. Template DNA is primed by mixing template at a DNA concentration of 0.167 g/liter with a 2.5-fold molar excess of synthetic oligonucleotide primer in 1 x SMT buffer (1 x SMT: 42.0 mM NaC1, 5.8 mM MgC12, 5.8 mM Tris-HCl at pH 7.5). The mixture is heated to 55 ° for 5 min, incubated at 37° for 2 hr, and then frozen on dry ice and stored at - 2 0 ° until use, when the primer is extended using the large fragment of DNA polymerase I. 5'-End Labeling of Probe Strand (Fig. 2, step II). Labeling of probe within regions complementary to the genomic sequence is confined to 10-
[74]
In Vivo FOOTPRINTING
743
¢o
-5 1
2
3
4
5
6
7
8
300
@
200
.Q I-0
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FIG. 3. Primed synthesis titrations with limiting [a-3~p]dNTPs. Pilot primed syntheses are performed as described on 0.75 p.g of ml9XB template using 3.52 pmol (lane l, 6-8), 4.22 pmol (lane 2), 5.06 pmol (lane 3), and 6.08 pmol of each [a-32P]dNTP. Lanes 1-4 are without a chase with excess unlabeled dNTPs. Lanes 5-8 are chased with unlabeled dNTPs and are then digested with different restriction endonucleases: Sinai (lane 5), BanI (lane 6), ApaI (lane 7), and MnlI (lane 8). The samples are denatured by heating in formamide and fractionated electrophoretically in a polyacrylamide gel of 12.5% acrylamide-0.625% bisacrylamide containing 45 mM Tris-borate (pH 8.3), 1.25 mM Na2EDTA, 8.5 M urea. A map of the region of primed synthesis is diagrammed at the far left, with the position of kinetic stops in the incorporation of label marked with arrows. The restriction standards (lanes 5-8) are correlated with the map by connecting lines. The 3' end of the mapped probe strand is shown cleaved at the HindIII site as during probe purification described in text. The sizes of the major kinetic stops are at 73 bases (37 bases of label at 5' end of genomic sequence) and I l0 bases. The restriction standards are at 25, 62, 74, 85, and 89 bases. The bands in lanes 6-8 illustrate radiolytic strand breakage of the probe, since a smear of breakage products appears 17 bases (the length of the unlabeled primer) below the major band. The intensity of the smear is proportional to the time between synthesis and electrophoresis, which for this pilot experiment was 12 hr.
744
IDENTIFICATION AND CHARACTERIZATION OF CLONES
[74]
40 bases near the 5' end by incubating first with limiting amounts of [o~32p]dNTPs and taking advantage of inherent kinetic blocks to polymerization, which provide stopping points for all templates we have tested (Fig. 3). We found it necessary to adjust labeled dNTP concentrations over a range of -2-fold for individual templates to obtain suitable end-labeling; the differences among templates are probably related to template secondary structures (e.g., self-complementary stem and loop structures).~ The mI9XB probe used in the analysis ofgenomic DNA shown in Figs. 4 and 5 is synthesized by adding primed MI3-m19XB template (0.75 ~g in 4.5/xl) to 16.7/zl of a mixture containing 3.33 pmol (10/xCi) each of o~-32p-labeled dATP, dCTP, dGTP, and dTTP (3000 Ci/mmol), 1.2x SMT, 0.15 mM 2mercaptoethanol, and 1.2 units of large-fragment DNA polymerase I (any polymerase preparation suitable for dideoxynucleotide-terminated DNA sequencing is acceptable in this application). Extension o f Probe Strand with Unlabeled Nucleotides (Fig. 2, step liD. After incubation at 20 ° for 1 hr, the probe strand is extended through the remaining genomic sequence and into adjacent MI3 vector sequence with greatly reduced incorporation of radioactivity (Fig. 2, step III) by adding 3.75/~1 of a mixture containing 2.0 nmol each of the four unlabeled dNTPs in 1 x SMT, 0.15 mM 2-mercaptoethanol, and 1.25 units of DNA polymerase I large fragment. Incubation is continued at 20° for 20 min, at 37° for 5 min, and finally at 68 ° for 10 min to denature polymerase. The extent of probe 5'-end label is approximately as in Fig. 3, lane 1.
Probe Purification Restriction Endonuclease Digestion of Probe-Template Complex (Fig. 2, step IV). The probe-template complex is digested with a restriction endonuclease chosen to maximize the size difference between the denatured single strands of the newly synthesized probe and its M13 template (Fig. 2, step IV). For the ml9XB probe, 10 units of HindlII endonuclease, 2.0/zl of buffer (0.5 M Tris-HCl at pH 8.0, 50 mM MgClz, bovine serum albumin at 1 g/liter, and water to 40/zl are added to the above reaction mixture and incubated at 37° for 1 hr. HindIII cleaves once, yielding a linearized template strand of -7500 bases and an end-labeled probe strand of - 3 5 0 bases including adjacent M13 sequences and primer (Fig. 3). The reaction is stopped by the addition of Na2EDTA (pH 8.0) to 15 mM final concentration, and the DNA purified by extraction with Trisneutralized phenol and chloroform. Traces of chloroform are removed by extraction with two volumes of n-butanol, the aqueous phase diluted with 2 ml of TE, and the sample concentrated and desalted by centrifugation in a
[74]
In Vil:o FOOTPRINTING
745
Centricon-30 disposable concentration device according to the manufacturer's directions (Amicon Corporation). Denaturing Gel Purification of Probe Strand (Fig. 2, step V). The retained sample, in a volume of 50-100/xl, is denatured by dilution to 1 ml with deionized formamide (plus 5 /xl 0.2 M NaEDTA at pH 8.0 and xylene cyanole FF dye to 0.01 g/l) and heated at 68 ° for 5 min prior to loading in a single slot on a 1.5-mM thick polyacrylamide gel (5% acrylamide, 0.25% bisacrylamide) containing 45 mM Tris-borate (pH 8.3), 1.25 mM Na2EDTA, 8.5 M urea. The gel is prerun prior to sample loading to heat it to - 5 0 °. Avoiding alcohol precipitation and keeping the sample dilute and warm prior to electrophoresis increase probe yield by decreasing rehybridization of the template and probe strands and by minimizing nonspecific losses on surfaces often incurred during precipitation. The sample is fractionated electrophoretically at 30-40 V/cm for 2 hr and the gel autoradiographed for about 15 sec to localize the probe. The probe band is excised and electroeluted from the gel slice for 1 hr at 125 V into a small volume of a high ionic strength solution (4.75 M NaC1, 25 mM HEPES at pH 7.5, 5 mM NaEEDTA, sonicated calf thymus DNA at 50 /zg/ml) in an electroelution apparatus (International Biotechnologies, Inc., catalog no. 46000) containing the Tris-borate-EDTA electrode buffer as above. The electroeluted sample is desalted and concentrated in a Centricon-30 by two or three cycles of dilution with TE followed by centrifugation as above. The probe is pure enough at this point to use for the study of restriction digests containing short unique chicken DNA sequences at 0.1 × genomic abundance (Fig. 4, lanes 1-5). Contaminating template sequences remain, however, and the signal-to-noise ratio obtained using this probe is improved (Fig. 4, lanes 6 - l l) by continuing the probe purification described below. The analysis of mixtures of restriction endonuclease-digested genomic DNA (as in Figs. 4 and 5) provides an important test of the validity of the mapping information provided by the technique. Each of the restriction endonuclease cleavages introduced into the genomic DNA should be mapped uniquely and at the correct distance with respect to the probe end label for the mapping information to be valid. This point will be covered in more detail in the discussion of Figs. 4 and 5 contained in the section on genomic footprinting and validation of results at the end of this chapter.
