MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2774-2786 0270-7306/90/062774-13$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 6

Developmental Regulation of Topoisomerase II Sites and DNase Hypersensitive Sites in the Chicken 1-Globin Locus

I-

MARC REITMAN AND GARY FELSENFELD* Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892 Received 22 November 1989/Accepted 23 February 1990

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken (-globin locus, which contains four globin genes (5Pp-H4,A-3P). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of p to just downstream of e. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the p-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of p, pH, and e. (v) A site 3' of pA and 5' of e was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo H sites. These data are consistent with the hypothesis that, in vivo, topo I preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.

The expression of eucaryotic genes is often influenced by loci on the DNA that are quite distant from the gene itself. This has been particularly well documented in the case of the globin gene families. Human alpha and beta thalassemias in some instances arise from deletions that end many kilobases from otherwise normal globin genes (57). Recent studies of the chromatin structure of globin genes have further focused attention on unusual features located many kilobases away. The DNA contained in these distant sites confers high-level, copy number-dependent expression of the associated gene in transgenic mice, independent of the position of integration. This behavior serves to define a new kind of control element, the dominant control or locus activation region, which presumably affects chromatin conformation and thereby gene expression over considerable distances in the genome (24). Chromatin configurational changes induced by a locus activation region may be permissive for optimal expression of all genes clustered in a domain, subject to the further control of local promoters and enhancers. The termini of the locus activation region may also function as delineators of the domain, preventing the further spread of the region of chromatin activation. DNase I digestion of DNA has proven a valuable tool for detection of alterations in chromatin structure that are associated with locus activation regions as well as with the more traditional enhancers and promoters (reviewed in references 14 and 23). Such regions are "hypersensitive" to nuclease digestion, a property that usually signifies the presence of nucleosome-free DNA (40). Typically, the histones are in part replaced by nonhistone, sequence-specific DNA-binding proteins. Hypersensitive sites are also found in other functionally interesting regions, including terminators, centromeres, telomeres, and silencers. In contrast, *

transcribed regions per se are not hypersensitive. Thus, accurate mapping of DNase I-hypersensitive sites can provide information about candidate control regions in large segments of the genome. Another way to study chromatin organization is to map the binding sites of topoisomerase II (topo II) in vivo. Topo II is a major component of the metaphase chromosome scaffold, with a location limited to the central chromosomal axis (13, 20). The drug teniposide can be used to map endogenous topo 1I-binding sites (6, 35). Such sites have been found in promoters, enhancers, and the 3' ends of genes (41, 47, 54). In this paper we apply these methods to the analysis of the chromatin organization of the chicken ,B-globin locus. This locus has been studied extensively; a considerable amount is known about the trans-acting factors involved in regulation of the four developmentally regulated globin genes (5'pH_p3H3A_E.31) that are clustered in a 15-kilobase (kb) segment of DNA within the domain (16). We present the results of mapping DNase I-hypersensitive sites and topo 1I-binding sites in 65 kb of this locus. We grouped the hypersensitive sites into five classes based on their tissue distribution, developmental specificity, and location. All strong topo II sites were found at DNase I-hypersensitive sites, suggesting that, in vivo, topo II acts on nucleosome-free DNA. Not all strongly DNase I-hypersensitive sites were topo II sites, suggesting regulation of topo II action. MATERIALS AND METHODS Preparation of nuclei. We prepared nuclei by two different methods, without detergent (10) and with nonionic detergent (40). Typical procedures are given below. All procedures after harvesting until DNase I digestion were performed on ice or at 4°C. Embryonated White Leghorn chicken eggs were obtained

Corresponding author. 2774

TOPOISOMERASE II AND DNase I HYPERSENSITIVITY

VOL. 10, 1990

2775

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-20 -10 0 10 20 30 40 50 FIG. 1. Strategy for DNase I hypersensitivity mapping of the chicken ,-globin locus. The genes (p, PH, pA, and E) are indicate by solid (exons) or open (introns) boxes. Transcription proceeds from left to right for all four genes. The top portion of the figure shows the probes (indicated by letters) over the corresponding restriction fragments (arrows) used in the hypersensitivity mapping. The probes are described in Materials and Methods. In the middle of the figure is a restriction map of the locus, based on Dolan et al. (11) and Villeponteau and Martinson (56) and our own work. The arbitrary numbering system of Villeponteau et al. (55), has been retained. Restriction sites are depicted by the following symbols: T , BamHI; I , EcoRI; V, HindIII; *, KpnI; 0, XbaI. We have not confirmed the previous mapping data except at the ends of the locus. The positions of subclones pCBG-D, pCBG-C, pCBG-A, pCBG28, and pCBG32 are indicated on the bottom (scale in kilobases).

