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Nuclease-Hypersensitive Regions in Chromosomes (Active Genes/Nuclease-Hypersensitive!Cleavage) M

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Nuclease-Hypersensitive Regions in Chromosomes (Active Genes/Nuclease-Hypersensitive!Cleavage) M Proc. Nati. Acad. Sci. USA Vol. 82, pp. 854-858, February 1985 Genetics Nick-translation of metaphase chromosomes: In vitro labeling of nuclease-hypersensitive regions in chromosomes (active genes/nuclease-hypersensitive!cleavage) M. TIEN Kuo* AND WILLIAM PLUNKETTt *Department of Pathology and tChemotherapy Research, The University of Texas, Houston, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030 Communicated by J. Herbert Taylor, October 4, 1984 ABSTRACT Chinese hamster metaphase chromosomes gions is also preferentially sensitive to single-stranded spe- were labeled by nick-translation, which involved pretreatment cific nuclease S1 (19, 20). However, whether there is single- of metaphase chromosomes with low levels of DNase I followed stranded DNA in these regions has not been settled. by incubation with DNA polymerase I and radioactively la- The present study was initiated to characterize the nature beled nucleotides. The labeled DNA was located on nuclease- of DNase-hypersensitive regions. We employed nick-trans- hypersensitive regions of the chromosomes, as suggested by lation to label metaphase chromosomes. Our data strongly the following observations. (i) The labeled DNA was hypersen- suggest that the nick-translation preferentially labels the sitive to the subsequent DNase I digestion. (ii) The labeled DNase-hypersensitive regions on chromosomes. Biochemi- DNA contained no nucleosomes. DNA reassociation kinetic cal characterizations of the hypersensitive regions are also analysis suggested that the labeled DNA was enriched in repet- presented. itive DNA sequences. Base composition analyses showed that the labeled DNA was highly enriched in guanine and adenine residues, suggesting that the nick-translation reaction was MATERIALS AND METHODS asymmetrical and the strand enriched in purine was preferen- Cell Culture, Cell Synchronization, and Preparation of tially translated. Autoradiographic analysis revealed that the Chromosomes. Chinese hamster ovary and male Chinese label was distributed on every chromosome, but there was a hamster fibroblast cells (Don line) were used for this study. lower grain density on the Y chromosome, which is hetero- Cells were grown in Dulbecco's modified Eagle's medium chromatic and exhibits a relatively low level of gene activity. supplied with 10% fetal calf serum. The locations of silver grains on the Y chromosomes were gen- Synchronization of cells was achieved by thymidine (7 erally consistent with that revealed by the in situ hybridization mM, 16 hr) block, released for 5 hr, followed by mitotic ar- using [3H]cDNA synthesized from the total Chinese hamster rest by Colcemid (0.04 gg/ml, 2 hr). The mitotic cells were messenger RNA. These observations suggest that a specific then gently shaken off. The harvested cells contained 98- subset of genomic DNA on active chromatin is the preferred 99% mitotic cells as monitored by phase-contrast microsco- site of the nick-translation. PY. Preparation of chromosomes were essentially the method It has been well established that active chromatin or poten- described previously (21) using chromosome solution de- tially active chromatin is preferentially sensitive to the diges- scribed by Blumenthal et al. (22). tion by nucleases (see reviews in refs. 1-3). In addition to Nick-Translation of Isolated Chromosomes. The isolated this general nuclease sensitivity in the transcribed regions, chromosomes were washed in DNase I-digesting buffer con- there are chromosome segments that are =10 times more taining 10 mM Tris HCl (pH 7.5), 10 mM NaCl, and 3 mM readily digested by the nucleases than the transcribed chro- MgCl2 and then were resuspended in the same buffer at A260 matin domain (4-6). These DNase-hypersensitive regions - ==0.5. The chromosomes were pretreated with DNase I have been determined almost exclusively by the indirect (0.2-1.0 ,ug/ml) for 10 min at 37°C. The pretreated chromo- end-labeling method (6), which employs a short, radioactive- somes were washed and resuspended in a nick-translation ly labeled probe to hybridize with the restricted DNA frag- buffer that contained 10 mM Tris HCl (pH 7.5), 5 mM ment isolated from low-level nuclease-digested nuclei. The MgCl2, 5 mM 2-mercaptoethanol, 20 ,uCi (1 Ci = 37 GBq) of nuclease-hypersensitive cleavage displays a subband on the [32P]dCTP (50 Ci/mmol, Amersham), 0.3 mg each of unla- autoradiograph with one end defined by the restriction en- beled dATP, dGTP, and dTTP per ml, and 70 units ofEsche- zyme site and the other by the DNase-hypersensitive site. richia coli DNA polymerase I. Three different DNA pQly- By using this method, DNase I-hypersensitive sites have merases (from Boehringer Mannheim) were used: DNA been assigned to certain regulatory sequences such as long polymerase I (Kornberg polymerase, nick-translation