Removal of Contaminating Template Strands by Sequential Self-Hybridization and Hydroxyapatite Chromatography (Fig. 2, steps VI and VII). The probe is purified further by self-hybridization and hydroxyapatite (HAP) chromatography to remove the small amount of contaminating template sequences. To the probe in 60/zl of TE is added 6.67/xl of 10x
746
IDENTIFICATION AND CHARACTERIZATIONOF CLONES
[74]
HB (10x HB: 3.18 M NaC1, 200 mM HEPES at pH 7.5, 1.0 mM Na2EDTA). This mixture is flame sealed in a glass capillary, heated to 107° for 5 rain in a saturated NaCI bath, and transferred directly to a 68° water bath for incubation overnight. During this self-hybridization, probe strands in excess will hybridize with contaminating template strands. The pure single-strand probes may be separated from these duplexes by their differential affinity for HAP which binds the duplex molecules more tightly than the single strands in 150 mM NaPO4 at pH 7. 23 Hydroxyapatite (Bio-Rad) is prepared by suspension and settling in 50 mM NaPO4 (pH 7.0). The HAP is washed once in 1 M NaPO4 (pH 7.0), and then equilibrated in 50 mM NaPO4 (pH 7.0). The self-hybridized probe is diluted into 0.2 ml of sonicated calf thymus DNA (0.2 g/liter) in 50 mM NaPO4 (pH 7.0), and applied to 0.1 ml of the gently packed HAP (centrifuged 1 min at 250 g in swinging bucket rotor) in a tightly capped polypropylene centrifuge tube. The HAP is suspended in the DNA solution by gentle vortexing or inversion and intermittently resuspended during a 15-min incubation at 68 °. The HAP is sedimented at 250 g for 1 min and washed six times with 2 ml of 50 mM NaPO4 (pH 7.0), at 68°. Greater than 90% of the radioactivity should bind to the HAP, and 32p in the wash buffer should reach a low constant value after three or four washes. The probe is eluted from the matrix with four 68° washes of 0.5 ml 150 mM NaPO4 (pH 7.0). Combined washes containing - 9 0 % of the eluted radioactivity (only a small percentage of the 32p should remain associated with HAP at this point) are filtered by centrifugation through 0.45-/.¢m pore size Centrex cellulose acetate filters (Schleicher & Schuell) to remove any stray HAP and the filtered probe desalted and concentrated by dilution with TE and centrifugation in a Centricon-30 device as before. Final Removal o f Radiolytic Degradation Products by Denaturing Gel Electrophoretic Purification o f Intact Probe Strand (Fig. 2, steps VII and VIII). The probe in - 5 0 ~l of TE is carefully concentrated to - 5 tzl by repeated extraction with 2 volumes of n-butanol and the sample diluted to I00 /zl with the same proportions of formamide, EDTA, and xylene cyanole FF used before. The sample is heated prior to electrophoresis in a polyacrylamide-urea-Tris-borate-EDTA gel as before with smaller well size to accommodate the smaller sample volume. The reason for performing this second gel purification of the probe is made clear upon autoradiography of the gel. It is apparent that during the time since electrophoresis ceased in the first gel ( - 2 0 hr) considerable radiolysis has occurred. Up to 50% of the probe will appear in a smear of radioactivity just below 23G. Bernardi, Procedures Nucleic Acid Res. 2, 455 (1971).
[74]
In Wioo FOOTPRINTING
747
the remaining tight band of intact probe. It is a conservative choice to excise only the intact probe band, but it thereby assures the integrity of the probe that now goes into the analysis of genomic DNA. The probe (Fig. 2, step VIII) is electroeluted, concentrated, and desalted as before and is now ready for hybridization to genomic DNA. The probe should be used immediately as storage only decreases the signal-to-noise ratio, eliminating the gains made by the second probe purification gel. Analysis of Genomic DNA
Hybridization of Genomic DNA with Probe (Fig. 1B, step 1) To make more efficient use of the effort expended in probe purification, we generally analyze 40-50 25-/zg samples at a time with the probe preparation scaled accordingly. Hybridization of single-stranded 5'-endlabeled probe with genomic DNA is accomplished by mixing 0.1 ng ( - 3 x 105 dpm) of probe with 25/zg of genomic DNA (a 30-fold molar excess of probe over its chicken genomic complement) in 20 /zl of 1 x HB. The mixture is flame sealed in a glass capillary, heat denatured (5 min, 107°), and directly transferred to a 68 ° bath for a 12-hr hybridization. Genomic DNA is 60-70% renatured and hybridization to probe is complete in this time. The reaction is stopped by expelling the mixture into 14/zl of TE and precipitating the DNA with 40/zl of isopropanol at room temperature. The pellet is washed in 70% ethanol, dried, and dissolved in 15.6/zl of 15 mM NaCI, 1.5 mM HEPES (pH 7.5).
Heat Denaturation of Nonspecific Hybrids Cross-hybridization of the probe to related sequences in the genome is eliminated by incubation in this low ionic strength solvent at 70° for 30 rain. The temperature of incubation is probe dependent as is discussed below. This heat denaturation step is necessary to eliminate high backgrounds of nonspecific hybridization, the magnitude of which varies as a function of probe sequence. Hybrids of probe with the specific genomic sequence are not denatured under these conditions. This step is analogous to the "high-stringency wash" step in Southern blotting ~6 and the considerations governing the choice of conditions are the same for both. Higher G + C content (the probe used here is - 7 0 % G + C) and greater homology to nonspecific genomic sequences dictate incubation at a higher temperature (e.g., 70° for the ml9XB probe). The temperature of incubation is chosen empirically by observation of the influence of temperature upon the signal-to-noise ratio obtained in the
748
IDENTIFICATION AND CHARACTERIZATION OF CLONES
[74]
analysis of standard genomic samples (e.g., a mixture of restriction endonuclease digests of genomic DNA as in Fig. 4). Since little is known in many cases about the extent of cross-reacting sequences within a genome, one can start such a survey of temperature effects near the melting temperature of the specific hybrid according to its content of G + C bases and its length, and work down in temperature until the background from nonspecific hybridization becomes unacceptable again. In practice, T (in degrees Centigrade) = 41 + 0.41 (% G + C bases in probe) is a reasonable upper limit, at which temperature both specific and nonspecific hybrids of less than about 50 base pairs are denatured, za,z5 Omission of the heat denaturing step is a potential source of artifact.
Digestion of Single-Strand DNA Regions After, heating, the hybridized samples are digested with a calibrated activity of mung bean nuclease 26 to trim off single-strand regions from the hybrids, as well as unhybridized single-strand probe and residual denatured genomic DNA (Fig. 1B, step 2). The pattern of mung bean nucleaseresistant fragments is relatively insensitive to 2-fold changes in enzyme activity but new batches of enzyme should be calibrated in the assay (Fig. 4). Overdigestion leads to nibbling in at the ends of duplex molecules; underdigestion leads to increasing levels of contamination with nonhybridized probe. The amount of nuclease used should be 1.5- to 2-fold more than the minimum necessary to degrade unhybridized probe molecules. This low nuclease activity (6.5 units of nuclease/25/xg of genomic DNA for the enzyme preparations we have used) will assure the elimination of the unhybridized species and the effective trimming of single-strand regions from the hybrids and yet minimize the nibbling in at the ends of hybrid molecules that can decrease resolution (Fig. 4, lane 6 vs. lane 7). A mixture of restriction endonuclease digests of genomic DNA is assayed in parallel with the other genomic samples to provide size standards that control for the effects of mung bean nuclease on fragment size (Fig. 5, lanes marked STD). A potential artifact can arise through the action of the single-strandspecific nuclease at sites where the probe is not complementary to the genomic sequence. Sequence mismatches in the hybrid might result from genomic sequence polymorphisms or inaccurate copying of template dur24 j. Marmur and P. Doty, J. Mol. Biol. 5, 109 (1962). 25 C. R. Cantor and P. R. Schimmel, "Biophysical Chemistry," Part III, Chapter 22. Freeman, San Francisco, California, 1980. 26 M. Laskowski, Sr., this series, Vol. 65, p. 263.
[74]
In Vioo FOOTPRINTING
749
ing probe synthesis (see discussion of alternative probe preparation strategies below). The nuclease can attack these defects in the duplex if they are large enough, and yield fragments unrelated to the genomic cleavage pattern under investigation. Digestion of single-strand regions is effected by adding 6.5 units of mung bean nuclease (P-L Biochemicals) in 10.4 p~l of 125 mM sodium acetate (pH 5.3), 2.5 mM magnesium acetate, 2.5 mM L-serine, 2.5 mM 2mercaptoethanol (added fresh), 0.0125% Triton X-100 (Sigma), 0.25 mM zinc acetate, and incubating at 40° for 60 min. The reaction is stopped by adding 3.5 /~1 of 2.5 M NaCI, 0.5 M Tris-HC1 (pH 8.5), followed by precipitation with 36/~1 2-propanol. The pellet is washed with 70% ethanol, dried, and redissolved in 4-8/zl of 10 mM Na2EDTA (pH 8.0), 6% Ficoll, 0.06% xylene cyanole FF to yield 25-12.5/xg genomic DNA equivalents of hybrid molecules per 4/zl to be loaded on the gel.