from Truslow Farms, Chestertown, Md. Erythrocytes were harvested into phosphate-buffered saline (PBS) containing 5 mM EDTA, washed twice with >20 volumes of PBS (200 x g, 5 min), allowed to swell in 20 volumes of 0.67x PBS (10 min), and pelleted. Cells were lysed in 5 volumes of buffer A (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] pH 7.9, 3 mM MgCl2 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM EGTA [ethylene glycol tetraacetic acid]) with 20 strokes of a type B Dounce homogenizer (lysis monitored by microscopy). The nuclei were pelleted (500 x g, 5 min), washed once with buffer A, and suspended in buffer A without EGTA and PMSF to 21 A260 units/ml. Brains (cerebral hemispheres) were removed from 12 16-day embryos, rinsed, minced with a razor blade, dispersed in 30 ml of PBS-5 mM EDTA-0.5 mM PMSF-0.5 mM dithiothreitol with five strokes of a Potter-Elvehjem homogenizer with a fluted pestle, centrifuged (1,875 x g, 8 min), allowed to swell in 50 ml of 0.67x PBS (10 min), and processed further as for erythrocytes. MSB cells, a chicken lymphoblastoid cell line (1), were supplied by Mark Minie of this laboratory. Cells (1.2 x 108, log-phase growth) were washed three times with PBS, suspended in 10 ml of buffer A, homogenized in a type B Dounce apparatus (40 strokes, lysis monitored by microscopy), and processed further as described for erythrocytes. Alternatively, 5.6 x 109 washed erythrocytes were suspended in 15 ml of buffer A containing 0.5% Nonidet P-40 (NP-40), homogenized in a type B Dounce apparatus, incubated on ice (10 min), rehomogenized, layered over 5 ml of buffer A containing 250 mM sucrose, and centrifuged (500 x g, 5 min). The pellet was suspended in 15 ml of sucrosebuffer A, centrifuged as before, and resuspended in buffer A without the PMSF and EGTA to 21 A260 units/ml. In comparing various preparations of nuclei, we noted that nuclei prepared without NP-40 progressed from no DNase I digestion to overdigestion over a narrower range of DNase I concentrations than did nuclei prepared with NP-40. Preliminary experiments showed the same hypersensitive sites with the two methods. The DNase I concentration dependence was also a function of how extensively the nuclei were washed: more washing expanded the useful concentration

range. A potential problem, a subset of nuclei remaining impermeable to DNase I, should give rise to significant persistence of the parental fragment even at high DNase I concentrations. This was not observed. DNase I treatment and isolation of DNA. DNase I (Worthington, catalog no. DPFF) digestion was initiated by adding 950 ,ul of nuclei (usually 20 A260 units) to 50 ,ul of 20 mM CaCl2-20 mM MgCl2-DNase I at 22°C. Unless noted otherwise, the final DNase I concentrations were 0, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 jig/ml. Incubations were stopped at 10 min by addition of 50 ,u of 500 mM EDTA and 20 ,ug of RNase A. After 15 min at 37°C, 4 ml of 1 M NaCl containing 250 pug of proteinase K was added. After 30 min at 37°C, 125 ,u1 of 10% sodium dodecyl sulfate (SDS) was added, and incubation was continued overnight. The samples were next extracted with phenol, phenol-chloroform, and chloroform, precipitated with 0.8 volume of isopropanol, washed with 70% ethanol, and dissolved in 10 mM Tris hydrochloride (Tris-HCl, pH 8.0)-i mM EDTA. Teniposide incubation and isolation of DNA. Cells were washed twice with L-15 medium (GIBCO) and resuspended at 3.3 x 107 cells per 0.5 ml. Teniposide (VM-26, epipodophyllotoxin; Bristol-Myers Co., Wallingford, Conn.) was freshly dissolved in dimethyl sulfoxide, and 2.5 pul of the appropriate concentration was added. After 30 min at 37°C, the cells were pelleted, 700 pul of 20 mM Tris-HCl (pH 8.0)-1% SDS was added, and the samples were mixed by inversion. One minute later, 30 pul of 500 mM EDTA (pH 8.0) was added and mixed by inversion, and 25 ,u1 of a 20-mg/ml solution of proteinase K was added. After overnight incubation at 37°C, the DNA was purified as described for the DNase I samples. Hybridization probes. A map of the chicken P-globin locus is shown in Fig. 1. The DNAs used in this study are derived from the laboratories of J. D. Engel (-9 to 8 map units), G. Felsenfeld (12 to 16.7 map units), and H. Martinson (16.7 to 35 map units) and were obtained as follows (39). BamHI fragments of XCPG3 (11) were inserted into pUC18, yielding pCBG-A (p495), pCBG-C (p501), and pCBG-D (p515) (Fig. 1). ppABE4.2 is the 4.2-kb BamHI-EcoRI fragment from XWES.CA,BG1 (22) cloned into pUC9 by J. Nickol. The BamHI fragments of XCBGv3 (56) were inserted into