terminal repeats of transcribed endogenous provirus (7) and grade), endonUclease-free DNA polyrnerase I, and large the 72-base-pair enhancer sequences of simian virus 40 (8- fragment of DNA polymerase I (Klenow enzyme). The reac- 10). A number of hypersensitive cleavage sites have been tion mixture (0.5 ml) was incubated at 14°C for 2-20 min, and assigned to the 5' side of the genes (11-13), some, but less the reaction was stopped by addition of EDTA (0.2 M) to a frequently (14, 15), at intervening gene sequences. final concentration of 10 mM. The nature of DNase-hypersensitive regions has not been Nick-Translation of Chromosomes in Mitotic Cells. The well characterized. It has been suggested, from restriction harvested mitotic cells were permeabilized with L-a-lyso- enzyme digestion (16, 17), electron microscopic observa- phosphatidylcholine (type 1, Sigma) according to the method tions (10), and "histone image" analysis (18) that these chro- described by Miller et al. (23). Cells were treated with hypo- matin stretches contain no histone. The DNA in these re- tonic solution (1:5 dilution of the regular medium with dis- tilled H20) at 22°C for 15 min, spun down, and resuspended The publication costs of this article were defrayed in part by page charge in a buffer containing 10 mM Tris HCl (pH 7.5), 10 mM payment. This article must therefore be hereby marked "advertisement" NaCl, 3 mM MgCl2, 0.5% Nonidet P-40 (NP-40), and 0.5% in accordance with 18 U.S.C. §1734 solely to indicate this fact. Triton X-100 at 4°C. After 30 min, the cells were washed two 854 Downloaded by guest on September 28, 2021 Genetics: Kuo and Plunkett Proc. Natl. Acad. Sci. USA 82 (1985) 855 times with the same buffer lacking NP-40 and Triton X-100 coccal nuclease digestion was somewhat different from that and were resuspended in the nick-translation buffer as de- of the DNase I digestion, it was also evident that the labeled scribed above containing 100 ,Ci of [3H]TTP (117 Ci/mmol, DNA was also highly preferentially sensitive to the micro- New England Nuclear) instead of [32P]dCTP. The reaction coccal nuclease digestion (not shown). Therefore, we con- was carried out at 14'C for 1 hr and stopped by the addition clude that the nick-translated DNA in chromosomes is hy- of EDTA (0.2 M) to a final concentration of 10 mM. Cells persensitive to the nuclease digestion. were washed twice with the nick-translation buffer, fixed We next investigated whether the nick-translated DNA with methanol/acetic acid, 3:1 (vol/vol), and used for air- contained a typical nucleosome structure. The nick-translat- dried preparation of metaphase chromosomes. ed chromosomes were digested with micrococcal nuclease, Metaphase chromosomes on glass slides were rinsed ex- which preferentially cuts the linker region of a nucleosome tensively with 0.3 M sodium chloride/0.03 M sodium citrate, array. DNA was extracted from the digested chromosomes, pH 7, followed by washes with 75% ethanol and 95% etha- separated by agarose gel electrophoresis, stained with ethidi- nol. Autoradiographs were prepared with Kodak thin film um bromide, visualized under UV light, and followed by AR-10 and developed in D-19B. The film was usually ex- autoradiography. A comparison between the autoradiogram posed for 1-2 weeks. The chromosomes were stained with (Fig. 1B) and the ethidium bromide-stained DNA pattern of Giemsa (10% prepared in 0.01 M sodium phosphate at pH the total genomic DNA (Fig. 1A) clearly shows that the nick- 6.8). translated DNA was much more sensitive to nuclease diges- Other Procedures. For DNA base composition determina- tion. Furthermore, no typical nucleosomal DNA repeat was tion, four labeled deoxyribonucleotides were used in the shown on the autoradiogram, whereas the ethidium bromide- nick-translation reaction. DNA (20-50 ,ug) was degraded stained pattern of total genomic DNA showed typical nu- with P1 nuclease and alkaline phosphatase (24). The result- cleosome repeats. These results suggest that the nick-trans- ing nucleosides were separated by reversed phase HPLC lated DNA does not contain a typical nucleosomal structure. (25). The retention times were 9.3 min for dCyd, 23 min for To determine whether the nick-translated DNA is a unique dThd, 25 min for dGuo, and 34.4 min for dAdo. Radioactiv- subset or a random representative of the total genomic ity associated with the eluents of each nucleoside was deter- DNA, we performed a DNA reassociation experiment. DNA mined by liquid scintillation spectroscopy. isolated from nick-translated chromosomes was mixed with Methods for digestion of nuclei with micrococcal nucle- unlabeled genomic DNA, sheared by sonication, denatured, ase, extraction of DNA, agarose gel electrophoresis of nu- and allowed to reassociate. The reassociated DNA was sepa- cleosomal DNA, DNA reassociation (26), and in situ hybrid- rated from the unreassociated DNA by hydroxyapatite col- ization of [3H]cDNA synthesized from total poly(A)+ RNA umn chromatography. Fig. 2 shows that the nick-translated (27) have been described.
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