Resolution of the Trimed Radioactive Hybrids on Nondenaturing Polyacrylamide Gels (Fig. 1B, step 3) The choice of resuspension volume depends on considerations of sensitivity and resolution. At 25 /zg/4 /xl, sensitivity is increased, but the higher load of DNA decreases the resolution, especially above 180 base pairs. Regardless of choice, the concentration of all experimental samples and the genomic restriction standards should be identical in order to equalize the effects of sample load on fragment migration during gel electrophoresis. Samples in 4/zl are loaded in 0.8 x 5 mm wells on a 0.8 x 330 x 380 nondenaturing gel (8% acrylamide-0.4% bisacrylamide containing electrophoresis buffer) and fractionated electrophoretically in a buffer containing 90 mM Tris-borate (pH 8.3), 2.5 mM Na2EDTA at 18 V/cm for 7 hr. Glass plates used to form the gel slab should be pretreated with dichlorodimethylsilane and rinsed with ethanol and water prior to gel casting. Nondenaturing gels are used to ensure the integrity of labeled fragments that may have been internally nicked by radiolysis or mung bean nuclease. After electrophoresis, the glass gel-casting plates are separated and a sheet of cellophane (Bio-Rad) that has been thoroughly wetted is closely apposed to the gel with care taken to eliminate air bubbles from the gelcellophane interface. A sheet of heavy filter paper is laid over the cellophane for mechanical support and the gel carefully freed from the glass plate by turning the glass plate side of the gel sandwich up and prying the gel-cellophane laminate free from the glass, allowing it to fall gently on the supporting heavy filter paper. A sheet of Mylar (0.0005 in. thick, or
750
IDENTIFICATION AND CHARACTERIZATION OF CLONES
IMPURE PROBE
PURIFIED PROBE I
1 2 3 4 5
|1
I
I
6 7 8 91011
--303 --228
--205 --200
--160 --142 --122
~
i!iii!i~I
[74]
[74]
In Vivo FOOTPRINTING
751
other transparent heat-stable film) is layered without air pockets over the upper naked gel surface, and the four-layered sandwich dried in a vacuum slab gel drying apparatus (e.g., Bio-Rad or Hoefer apparatus) at 80°. The transparent dried cellophane-gel-Mylar laminate (heavy filter paper removed) is sandwiched between two Dupont Cronex Lightening Plus intensifying screens and exposed to a sheet of flashed 27 (also this volume [7]) Kodak XAR-5 film for - 1 4 days at -80 °. The use of a transparent gel backing (cellophane) and two intensifying screens effectively doubles the sensitivity of the method over that previously described. 1 Potential Modifications to Mapping Methods Further gains in sensitivity could be obtained if necessary by prior partial purification of the genomic sequences to be probed (e.g., by gel electrophoretic size fractionation of restriction endonuclease-digested genomic samples). This sequence purification has the potential to dramatically increase the signal-to-noise ratio by greatly decreasing the complexity of the analyzed genomic sample and by allowing a much greater specific sequence load on the final analytical gel. The basic steps of this analysis (i.e., end-labeled single-strand probe synthesis and purification, hybridization, trimming of the hybrids with single-strand-specific nuclease and gel electrophoresis) might be accomplished using variations of the mapping technique described here. Some of these variations may represent improvements in ease of probe purification or increased resolution of the genomic analysis, but for the most part they remain speculative and untested. 27 R. Lasky, this series, Vol. 65, p. 363.
FIG. 4. Mung bean nuclease digestion titrations of mI9XB probe-chicken DNA restriction mix hybrids. Single-strand probe hybridized with a mixture of chicken DNA restriction endonuclease digests is processed as described in the text with the exception that different activities of mung bean nuclease are used to trim the hybrids. Two different purities of probe are used in the hybridization: impure probe (lanes 1-5) used directly after the first denaturing gel and highly purified probe (lanes 6-11) that has gone through the entire purification scheme. Mung bean nuclease concentrations used are 6.5 units (lanes 1, 6, and 11), 13 units (lanes 2 and 10), 26 units (lanes 3 and 9), 52 units (lanes 4 and 8), and 104 units (lanes 5 and 7). The position of the restriction endonuclease cuts in the genomic sequence are given at the right with respect to the start site of transcription. The actual lengths of the trimmed hybrid fragments extending from the probe 5' end at - 4 to the genomic restriction positions marked at the right of the figure are 118, 138, 156, 196,201,224, and 299 base pairs from bottom to top radioactive fragment [e.g., (-122) - ( - 4 ) = 118 base pairs].
752
IDENTIFICATION AND CHARACTERIZATION OF CLONES
[743
A 0 ~0 -H
0 Z
C
CO
r-
"4
p
1234
-228
-460
•. ~ t
g
-.122 ~ 8. ~ ~ , ~
~
j~ . DNALane2 V ~ ~ ADULT Lane3
FIG. S. Analysis of genomic DNase I cleavage patterns. (A) Genomic protein-free DNA samples (lanes 1 and 2) or purified nuclei from adult erytbrocytes (lanes 3 and 4) were digested with DNase I. D N a s e I digestion increases by 2.5-fold going from right to left within each pair of samples. The genomic DNA in the standard lanes (STD) is as described in Fig. 3. Samples are analyzed with the mlgXB probe as described in text. A region protected from DNas¢ I cleavage in adult'erythrocytes is mapped to the sequence on the left which, along with the standards, are numbered with respect to the initiation site for transcription on the chicken/3^-globin gene. Enhanced cleavage is indicated by the small arrow, and the protection adjacent to the 5' edge of the hypersensitive region by the large open arrow. (B) Densitometric scans of the indicated lanes from (A).
[74]
In Vioo FOOTPRINTING
753
Probe purification could be effected by the incorporation of an affinity label (e.g., Hg-dUTP 28 or biotinylated dUTP) during probe synthesis followed by the separation of denatured probe and template strands by chromatography on matrices containing covalently bound sulfhydryl or streptavidin moieties. Alternatively, an RNA template for probe synthesis could be transcribed from a genomic clone in one of the vector systems utilizing phage RNA polymerase promoters. 21,22Treatment with DNase I would yield pure RNA template, which could then serve as substrate for the synthesis and purification of probe using sequential primed synthesis with reverse transcriptase followed by alkaline hydrolysis of the RNA template. Direct end labeling of the RNA copy would provide even more facile access to a probe which, after genomic hybridization and nucleolytic trimming might provide a further advantage in the potential for elimination of gel overloading problems by DNase I treatment of hybridized, single-strand trimmed samples. In practice, the incorporation of an affinity label is probably only to be preferred for the preparation of long (2-kb) probes for which gel purification is inefficient. The use of an RNA template is an intriguing possibility, but might suffer from the inherently lower fidelity of the RNA polymerase and reverse transcriptase reactions relative to those catalyzed by DNA polymerase I large fragment, which possesses a 3'-5' exonucleolytic "editing" activity that limits misincorporation of bases. 29 Inaccurate transcription of the templates during probe synthesis would result in sequence mismatches in the probe-genomic DNA hybrid that could be recognized by mung bean nuclease or other single-strand-specific nucleases used to trim hybrids. In addition, altering the chemistry of the probe or using nucleases other than mung bean to trim the hybrids can introduce novel artifacts arising from the varying propensity of the nucleases for exo- or endonucleolytic attack upon duplex substrates. For example, the substitution of Si nuclease for mung bean nuclease to trim the hybrids has consistently led to poorer signal-to-noise ratios in our hands. The problems to be overcome in the development of an alternative scheme for genomic mapping are obvious: signals introduced by errors in probe synthesis or by enzymatic degradation of the hybrid duplex must be small relative to the miniscule signals arising from the genomic cleavages to be mapped. Any alternative that can solve these problems can be used for high resolution genomic mapping when used in conjunction with a heat denaturation step to decrease signals due to cross-hybridization to related genomic sequences. P. D. Jackson and G. Felsenfeld, unpublished observations. 29 D. Brutlag and A. Kornberg, J. Biol. Chem. 247, 241 (1972).
754
IDENTIFICATION AND CHARACTERIZATION OF CLONES
[74]
Genomic Footprint and Validation of Mapping Results
The genomic restriction standards shown in Figs. 4 and 5A are mixtures of several different restriction endonuclease digests. Each digest should give a single band in the autoradiogram corresponding in length to the distance between the probe Y-end label and the closest restriction endonuclease site in the complementary genomic strand. In examining these standard lanes, the validity of the mapping information gained from this technique becomes apparent. Approximately 95% of the radioactivity migrates in bands corresponding to the predicted lengths of the end-labeled hybrid molecules for each of the individual restriction digests in the standard. This demonstrates that the signals observed in an analysis of DNase I-cleaved genomic DNA (e.g., Fig. 5A, lanes 1-4) can be mapped with confidence onto the genomic DNA sequence with little interference from internal fragments (Fig. 1B, molecules labeled II) or from crosshybridizing genomic sequences. The probe used in this example of intranuclear genomic footprinting is complementary to the region extending from - 6 to -303 bases from the site of transcription initiation in the chicken/3A-globin gene. The mapped genomic strand contains a string of 16 G residues extending from - 181 to - 196. A region protected from DNase I attack in adult erythrocyte nuclei can be seen (Fig. 5); it includes the sequence from -130 to -156. Enhanced cutting is observed at -162 on this sequence and depressed cutting at - - 2 3 0 , the 5' margin of the nuclease hypersensitive region. A densitometric scan (Fig. 5B) of lanes of comparably digested protein-flee DNA (lane 2) and adult erythrocyte nuclei (lane 3) emphasizes the protection from DNase I attack afforded by proteins site-specifically bound to this sequence in adult erythrocyte nuclei. The existence of this site-specific protein DNA complex in vivo is supported by the observation of similar nuclease protection in this region shown by genomic mapping of the complementary genomic strand 1 and by in vitro nuclease protection experiments using partially purified nuclear factors reconstituted on cloned DNA. 3° Genomic DNA marked with DNA cleavage reagents in either whole cells, cell nuclei, or protein-flee DNA can be analyzed with this method to display high resolution maps of specific protein-DNA interactions. This provides valuable information about developmental changes in the structure of genetic regulatory elements in viuo. The structural information obtained about various functional states of a gene can be a powerful 30 B. E. Emerson, C. D. Lewis, and G. Felsenfeld, Cell (Cambridge, Mass.) 41, 21 (1985).