2776

REITMAN AND FELSENFELD

pUC18, yielding pCBG23 (p556) and pCBG32 (p527) (Fig. 1). Restriction sites at map units below -9 or above 35 are based on Southern blots and therefore may not be complete. The unique probes of the chicken 03-globin locus are detailed below, in the following format: letter name (used in this article), our laboratory designation, map location, defining restriction sites, and the plasmid of origin. A, 183, -8.3 to -7.9, NcoI to NcoI, pCBG-D; B, 199, -5.4 to -5.0, PvuII to Sacl, pCBg-C; C, 192, 2.1 to 2.5, BamHI to EcoRI, pCBG-A; F, 1142, 15.9 to 16.3, BglII to KpnI, ppABE4.2; G, 1144, 16.7 to 17.2, BamHI to NcoI, pCBG17 (55); H, 1125, 18.4 to 18.6, EcoRI to BanI, pCBG17; K, 1112, 23.3 to 24.3, KpnI to PstI, pCBG23; and L, 187, 31.6 to 32.1, NcoI to NcoI, pCBG32. Blots made with these probes showed no extra hybridization bands at 10- to 50-fold overexposure. A chicken 32-tubulin clone (38) was provided by D. W. Cleveland. The -0.45-kb PstI-EcoRI fragment just 3' of the 132 gene was used as the probe. It does not hybridize to other tubulin genes under the conditions chosen. Restriction enzyme digestion and Southern blotting. Genomic DNA was cut with restriction enzymes (New England BioLabs) and electrophoresed (5 jig of DNA per 2.5-mmwide lane) in 0.8% agarose. After acid depurination, the DNA was transferred to GeneScreen Plus (Du Pont) and hybridized (formamide protocol, with sonicated calf thymus DNA at 100 pg/ml) as recommended by the manufacturer. 32P-labeled probes were made by the random primer method (17) (Boehringer Mannheim) to a specific activity of 3 x 109 to 5 x 109 cpm/p.g and used at 1 to 2 ng/ml. Blots were washed as recommended, except that the second 65°C wash was done with 0.1% SDS-0.1 x SSC (I x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Blots were exposed for 1 to 14 days with intensifying screens at -70°C. All analyses included examination of overexposed autoradiograms, which facilitated detection of weak sites and helped to distinguish artifacts resulting from uneven washing of a blot. RESULTS Strategy for mapping the chicken ,-globin locus. To localize precisely the DNase I-hypersensitive sites in the 1-globin locus, we used the indirect end-labeling method (43, 60). DNA from DNase I-digested nuclei was cut to completion with a suitable restriction enzyme and analyzed by Southern hybridization with a short probe that hybridizes to one end of the restriction fragment. To map the hypersensitive sites, it was first necessary to construct a more complete restriction site map of the locus (Materials and Methods and Fig. 1). Next, we obtained probes from regions near the restriction sites that did not contain repetitive sequences. The nine probe-restriction enzyme combinations used for DNase I site mapping are shown in Fig. 1. The combinations were chosen to include overlaps so that all potential hypersensitive sites would be detected and to give redundancy, allowing checks for internal consistency. Hypersensitivity mapping. We mapped DNase I-hypersensitive sites in seven different sample types. Four erythroid samples were tested (summarized at the top of Fig. 8) (4, 37): (i) circulating 5-day embryonic cells, which are of the primitive lineage and express the p- and e-globin genes but not PH_ or 3A-globin; (ii) circulating 10-day embryonic cells, a mixed population containing 85 to 90% definitive cells, which express PH_ and 3A-globin but not p or £; the other 10 to 15% of 10-day cells are of the primitive lineage; (iii) 15-day embryonic cells, which are solely definitive erythrocytes;