[74]
In Vil)o FOOTPRINTING
755
adjunct in the design and interpretation of both in vitro reconstitution and transcriptional studies, and in vioo transcriptional analyses. Acknowledgments We thank Byron Kemper for introducing us to the use of pTZ plasmids in this application, and Betty Canning for her superb help in preparing the manuscript.
In Vivo FOOTPRINTING
735
[Editors' Note. In Chapter 73, methods were presented for determining sequence specific binding of proteins in vitro to cloned regulatory elements of DNA. Chapter 74 contains high resolution methods for mapping the location of proteins bound to DNA in vivo; the difference in sensitivity to DNase I of DNA in chromatin compared with naked DNA is used to predict specific protein-DNA interactions. A different version L2 of this technique involves digestion of chromatin with DNase I, isolation of DNA, and complete cleavage with a restriction endonuclease that cuts rarely, as in [74]. Then, the fragments are separated electrophoretically in gels [8], transferred to nitrocellulose [61] and hybridized to a 32p-labeled probe [43, 45] that abuts the particular restriction site chosen as the point of reference. The visualization step is called indirect end labeling because the end is generated by a restriction enzyme and "labeling" of all subfragments sharing that end is achieved by hybridization. Improvements in Chapter 74 include solution hybridization and the use of short, highly radioactive, highly purified probes.] S. A. Nedospasov and G. P. Georgiev, Biochem. Biophys. Res. Commun. 92, 532 (1980). 2 C. Wu, Nature (London) 286, 854 (1980).
[74] I n V i v o F o o t p r i n t i n g o f Specific P r o t e i n - D N A Interactions By
P. D A V I D JACKSON a n d GARY FELSENFELD
We describe in this chapter a method for mapping the pattern of cleavages within specific unique DNA sequences in a complex genome at near single base resolution. The method has been used to accurately map the sites of sequence-specific protein-nucleic acid interactions ("footprints"; Fig. 1A) upon regulatory sequences in various members of the a- and/3globin gene families within chicken cells and cell nuclei.t-3 We examined the pattern of cleavages introduced into the genomic copies of/3- and aglobin gene promoters by exogenously added nuclease, but any reagent (endogenous as well as exogenous) that cleaves DNA is a candidate for this analysis. The method can be applied to the study of any gene for which in vivo structural information is desirable. The method described here is an improved version of that previously published) The method (Fig. 1B) is related to that of Berk and Sharp for mapping mRNA cap sites 4 with modifications that increase sensitivity and specifici p. 2 B. 3 p. 4 A.
D. Jackson and G. Felsenfeld, Proc. Natl. Acad. Sei. U.S.A. 82, 2296 (1985). Kemper, P. D. Jackson, and G. Felsenfeld, Molec. Cell. Biol. 7, 2059 (1987). D. Jackson, B. E. Emerson, and G. Felsenfeld, unpublished observation. J. Berk and P. A. Sharp, Cell (Cambridge, Mass.) 12, 721 (1977).
METHODS IN ENZYMOLOGY,VOL. 152
736
IDENTIFICATION
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CHARACTERIZATION
OF
[74]
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o
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ity. It (along with other mapping methods 5,6) also adapts the "indirect end-labeling" concept 7 to allow the high-resolution mapping of genomic cleavage sites with respect to an experimentally introduced reference point, the "indirect end label. ''8 Genomic DNA in cell nuclei or in whole cells is treated with nucleases or other DNA cutting agents to delineate the DNA contact points of sitespecific DNA binding proteins and other chromatin structures that protect DNA from attack (Fig. 1A). The partially digested genomic DNA (Fig. 1B, top) is then purified, cut with a restriction endonuclease to reduce the size of the DNA fragment containing the target sequence, and hybridized to a highly purified 5'-end-labeled single-strand DNA probe complementary to the site of interest (Fig. 1B, step 1). The probe is prepared by primed synthesis on a single-strand template (Fig. 2). The probe-genomic DNA hybrids are heat treated at low ionic strength to eliminate nonspecific hybridization and are then digested with a single-strand-specific nu5 C. Wu, Nature (London) 309, 229 (1984). 6 G. M. Church and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 81, 1991 (1984). 7 C. Wu, Nature (London) 286, 854 (1980). 8 "Indirect end label" concept as used here is diagrammed in Fig. 1B. Briefly, cleavages in the target genomic strand are mapped with respect to the "end label" on the probe strand hybridizing to the target strand. As the end label reference point is not covalently attached to the mapped strand, the label is referred to as "indirect." FIG. 1. (A) Footprint concept. Binding of proteins to DNA affects the accessibility of DNA sequences at the binding site to digestion with nucleases or other DNA cleavage reagents. Partial digestion of protein-DNA complexes and protein-free DNA reveals a site that is normally cleaved in protein-free DNA but is protected from cleavage by protein binding (site b) as well as sites that are unaffected by protein binding (sites a and c). The protection against cleavage at site b constitutes the footprint of the protein bound to its recognition site on the DNA molecule. (B) Genomic mapping. Information on the patterns of cleavage and protection along a genomic DNA sequence that can identify footprints of in vivo protein-DNA interactions is obtained by mapping the cleavage sites with respect to an experimentally introduced indirect end-label reference point. Genomic DNA from nucleic or from whole cells is partially digested with the DNA cleavage reagent of choice. Protein-free DNA from the same source is treated similarly. Both samples are subsequently purified and further digested (step 1) with restriction endonucleases (R in diagram) chosen to uncouple the sequence of interest from the extended adjacent lengths of genomic DNA that might otherwise interfere with hybridization. The restriction endonuclease-cleaved DNA is then hybridized (step i) to a single-strand 5'-end-labeled DNA probe complementary to the sequence to be mapped. After hybridization, the hybridization mixture is heated to denature nonspecific hybridization products and the single-strand tails trimmed from the hybrids and unhybridized probe and genomic strands degraded by digestion with a single-strand-specific nuclease (step 2). The resultant mixture is resolved by electrophoresis in nondenaturing polyacrylamide gels (step 3) and the indirect end-labeled hybrid bands (molecule 1) visualized by autoradiography.
738
IDENTIFICATION
primer i~
fR
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-
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OF CLONES
end-label i~
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I
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AND CHARACTERIZATION
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-vf
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Single-Stranded End-Labeled Probe
FIG. 2. Preparation of highly purified single-strand 5'-end-labeled DNA probe. Singlestrand end-labeled probe is made by primed synthesis on a single-strand template. Singlestrand template is primed (I) by hybridization of a short oligonucleotide (primer) to vector sequences (solid semicircle) directly adjacent to the template sequence for the genomic probe to be synthesized (dashed semicircle). 5'-End-label is introduced (II) by extending the primer using large-fragment DNA polymerase I and limiting [a-32P]dNTP such that label is extended only into the first 10-30 nucleotides of the nascent probe sequence complementary to the genome. Probe synthesis is completed (III) by continuing the synthesis with a massive excess of unlabeled nucleotides on through the rest of the probe template sequence (dashed semicircle) and into the adjacent vector template sequences (solid semicircle). The probetemplate complex is digested (IV) with a restriction endonuclease (R) (HindlII in the example in the text) chosen to maximize the size difference between the subsequently denatured probe and template strands, and the probe strand purified away from the template by denaturing gel electrophoresis (V). The probe is further purified to remove the few remaining template strands (and template fragments) by sequential self-hybridization (VI) and hydroxyapatite (HAP) chromatography (VII) which separates the pure single-strand probe from contaminating template-probe duplex molecules. Intact probe molecules (VIII) are separated from the radiolytically degraded probe molecules that accumulate during probe purification by a second round of denaturing gel electrophoresis (VII) to yield single-strand end-labeled probe (VIII) for genomic mapping and footprinting (Fig. 1, A and B). clease (Fig. 1B, step 2). This e n z y m a t i c step d e g r a d e s u n h y b r i d i z e d p r o b e m o l e c u l e s and trims single-strand tails f r o m the h y b r i d s leaving a population o f r a d i o a c t i v e h y b r i d m o l e c u l e s that e x t e n d f r o m the p r o b e 5' end label to the p r o x i m a l cut in the c o m p l e m e n t a r y g e n o m i c strand, t h e r e b y allowing the m a p p i n g o f these g e n o m i c cuts with r e s p e c t to the end label r e f e r e n c e point (Fig. 1B, m o l e c u l e I). T h e p o p u l a t i o n o f labeled h y b r i d m o l e c u l e s thus g e n e r a t e d are sized by e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e gels u n d e r n o n d e n a t u r i n g c o n d i t i o n s (Fig. 1B, step 3) and the gels dried
[74]
In Vioo FOOTPRINTING
739
and autoradiographed to display the genomic cutting pattern comprising the footprint relic of in oioo DNA-protein interactions. Cleavage and Subsequent Preparation of Genomic DNA The choice of whether to perform the footprint analysis on whole cells or cell nucleic depends to some extent on the nature of the DNA cleavage reagent employed. Nuclei are permeable to most cleavage reagents whereas cells are more selective. Reagents such as dimethyl sulfate, 6 psoralen, 9 neocarcinostatin, 1°-~3 and related drugs can be used to cleave DNA in intact cells. We have used nucleases ~-3 as probes of chromatin structure in isolated nuclei and in concentrated lysed whole cells and we have used neocarcinostatin chromophore ~4,~5as a structural probe in intact cells. Nuclease digestion of isolated nuclei is technically the easiest experiment, but it is important to appreciate the lability of some chromatin structures in the design and interpretation of experiments on isolated nuclei, since structural components can be lost during isolation procedures. The exact procedure to be used in cell or nuclear preparation will vary with the tissue or cell type to be analyzed but in general the samples should be handled gently and quickly, and kept cold and protease free to stabilize nuclear structures. Aliquots of the preparation are treated with a series of concentrations of the cleavage reagent designed to produce one or two cuts per 500 bases within the sequence of interest. For DNase I digestion of protein-free DNA or nuclease hypersensitive sites in isolated nuclei these concentrations are in the range 5-200 ng enzyme/mg DNA for 30 min at 20° in 50 mM NaCI, 20 mM HEPES (pH 7.5), 5 mM MgCl2, and 1 mM CaCI2 with protease inhibitors when necessary. Digestions are terminated by the addition of NaEEDTA (pH 8.0), to 10 mM, NaC1 to 0.5 M, and protease K to 100 /zg/ml. After 30 min at 37° sodium dodecyl sulfate is added to 0.25% and protease K to 200 /zg/ml and digestion allowed to proceed for 24 hr at 37°. Cutting frequency can be assayed by Southern blotting ~6to follow the appearance of cutting within a hypersen-
9 M. M. Becker and J. C. Wang, Nature (London) 309, 682 (1984). l0 L. S. Kappen, I. H. Goldberg, and T. S. A. Samy, Biochemistry 18, 5123 (1979). 11 T. Hatayama, I. H. Goldberg, M. Takeshita, and A. P. Grollman, Proc. Natl. Acad. Sci. U.S.A. 75, 3603 (1978). 12 p. D. Jackson and G. Felsenfeld, unpublished observations. t3 L. S. Kappen, M. A. Napier, I. H. Goldberg, and T. S. A. Samy, Biochemistry 19, 4780 (1980). 14 L. S. Kappen, M. A. Napier, and I. H. Goldberg, Proc. Natl. Acad. Sci. U.S.A. 77, 1970 (1980). 15 L. S. Kappen and I. H. Goldberg, Biochemistry 19, 4786 (1980). ~6E. M. Southern, this series, Vol. 68, p. 152.
740
IDENTIFICATION AND CHARACTERIZATION OF CLONES
[74]
sitive site 7,17 of interest or to follow the disappearance of a short (-500 bp)
restriction fragment containing the site of interest. The indirect end-label Southern blotting method for low-resolution mapping of cutting within hypersensitive sites is well documented. 7,16,~7 Briefly, purified genomic DNA froM, e.g., nuclease-digested nuclei is digested with restriction endonucleases that will cut at least 500 and not more than 4000-6000 base pairs away from possible nuclease-hypersensitive sites near the gene of interest. The DNA is resolved by electrophoresis in agarose gels (see this volume [8]), transferred to a solid support [61], and hybridized to a radioactive probe [45]. TM The 300- to 600-bp probe is chosen such that it hybridizes specifically with the first 300-600 bases adjacent to the restriction endonuclease cut at one end of the genomic restriction fragment and such that it does not overlap the restriction site. The combination of restriction endonuclease digestion of genomic DNA and hybridization to short probes specific for one end of the genomic restriction fragment allows the mapping of genomic cleavage sites (e.g., hypersensitive sites) with respect to the apparent end label (indirect end label) provided by the short probe. Deproteinized, unrestricted genomic DNA is used to prepare controls displaying the cleavage pattern of the cleavage reagent on protein-free DNA and to make up a mixture of various restriction endonuclease-digested samples to act as size standards (Fig. 5, lanes 1-2 and lanes marked STD, respectively). We have found that genomic DNA serves quite well as substrate for the preparation of protein-free DNA controls, obviating the need to use cloned sequences for this purpose as was previously described. Genomic DNA is prepared for high-resolution mapping by deproteinization following by restriction endonuclease digestion and treatment with ribonuclease A to eliminate endogenous RNA that would otherwise compete for radioactive probe during hybridization. The deproteinized genomic DNA is treated with 1/xg of heated ribonuclease A (100 ° for 15 min, then cooled to room temperature) per 20/xg of genomic DNA in 10 mM Tris-HC1 (pH 8.0), 1 mM Na2EDTA (TE buffer) at 37° for 30 min and followed directly by restriction endonuclease digestion, phenol-chloroform extractions to remove protein (this volume [4]), alcohol precipitation (this volume [5]), and resuspension of the DNA to 2 g/liter in TE. The restriction endonuclease digestion is chosen to isolate the genomic DNA sequence of interest on a small (300-1000 bp) fragment within the genomic digest. In the example of the mapping method presented here, digestion with PvulI serves to center the -200-bp 5' nucle17 j. D. McGhee, W. I. Wood, M. Dolan, J. D. Engel, and G. Felsenfeld, Cell (Cambridge, Mass.) 27, 45 (1981).
[74]
In Vivo FOOTPRINTING
741
ase-hypersensitive region of the chicken erythrocyte/3 A-globin gene on a 580-bp fragment. Having the target sequence on such a small genomic DNA fragment limits the ability of otherwise long genomic DNA to compete kinetically with single-strand probe for hybridization to the complementary genomic strand, TM and similarly lowers the initiation rate of strand displacement reactions which would favor the longer, more stable genomic renaturation products over the shorter probe-genomic DNA hybrids at thermodynamic equilibrium. The loss of signal due to these phenomena is further limited by driving the hybridization reaction with a -30-fold molar excess of probe over genomic complement. The preferential stability of genomic renaturation products and the resulting negative effects of strand displacement on signal intensity can be completely eliminated by choosing the 5' end of the genomic restriction fragment to coincide with that of the probe. Probe Preparation and Purification Construction o f Probe Template
5'-End-labeled single-strand probe is prepared by primed synthesis on a single-strand template followed by isolation and purification of the newly synthesized probe strand. The basic procedure is as previously described I (Fig. 2) with technical modifications that increase probe yield. The template consists of ->300 bp of the genomic sequence of interest cloned into a vector which yields filamentous phage particles containing single-strand DNA copies during infectious growth in bacterial cultures. We have used the M13 phage vectors developed by Messing 1,3A9as well as pTZ plasmid vectors (P-L Biochemicals) bearing filamentous phage origins of replication 2,2°-22with equal success for the preparation of template. Recombinant phage are prepared from infected bacterial cultures ~9-2~(see this volume [13]) and the single-strand DNA template purified from the phage by phenol-chloroform extraction and alcohol precipitation. The choice of genomic fragment to be cloned and how to orient it within the vector sequence is made on the basis of two criteria: (1) each template will produce a probe that is strand specific (i.e., two templates will be required to examine completely the cutting pattern on both strands ~8j. G. Wetmurand N. Davidson,J. Mol. Biol. 31, 349 (1968). t9j. Messing, this series, Vol. 101, p. 20. 20L. Dente, G. Cesareni, and R. Cortese, Nucleic Acids Res. 11, 1645(1983). 21B. P. H. Peeters, J. G. G. Schoenmakers,and R. N. H. Konings,Gene 46, 269 (1986). 22D. A. Mead and B. Kemper, in "Vectors: A Survey of MolecularCloningVectors and Their Uses" (D. T. Denhardtand R. L. Rodriguez,eds.). Butterworth,Boston, in press.
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I D E N T I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF CLONES
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within a given genomic sequence), and (2) the mapping technique has best resolution 60-220 bases from the 5'-end-labeled region of the probe complementary to the genome (Fig. 3). As with any mapping technique, it is desirable to choose the end-labeled region such that it will avoid intense cutting sites in the genome. In the worst case, in which 100% of the genomic DNA is cut within the region complementary to the 5'-endlabeled stretch of the probe, it is very difficult to map the other genomic cut sites properly. Placement of the 5'-end label so that it is complementary to areas less heavily cut just adjacent to hypersensitive regions in chromatin eliminates this problem. The ml9XB probe used here (Figs. 35) is about 300 bases long and extends at each end about 50 bases beyond the sequence of the 200-bp 5'-nuclease-hypersensitive region of the flA_ globin gene. We have used synthetic primers complementary either to vector sequences directly adjacent to the cloned genomic sequences (e.g., 17-base "universal sequencing primer" used as primer for the ml9XB probe), or to regions within the cloned genomic sequences. The amount of [a32p]dNTPs necessary to extend label into probe sequences complementary to genomic DNA is directly proportional to the distance between the 3' end of the primer and the 5' end of genomic complement in the probe sequence. It is a consideration of economy to minimize this distance when designing a cloning strategy. A related point is that shorter labeled stretches will suffer less radiolytic damage and thereby increase probe yield, as will become obvious in descriptions of probe purification below.