MOL. CELL. BIOL.

and (iv) adult erythrocytes, which no longer actively express any genes, but which expressed PA, but not PH, before maturation. The two nonerythroid samples tested were MSB cells (a lymphoid line) and 16-day embryonic brain cells. Note that erythrocyte contamination of the brain tissue would give a hypersensitivity pattern similar to that of the 15-day erythrocytes. No significant contamination was detected. Free DNA was also examined for the presence of intrinsic DNase I-hypersensitive sites; none was found. Three examples of the blots showing the DNase I-hypersensitive sites are presented. Figure 2 shows the region starting 11 kb upstream of the p-globin gene examined with probe B on KpnI-digested DNA. The hypersensitive sites were thus measured from the KpnI site at -5.9 map units and going rightward (Fig. 1). This blot showed a strong cleavage site (denoted site 1) in nuclei from all tissues, but not in free DNA, at 1.05 to 1.3 kb. Site 1 consisted of two regions of hypersensitivity separated by a less sensitive region. In brain cells, the two hypersensitive components were of about equal intensity, but in the erythroid cells the larger fragment predominated. Additional strong erythroidspecific sites were centered at 4.9 (site 2), 7.7 (site 3), and 9 (site 4) kb. (The 9-kb and larger sites are seen more clearly in Fig. 3.) This blot also contained a number of weakly hypersensitive sites at 1.8 to 2.0 kb in all tissues, at 2.9 to 4.5 kb in all erythroid samples, and at 6.7 kb in brain cells only. In Fig. 3, the region overlapping the 3' end of that shown in Fig. 2 is presented. Probe C was used to detect an EcoRI restriction fragment beginning at 2.1 map units and going rightward. At 1.6 to 1.9 kb was a weak site seen only in brain tissue. At 1.9 to 2.1 kb (site 4) there was a strong site present in 5-, 10-, and 15-day embryonic erythroid cells. This site was present but weaker in adult erythrocytes. Hypersensitivity at 1.9 to 2.1 kb was not observed in DNA from 16-day embryonic brain tissue, MSB cells, or free DNA. Thus the site at 1.9 to 2.0 kb is erythroid cell specific and does not correlate with the expression of any single gene. The band at 2.1 kb is probably a contaminant, as its appearance was sporadic, and it was occasionally found in the marker lanes. At 2.9 to 3.3 kb (site 5), a strongly hypersensitive site was present in 5-day embryonic erythroid cells. It was much weaker in 10-day embryonic erythroid cells and was not seen in any other sample tested. This site is the region of the p-globin promoter. At 4.1 to 4.8 kb (site 6), a hypersensitive region was found with the same developmental and tissue specificity as the site at 2.9 to 3.3 kb. (With 10-day embryonic erythrocytes, a longer exposure was needed to see this site.) Another site, present only in brain tissue and adult erythrocytes, was at 4.8 to 6.0 kb. At 7.2 to 7.7 kb (site 7) was a site present only in 10- and 15-day embryonic erythroid cells. The 7.2 to 7.7 kb site is the p3H-globin promoter. The DNase I cleavage pattern near the pA and E genes is shown in Fig. 4. This blot showed hypersensitivity at 1.8 to 2.2 kb (site 11), the E-globin promoter, in 5- and 10-day erythroid cells. A weak site at 3.0 to 3.4 kb was seen in brain and the erythroid cell samples. Nearby is the pA/E enhancer (site 10), which was hypersensitive in all erythroid nuclei. Interestingly, in the brain sample, site 10 (or a region just 3' to it) was present, albeit faint. At 5.2 to 5.8 kb is the pA promoter (site 9), hypersensitive only in 10-day, 15-day, and adult erythroid cells. Visible as a smear at the top of site 9 was a weaker site (site 8), 3' of pH. This site, present in 10-day, 15-day, and adult cells, was seen more clearly on higher-resolution blots (29). Although too large for accurate

VOL. 10, 1990

M

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TOPOISOMERASE II AND DNase I HYPERSENSITIVITY

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0 0.87- v FIG. 2. DNase I-hypersensitive sites at the 5' end of the ,-globin locus. DNA from DNase I-treated nuclei was digested with KpnI and detected with probe B. The samples are labeled at the top, with the arrows showing the direction of increasing DNase I digestion. Lanes marked M contain size standards; sizes (in kilobases) are indicated at the left. Between the two blots is a diagram of the locus showing the approximate position of the p-globin gene, sites 1, 2, 3, and 4, and a brain tissue-specific site. DNase I digestions were performed as described in the text, with the following changes: 5-day embryonic erythrocytes (Sd RBC), extra sample at 10 ,ug of DNase I per ml; 10-day embryonic erythrocytes (lOd RBC), extra sample at 0.02 ,ug/ml; 15-day embryonic erythrocytes (15d RBC), DNA at 50 A260 units/ml; MSB cells, DNA at 6.9 A260 units/ml; and free DNA (at 2 mg/ml), DNase I at 0, 0.1, 0.3, 1, and 3 ng/ml. Ad RBC, Adult erythrocytes. sizing on this blot, site 7, the 1H promoter, was clearly visible in 10- and 15-day but not adult erythroid cells. It should be noted that quantification of the degree of sensitivity of a hypersensitive site is subjective. The more distant a hypersensitive site is from the probe (and thus the larger the resulting fragments), the more compact, and therefore intense, the band on the blot will be. At higher DNase I concentrations, at which each fragment is likely to have been cleaved at more than one hypersensitive site, only the cleavage nearest to the probe will be detected. In addition, how the nuclei are prepared determines the observed, apparent DNase I sensitivity (see Materials and Methods). Thus, caution should be used in the quantitative interpretation of hypersensitivity data, especially between series of digestions. A complete tabulation of the DNase I-hypersensitive sites in 65 kb of the chicken ,-globin locus is shown in Table 1. The data are from blots such as those shown in Fig. 2, 3, and 4, with probes and restriction enzymes as shown in Fig. 1. We report the weakly hypersensitive sites in the hope that their identification will prove valuable in future studies of this locus. Table 1 gives the location of the DNase I sites in map units (Fig. 1) and by the size of the fragment on the blot. The latter measure gives an estimate of the uncertainty of the measurement (about 10% of the size of the fragment) and is independent of possible mapping errors. Twelve of the hypersensitive sites were defined as strong (usually because they were visible in the least-digested samples) and were clustered in 25 kb near the transcription units. Figure 8 (middle) is a map of the locus showing the

strongly hypersensitive sites. Possible functions for these sites are discussed below. Localizing teniposide-induced cleavage sites in the ,I-globin locus. We used teniposide to map topo II sites in intact cells. This drug traps a putative intermediate in the topo II catalytic cycle, in which the DNA is cut and covalently bound to topo 11 (6). Unlike other drugs used to trap topo II complexes, teniposide neither intercalates into nor binds to DNA (35). After denaturation and proteolytic removal of the topo II, the breaks in the DNA are mapped, as was done for the DNase I cleavages. Figure 5 shows the teniposide-induced cuts near the p-globin gene. At the DNA position corresponding to DNase I-hypersensitive site 4, teniposide cuts were found in 5-, 10-, and 15-day embryonic erythroid cells, but not in adult erythrocytes, brain, or MSB cells. More teniposide cuts were found in the 5-day embryonic erythroid cells at hypersensitive sites 5 and 6 (5' and 3' of p, respectively). None of the other tissues showed significant cleavage at sites 5 and 6. These data present a remarkable correlation between the location of DNase I-hypersensitive sites and teniposideinduced DNA cleavage. At three regions (hypersensitive sites 4, 5, and 6) in five tissues (5-, 10-, and 15-day erythroid, brain, and MSB cells), there was exact correspondence between DNase I hypersensitivity and teniposide-induced cutting. The lack of teniposide-induced cleavage in adult chicken erythrocytes has been observed previously (41) and may be explained by the lack of immunoreactive topo II in these cells (26). To examine the generality of these observations, we

REITMAN AND FELSENFELD

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FIG. 4. DNase I-hypersensitive sites of the P-globin locus near the PA_ and e-globin genes. DNA from DNase I-treated nuclei was digested with XbaI, electrophoresed, blotted, and detected with probe H. The diagram between the two blots shows the positions of the pA, and pH genes and sites 11, 10, and 9. Other labels and experimental details are as described in the legend to Fig. 2. e,

TOPOISOMERASE II AND DNase I HYPERSENSITIVITY

VOL. 10, 1990

TABLE 1. Sites of increased DNase I sensitivity in the chicken Region Probe

A

Enzyme

Base pairs Sd Map units

4800-5200 4000-4300 3400-3700 2800-3000

KpnI KpnI

2200-2400 1450-1750

-12.0 to -12.4 -11.2 to-11.5 -10.6 to -10.9 -10.0 to -10.2 -9.4 to -9.6 -8.7 to -9.0