Probe Synthesis Priming of Single-Strand Template (Fig. 2, step I). Approximately 0.75/xg of M13 phage DNA or 0.35 ~g of single-strand pTZ DNA with a 300-base insert is required for each 250/.Lg of genomic DNA analyzed. This starting amount represents a 1000-fold molar excess of the cloned template sequence over its chicken genomic complement and compensates for radiolytic and other losses incurred during probe purification. Template DNA is primed by mixing template at a DNA concentration of 0.167 g/liter with a 2.5-fold molar excess of synthetic oligonucleotide primer in 1 x SMT buffer (1 x SMT: 42.0 mM NaC1, 5.8 mM MgC12, 5.8 mM Tris-HCl at pH 7.5). The mixture is heated to 55 ° for 5 min, incubated at 37° for 2 hr, and then frozen on dry ice and stored at - 2 0 ° until use, when the primer is extended using the large fragment of DNA polymerase I. 5'-End Labeling of Probe Strand (Fig. 2, step II). Labeling of probe within regions complementary to the genomic sequence is confined to 10-
[74]
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¢o
-5 1
2
3
4
5
6
7
8
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200
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FIG. 3. Primed synthesis titrations with limiting [a-3~p]dNTPs. Pilot primed syntheses are performed as described on 0.75 p.g of ml9XB template using 3.52 pmol (lane l, 6-8), 4.22 pmol (lane 2), 5.06 pmol (lane 3), and 6.08 pmol of each [a-32P]dNTP. Lanes 1-4 are without a chase with excess unlabeled dNTPs. Lanes 5-8 are chased with unlabeled dNTPs and are then digested with different restriction endonucleases: Sinai (lane 5), BanI (lane 6), ApaI (lane 7), and MnlI (lane 8). The samples are denatured by heating in formamide and fractionated electrophoretically in a polyacrylamide gel of 12.5% acrylamide-0.625% bisacrylamide containing 45 mM Tris-borate (pH 8.3), 1.25 mM Na2EDTA, 8.5 M urea. A map of the region of primed synthesis is diagrammed at the far left, with the position of kinetic stops in the incorporation of label marked with arrows. The restriction standards (lanes 5-8) are correlated with the map by connecting lines. The 3' end of the mapped probe strand is shown cleaved at the HindIII site as during probe purification described in text. The sizes of the major kinetic stops are at 73 bases (37 bases of label at 5' end of genomic sequence) and I l0 bases. The restriction standards are at 25, 62, 74, 85, and 89 bases. The bands in lanes 6-8 illustrate radiolytic strand breakage of the probe, since a smear of breakage products appears 17 bases (the length of the unlabeled primer) below the major band. The intensity of the smear is proportional to the time between synthesis and electrophoresis, which for this pilot experiment was 12 hr.
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IDENTIFICATION AND CHARACTERIZATION OF CLONES
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40 bases near the 5' end by incubating first with limiting amounts of [o~32p]dNTPs and taking advantage of inherent kinetic blocks to polymerization, which provide stopping points for all templates we have tested (Fig. 3). We found it necessary to adjust labeled dNTP concentrations over a range of -2-fold for individual templates to obtain suitable end-labeling; the differences among templates are probably related to template secondary structures (e.g., self-complementary stem and loop structures).~ The mI9XB probe used in the analysis ofgenomic DNA shown in Figs. 4 and 5 is synthesized by adding primed MI3-m19XB template (0.75 ~g in 4.5/xl) to 16.7/zl of a mixture containing 3.33 pmol (10/xCi) each of o~-32p-labeled dATP, dCTP, dGTP, and dTTP (3000 Ci/mmol), 1.2x SMT, 0.15 mM 2mercaptoethanol, and 1.2 units of large-fragment DNA polymerase I (any polymerase preparation suitable for dideoxynucleotide-terminated DNA sequencing is acceptable in this application). Extension o f Probe Strand with Unlabeled Nucleotides (Fig. 2, step liD. After incubation at 20 ° for 1 hr, the probe strand is extended through the remaining genomic sequence and into adjacent MI3 vector sequence with greatly reduced incorporation of radioactivity (Fig. 2, step III) by adding 3.75/~1 of a mixture containing 2.0 nmol each of the four unlabeled dNTPs in 1 x SMT, 0.15 mM 2-mercaptoethanol, and 1.25 units of DNA polymerase I large fragment. Incubation is continued at 20° for 20 min, at 37° for 5 min, and finally at 68 ° for 10 min to denature polymerase. The extent of probe 5'-end label is approximately as in Fig. 3, lane 1.
Probe Purification Restriction Endonuclease Digestion of Probe-Template Complex (Fig. 2, step IV). The probe-template complex is digested with a restriction endonuclease chosen to maximize the size difference between the denatured single strands of the newly synthesized probe and its M13 template (Fig. 2, step IV). For the ml9XB probe, 10 units of HindlII endonuclease, 2.0/zl of buffer (0.5 M Tris-HCl at pH 8.0, 50 mM MgClz, bovine serum albumin at 1 g/liter, and water to 40/zl are added to the above reaction mixture and incubated at 37° for 1 hr. HindIII cleaves once, yielding a linearized template strand of -7500 bases and an end-labeled probe strand of - 3 5 0 bases including adjacent M13 sequences and primer (Fig. 3). The reaction is stopped by the addition of Na2EDTA (pH 8.0) to 15 mM final concentration, and the DNA purified by extraction with Trisneutralized phenol and chloroform. Traces of chloroform are removed by extraction with two volumes of n-butanol, the aqueous phase diluted with 2 ml of TE, and the sample concentrated and desalted by centrifugation in a
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Centricon-30 disposable concentration device according to the manufacturer's directions (Amicon Corporation). Denaturing Gel Purification of Probe Strand (Fig. 2, step V). The retained sample, in a volume of 50-100/xl, is denatured by dilution to 1 ml with deionized formamide (plus 5 /xl 0.2 M NaEDTA at pH 8.0 and xylene cyanole FF dye to 0.01 g/l) and heated at 68 ° for 5 min prior to loading in a single slot on a 1.5-mM thick polyacrylamide gel (5% acrylamide, 0.25% bisacrylamide) containing 45 mM Tris-borate (pH 8.3), 1.25 mM Na2EDTA, 8.5 M urea. The gel is prerun prior to sample loading to heat it to - 5 0 °. Avoiding alcohol precipitation and keeping the sample dilute and warm prior to electrophoresis increase probe yield by decreasing rehybridization of the template and probe strands and by minimizing nonspecific losses on surfaces often incurred during precipitation. The sample is fractionated electrophoretically at 30-40 V/cm for 2 hr and the gel autoradiographed for about 15 sec to localize the probe. The probe band is excised and electroeluted from the gel slice for 1 hr at 125 V into a small volume of a high ionic strength solution (4.75 M NaC1, 25 mM HEPES at pH 7.5, 5 mM NaEEDTA, sonicated calf thymus DNA at 50 /zg/ml) in an electroelution apparatus (International Biotechnologies, Inc., catalog no. 46000) containing the Tris-borate-EDTA electrode buffer as above. The electroeluted sample is desalted and concentrated in a Centricon-30 by two or three cycles of dilution with TE followed by centrifugation as above. The probe is pure enough at this point to use for the study of restriction digests containing short unique chicken DNA sequences at 0.1 × genomic abundance (Fig. 4, lanes 1-5). Contaminating template sequences remain, however, and the signal-to-noise ratio obtained using this probe is improved (Fig. 4, lanes 6 - l l) by continuing the probe purification described below. The analysis of mixtures of restriction endonuclease-digested genomic DNA (as in Figs. 4 and 5) provides an important test of the validity of the mapping information provided by the technique. Each of the restriction endonuclease cleavages introduced into the genomic DNA should be mapped uniquely and at the correct distance with respect to the probe end label for the mapping information to be valid. This point will be covered in more detail in the discussion of Figs. 4 and 5 contained in the section on genomic footprinting and validation of results at the end of this chapter.