HindIll KpnI KpnI KpnI

2000-2600 1050-1300 1850-2050 2950-4650

-6.8 to -7.4 -4.9 to-4.6 -4.1 to -3.9 -3.0 to -1.3

BamHI

3100-3400

BamHI

1600-1800 1000-1300 1600-1850 1850-2050 2900-3250 4100-4750 4750-5100 5100-5500

-0.6 to -1.0 0.7 to 0.9 1.2to 1.5 3.7 to 4.0 4.0 to 4.2 4.9 to 5.4 6.2 to 6.9 6.9 to 7.2 7.2 to 7.6

KpnI KpnI

5900-6200 4100-4300 2900-3300 1050-1350

10.4 to 12.2 to 13.4to 15.3 to

H

XbaI

3050-3400

16.5 to 16.1

G

BamHI BamHI BamHI

750-1000 2100-3000 3250-3800

BamHI

4050-4700 5250-5900

17.4 to 18.8 to 19.9 to 20.7 to 21.9 to 23.1 to

C

BamHI EcoRI EcoRI EcoRI EcoRI EcoRI EcoRI F

Kpnle

Kpnlf

BamHI BamHI

64507500

P-globin locusa

DNase I hypersensitivityc

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B

2779

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17.7 19.7 20.5 21.4 22.6 24.2

SiteEytoicel Free Brain MSB no.b no. Free Erythroid cellsS DNA cells 5-day 15-day Adult tissue 10-day ? -

1

2 3 4 5 6

7 8 9 10

11 12

-

+ +

+ + + +

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+? +? +? +? +

+ + +? -? +

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+?

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+

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+ Hindlll 4700-5400 ? 28.6 to 29.3 + +? +? ? a Each row shows data for one site where increased DNase I sensitivity was found. The probe (see text) and restriction enzyme that most accurately located the site are listed. b Only strongly hypersensitive sites are numbered. c Symbols: +++, strong site, usually seen in even the least-digested samples; + +, moderate site; +, weak site, definitely present but usually detected only in highly digested samples; +?, weak site probably present; -?, weak site probably not present; ?, unable to determine whether a weak site is present or not. d Distance from the probe end of the restriction fragment to the region of increased DNase I digestion. In some blots this site coincided with a contaminant band. f Based also on data from reference 29.

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mapped teniposide-induced cleavages over 65 kb of the P-globin cluster (using the same strategy as shown in Fig. 1 for DNase I mapping) in the six tissues mentioned above. All the strong teniposide-mediated cleavage sites coincided with DNase I-hypersensitive sites. These data are summarized in Fig. 8 (bottom). Again, no cleavage was observed in the adult erythrocytes. We also observed that actively transcribed chromatin shows a slight increase in teniposide-induced cleavage compared with nontranscribed DNA. The transcribed region of the p-globin gene (between sites 5 and 6 in 5-day cells) showed slightly more teniposide-induced cleavage than the surrounding DNA, which was neither transcribed nor hypersensitive (Fig. 5). This is more clearly visible in Fig. 6, which shows that teniposide treatment of 5-day erythroid cells produced strong cleavage at the 5' and 3' ends of the e-globin gene (within DNase I-hypersensitive sites 11 and 12), but also weaker cleavage within the gene. Increased teniposide-

mediated cleavage was also observed during transcription of the pH and pA genes (data not shown). The shape of teniposide-induced and DNase I-hypersensitive bands was often slightly different. For example, site 6 (Fig. 5) was both broader and more intense with teniposide than with DNase I. (Note that these comparisons should only be made under conditions where the digestions obey single-hit kinetics, such as in the first four DNase I lanes of the 5-day sample, or any of the teniposide lanes.) Teniposide-induced cleavage of the 12-tubulin gene. To test further the rules governing topo II cleavage sites, we chose the chicken P2-tubulin gene. This gene is transcribed in brain and MSB cells but not in erythroblasts from adult chickens (42, 51). The promoter is DNase I hypersensitive in MSB cells (51). As a reference, we first determined the developmental pattern of DNase I hypersensitivity of the P2-tubulin gene. Figure 7 (left) shows a single hypersensitive site at -3.1 kb in all tissues tested (5-day, 10-day, 15-day, and adult

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Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5...
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