Removal of Contaminating Template Strands by Sequential Self-Hybridization and Hydroxyapatite Chromatography (Fig. 2, steps VI and VII). The probe is purified further by self-hybridization and hydroxyapatite (HAP) chromatography to remove the small amount of contaminating template sequences. To the probe in 60/zl of TE is added 6.67/xl of 10x
746
IDENTIFICATION AND CHARACTERIZATIONOF CLONES
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HB (10x HB: 3.18 M NaC1, 200 mM HEPES at pH 7.5, 1.0 mM Na2EDTA). This mixture is flame sealed in a glass capillary, heated to 107° for 5 rain in a saturated NaCI bath, and transferred directly to a 68° water bath for incubation overnight. During this self-hybridization, probe strands in excess will hybridize with contaminating template strands. The pure single-strand probes may be separated from these duplexes by their differential affinity for HAP which binds the duplex molecules more tightly than the single strands in 150 mM NaPO4 at pH 7. 23 Hydroxyapatite (Bio-Rad) is prepared by suspension and settling in 50 mM NaPO4 (pH 7.0). The HAP is washed once in 1 M NaPO4 (pH 7.0), and then equilibrated in 50 mM NaPO4 (pH 7.0). The self-hybridized probe is diluted into 0.2 ml of sonicated calf thymus DNA (0.2 g/liter) in 50 mM NaPO4 (pH 7.0), and applied to 0.1 ml of the gently packed HAP (centrifuged 1 min at 250 g in swinging bucket rotor) in a tightly capped polypropylene centrifuge tube. The HAP is suspended in the DNA solution by gentle vortexing or inversion and intermittently resuspended during a 15-min incubation at 68 °. The HAP is sedimented at 250 g for 1 min and washed six times with 2 ml of 50 mM NaPO4 (pH 7.0), at 68°. Greater than 90% of the radioactivity should bind to the HAP, and 32p in the wash buffer should reach a low constant value after three or four washes. The probe is eluted from the matrix with four 68° washes of 0.5 ml 150 mM NaPO4 (pH 7.0). Combined washes containing - 9 0 % of the eluted radioactivity (only a small percentage of the 32p should remain associated with HAP at this point) are filtered by centrifugation through 0.45-/.¢m pore size Centrex cellulose acetate filters (Schleicher & Schuell) to remove any stray HAP and the filtered probe desalted and concentrated by dilution with TE and centrifugation in a Centricon-30 device as before. Final Removal o f Radiolytic Degradation Products by Denaturing Gel Electrophoretic Purification o f Intact Probe Strand (Fig. 2, steps VII and VIII). The probe in - 5 0 ~l of TE is carefully concentrated to - 5 tzl by repeated extraction with 2 volumes of n-butanol and the sample diluted to I00 /zl with the same proportions of formamide, EDTA, and xylene cyanole FF used before. The sample is heated prior to electrophoresis in a polyacrylamide-urea-Tris-borate-EDTA gel as before with smaller well size to accommodate the smaller sample volume. The reason for performing this second gel purification of the probe is made clear upon autoradiography of the gel. It is apparent that during the time since electrophoresis ceased in the first gel ( - 2 0 hr) considerable radiolysis has occurred. Up to 50% of the probe will appear in a smear of radioactivity just below 23G. Bernardi, Procedures Nucleic Acid Res. 2, 455 (1971).
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the remaining tight band of intact probe. It is a conservative choice to excise only the intact probe band, but it thereby assures the integrity of the probe that now goes into the analysis of genomic DNA. The probe (Fig. 2, step VIII) is electroeluted, concentrated, and desalted as before and is now ready for hybridization to genomic DNA. The probe should be used immediately as storage only decreases the signal-to-noise ratio, eliminating the gains made by the second probe purification gel. Analysis of Genomic DNA
Hybridization of Genomic DNA with Probe (Fig. 1B, step 1) To make more efficient use of the effort expended in probe purification, we generally analyze 40-50 25-/zg samples at a time with the probe preparation scaled accordingly. Hybridization of single-stranded 5'-endlabeled probe with genomic DNA is accomplished by mixing 0.1 ng ( - 3 x 105 dpm) of probe with 25/zg of genomic DNA (a 30-fold molar excess of probe over its chicken genomic complement) in 20 /zl of 1 x HB. The mixture is flame sealed in a glass capillary, heat denatured (5 min, 107°), and directly transferred to a 68 ° bath for a 12-hr hybridization. Genomic DNA is 60-70% renatured and hybridization to probe is complete in this time. The reaction is stopped by expelling the mixture into 14/zl of TE and precipitating the DNA with 40/zl of isopropanol at room temperature. The pellet is washed in 70% ethanol, dried, and dissolved in 15.6/zl of 15 mM NaCI, 1.5 mM HEPES (pH 7.5).
Heat Denaturation of Nonspecific Hybrids Cross-hybridization of the probe to related sequences in the genome is eliminated by incubation in this low ionic strength solvent at 70° for 30 rain. The temperature of incubation is probe dependent as is discussed below. This heat denaturation step is necessary to eliminate high backgrounds of nonspecific hybridization, the magnitude of which varies as a function of probe sequence. Hybrids of probe with the specific genomic sequence are not denatured under these conditions. This step is analogous to the "high-stringency wash" step in Southern blotting ~6 and the considerations governing the choice of conditions are the same for both. Higher G + C content (the probe used here is - 7 0 % G + C) and greater homology to nonspecific genomic sequences dictate incubation at a higher temperature (e.g., 70° for the ml9XB probe). The temperature of incubation is chosen empirically by observation of the influence of temperature upon the signal-to-noise ratio obtained in the
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analysis of standard genomic samples (e.g., a mixture of restriction endonuclease digests of genomic DNA as in Fig. 4). Since little is known in many cases about the extent of cross-reacting sequences within a genome, one can start such a survey of temperature effects near the melting temperature of the specific hybrid according to its content of G + C bases and its length, and work down in temperature until the background from nonspecific hybridization becomes unacceptable again. In practice, T (in degrees Centigrade) = 41 + 0.41 (% G + C bases in probe) is a reasonable upper limit, at which temperature both specific and nonspecific hybrids of less than about 50 base pairs are denatured, za,z5 Omission of the heat denaturing step is a potential source of artifact.
Digestion of Single-Strand DNA Regions After, heating, the hybridized samples are digested with a calibrated activity of mung bean nuclease 26 to trim off single-strand regions from the hybrids, as well as unhybridized single-strand probe and residual denatured genomic DNA (Fig. 1B, step 2). The pattern of mung bean nucleaseresistant fragments is relatively insensitive to 2-fold changes in enzyme activity but new batches of enzyme should be calibrated in the assay (Fig. 4). Overdigestion leads to nibbling in at the ends of duplex molecules; underdigestion leads to increasing levels of contamination with nonhybridized probe. The amount of nuclease used should be 1.5- to 2-fold more than the minimum necessary to degrade unhybridized probe molecules. This low nuclease activity (6.5 units of nuclease/25/xg of genomic DNA for the enzyme preparations we have used) will assure the elimination of the unhybridized species and the effective trimming of single-strand regions from the hybrids and yet minimize the nibbling in at the ends of hybrid molecules that can decrease resolution (Fig. 4, lane 6 vs. lane 7). A mixture of restriction endonuclease digests of genomic DNA is assayed in parallel with the other genomic samples to provide size standards that control for the effects of mung bean nuclease on fragment size (Fig. 5, lanes marked STD). A potential artifact can arise through the action of the single-strandspecific nuclease at sites where the probe is not complementary to the genomic sequence. Sequence mismatches in the hybrid might result from genomic sequence polymorphisms or inaccurate copying of template dur24 j. Marmur and P. Doty, J. Mol. Biol. 5, 109 (1962). 25 C. R. Cantor and P. R. Schimmel, "Biophysical Chemistry," Part III, Chapter 22. Freeman, San Francisco, California, 1980. 26 M. Laskowski, Sr., this series, Vol. 65, p. 263.
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ing probe synthesis (see discussion of alternative probe preparation strategies below). The nuclease can attack these defects in the duplex if they are large enough, and yield fragments unrelated to the genomic cleavage pattern under investigation. Digestion of single-strand regions is effected by adding 6.5 units of mung bean nuclease (P-L Biochemicals) in 10.4 p~l of 125 mM sodium acetate (pH 5.3), 2.5 mM magnesium acetate, 2.5 mM L-serine, 2.5 mM 2mercaptoethanol (added fresh), 0.0125% Triton X-100 (Sigma), 0.25 mM zinc acetate, and incubating at 40° for 60 min. The reaction is stopped by adding 3.5 /~1 of 2.5 M NaCI, 0.5 M Tris-HC1 (pH 8.5), followed by precipitation with 36/~1 2-propanol. The pellet is washed with 70% ethanol, dried, and redissolved in 4-8/zl of 10 mM Na2EDTA (pH 8.0), 6% Ficoll, 0.06% xylene cyanole FF to yield 25-12.5/xg genomic DNA equivalents of hybrid molecules per 4/zl to be loaded on the gel.
Resolution of the Trimed Radioactive Hybrids on Nondenaturing Polyacrylamide Gels (Fig. 1B, step 3) The choice of resuspension volume depends on considerations of sensitivity and resolution. At 25 /zg/4 /xl, sensitivity is increased, but the higher load of DNA decreases the resolution, especially above 180 base pairs. Regardless of choice, the concentration of all experimental samples and the genomic restriction standards should be identical in order to equalize the effects of sample load on fragment migration during gel electrophoresis. Samples in 4/zl are loaded in 0.8 x 5 mm wells on a 0.8 x 330 x 380 nondenaturing gel (8% acrylamide-0.4% bisacrylamide containing electrophoresis buffer) and fractionated electrophoretically in a buffer containing 90 mM Tris-borate (pH 8.3), 2.5 mM Na2EDTA at 18 V/cm for 7 hr. Glass plates used to form the gel slab should be pretreated with dichlorodimethylsilane and rinsed with ethanol and water prior to gel casting. Nondenaturing gels are used to ensure the integrity of labeled fragments that may have been internally nicked by radiolysis or mung bean nuclease. After electrophoresis, the glass gel-casting plates are separated and a sheet of cellophane (Bio-Rad) that has been thoroughly wetted is closely apposed to the gel with care taken to eliminate air bubbles from the gelcellophane interface. A sheet of heavy filter paper is laid over the cellophane for mechanical support and the gel carefully freed from the glass plate by turning the glass plate side of the gel sandwich up and prying the gel-cellophane laminate free from the glass, allowing it to fall gently on the supporting heavy filter paper. A sheet of Mylar (0.0005 in. thick, or
750
IDENTIFICATION AND CHARACTERIZATION OF CLONES
IMPURE PROBE
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other transparent heat-stable film) is layered without air pockets over the upper naked gel surface, and the four-layered sandwich dried in a vacuum slab gel drying apparatus (e.g., Bio-Rad or Hoefer apparatus) at 80°. The transparent dried cellophane-gel-Mylar laminate (heavy filter paper removed) is sandwiched between two Dupont Cronex Lightening Plus intensifying screens and exposed to a sheet of flashed 27 (also this volume [7]) Kodak XAR-5 film for - 1 4 days at -80 °. The use of a transparent gel backing (cellophane) and two intensifying screens effectively doubles the sensitivity of the method over that previously described. 1 Potential Modifications to Mapping Methods Further gains in sensitivity could be obtained if necessary by prior partial purification of the genomic sequences to be probed (e.g., by gel electrophoretic size fractionation of restriction endonuclease-digested genomic samples). This sequence purification has the potential to dramatically increase the signal-to-noise ratio by greatly decreasing the complexity of the analyzed genomic sample and by allowing a much greater specific sequence load on the final analytical gel. The basic steps of this analysis (i.e., end-labeled single-strand probe synthesis and purification, hybridization, trimming of the hybrids with single-strand-specific nuclease and gel electrophoresis) might be accomplished using variations of the mapping technique described here. Some of these variations may represent improvements in ease of probe purification or increased resolution of the genomic analysis, but for the most part they remain speculative and untested. 27 R. Lasky, this series, Vol. 65, p. 363.
FIG. 4. Mung bean nuclease digestion titrations of mI9XB probe-chicken DNA restriction mix hybrids. Single-strand probe hybridized with a mixture of chicken DNA restriction endonuclease digests is processed as described in the text with the exception that different activities of mung bean nuclease are used to trim the hybrids. Two different purities of probe are used in the hybridization: impure probe (lanes 1-5) used directly after the first denaturing gel and highly purified probe (lanes 6-11) that has gone through the entire purification scheme. Mung bean nuclease concentrations used are 6.5 units (lanes 1, 6, and 11), 13 units (lanes 2 and 10), 26 units (lanes 3 and 9), 52 units (lanes 4 and 8), and 104 units (lanes 5 and 7). The position of the restriction endonuclease cuts in the genomic sequence are given at the right with respect to the start site of transcription. The actual lengths of the trimmed hybrid fragments extending from the probe 5' end at - 4 to the genomic restriction positions marked at the right of the figure are 118, 138, 156, 196,201,224, and 299 base pairs from bottom to top radioactive fragment [e.g., (-122) - ( - 4 ) = 118 base pairs].
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IDENTIFICATION AND CHARACTERIZATION OF CLONES
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FIG. S. Analysis of genomic DNase I cleavage patterns. (A) Genomic protein-free DNA samples (lanes 1 and 2) or purified nuclei from adult erytbrocytes (lanes 3 and 4) were digested with DNase I. D N a s e I digestion increases by 2.5-fold going from right to left within each pair of samples. The genomic DNA in the standard lanes (STD) is as described in Fig. 3. Samples are analyzed with the mlgXB probe as described in text. A region protected from DNas¢ I cleavage in adult'erythrocytes is mapped to the sequence on the left which, along with the standards, are numbered with respect to the initiation site for transcription on the chicken/3^-globin gene. Enhanced cleavage is indicated by the small arrow, and the protection adjacent to the 5' edge of the hypersensitive region by the large open arrow. (B) Densitometric scans of the indicated lanes from (A).
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Probe purification could be effected by the incorporation of an affinity label (e.g., Hg-dUTP 28 or biotinylated dUTP) during probe synthesis followed by the separation of denatured probe and template strands by chromatography on matrices containing covalently bound sulfhydryl or streptavidin moieties. Alternatively, an RNA template for probe synthesis could be transcribed from a genomic clone in one of the vector systems utilizing phage RNA polymerase promoters. 21,22Treatment with DNase I would yield pure RNA template, which could then serve as substrate for the synthesis and purification of probe using sequential primed synthesis with reverse transcriptase followed by alkaline hydrolysis of the RNA template. Direct end labeling of the RNA copy would provide even more facile access to a probe which, after genomic hybridization and nucleolytic trimming might provide a further advantage in the potential for elimination of gel overloading problems by DNase I treatment of hybridized, single-strand trimmed samples. In practice, the incorporation of an affinity label is probably only to be preferred for the preparation of long (2-kb) probes for which gel purification is inefficient. The use of an RNA template is an intriguing possibility, but might suffer from the inherently lower fidelity of the RNA polymerase and reverse transcriptase reactions relative to those catalyzed by DNA polymerase I large fragment, which possesses a 3'-5' exonucleolytic "editing" activity that limits misincorporation of bases. 29 Inaccurate transcription of the templates during probe synthesis would result in sequence mismatches in the probe-genomic DNA hybrid that could be recognized by mung bean nuclease or other single-strand-specific nucleases used to trim hybrids. In addition, altering the chemistry of the probe or using nucleases other than mung bean to trim the hybrids can introduce novel artifacts arising from the varying propensity of the nucleases for exo- or endonucleolytic attack upon duplex substrates. For example, the substitution of Si nuclease for mung bean nuclease to trim the hybrids has consistently led to poorer signal-to-noise ratios in our hands. The problems to be overcome in the development of an alternative scheme for genomic mapping are obvious: signals introduced by errors in probe synthesis or by enzymatic degradation of the hybrid duplex must be small relative to the miniscule signals arising from the genomic cleavages to be mapped. Any alternative that can solve these problems can be used for high resolution genomic mapping when used in conjunction with a heat denaturation step to decrease signals due to cross-hybridization to related genomic sequences. P. D. Jackson and G. Felsenfeld, unpublished observations. 29 D. Brutlag and A. Kornberg, J. Biol. Chem. 247, 241 (1972).
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Genomic Footprint and Validation of Mapping Results
The genomic restriction standards shown in Figs. 4 and 5A are mixtures of several different restriction endonuclease digests. Each digest should give a single band in the autoradiogram corresponding in length to the distance between the probe Y-end label and the closest restriction endonuclease site in the complementary genomic strand. In examining these standard lanes, the validity of the mapping information gained from this technique becomes apparent. Approximately 95% of the radioactivity migrates in bands corresponding to the predicted lengths of the end-labeled hybrid molecules for each of the individual restriction digests in the standard. This demonstrates that the signals observed in an analysis of DNase I-cleaved genomic DNA (e.g., Fig. 5A, lanes 1-4) can be mapped with confidence onto the genomic DNA sequence with little interference from internal fragments (Fig. 1B, molecules labeled II) or from crosshybridizing genomic sequences. The probe used in this example of intranuclear genomic footprinting is complementary to the region extending from - 6 to -303 bases from the site of transcription initiation in the chicken/3A-globin gene. The mapped genomic strand contains a string of 16 G residues extending from - 181 to - 196. A region protected from DNase I attack in adult erythrocyte nuclei can be seen (Fig. 5); it includes the sequence from -130 to -156. Enhanced cutting is observed at -162 on this sequence and depressed cutting at - - 2 3 0 , the 5' margin of the nuclease hypersensitive region. A densitometric scan (Fig. 5B) of lanes of comparably digested protein-flee DNA (lane 2) and adult erythrocyte nuclei (lane 3) emphasizes the protection from DNase I attack afforded by proteins site-specifically bound to this sequence in adult erythrocyte nuclei. The existence of this site-specific protein DNA complex in vivo is supported by the observation of similar nuclease protection in this region shown by genomic mapping of the complementary genomic strand 1 and by in vitro nuclease protection experiments using partially purified nuclear factors reconstituted on cloned DNA. 3° Genomic DNA marked with DNA cleavage reagents in either whole cells, cell nuclei, or protein-flee DNA can be analyzed with this method to display high resolution maps of specific protein-DNA interactions. This provides valuable information about developmental changes in the structure of genetic regulatory elements in viuo. The structural information obtained about various functional states of a gene can be a powerful 30 B. E. Emerson, C. D. Lewis, and G. Felsenfeld, Cell (Cambridge, Mass.) 41, 21 (1985).
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adjunct in the design and interpretation of both in vitro reconstitution and transcriptional studies, and in vioo transcriptional analyses. Acknowledgments We thank Byron Kemper for introducing us to the use of pTZ plasmids in this application, and Betty Canning for her superb help in preparing the manuscript.