Hypomethylation of human heterochromatin detected by restriction enzyme nick translation

§HORT NOT omethylation of Human HeterQchrQm~ti by Restriction Enzyme Nick Tran~~~ti A.R. MRC

f-fwnun

Genetics

Unit,

Western

General

MITCHELL

Hospital,

Using the restrmtion enzymes MspI and HpaII in the nick translation procedure it has been shown that decondensation of the paracentric heterochromatin of chromosome 9 during human spermatogenesis is associated with bypomethylation of the DNA sequences in this domain. Somatic cells treated with S’azaeytidine also showed de~o~~ensation of eentromerk heterochromatin. Irr this instance, bowever, hypomet~ylation is detected both in’the extended heterochromatin at the centromeres aml in the euehromatin of the chromosome arms. cz 1992 Aoademic I%ess, Inc.

It is now generally accepted that the enzyme DNase I preferentially cleaves tra~scriptionally active regions of ~hromatin [l]. ‘Ihis property of DNase I can be extended to meta~hase chromosomes conventionally fixed by methanol-acetic acid and spread on microscope slides [Z-4]. The term D-bands was proposed by Kerem et &. ]5] to describe the type of banding pattern produced by DNase I-sensitive regions along the length of Chinese hamster chromosomes. Using this approach it should be possible to demonstrate the existence of domains within chromatin which are DNase I sensitive and therefore potentially transcriptionally active. In a similar approach restriction endonucleases have been used to study the distribution of repetitive DNA sequences in methanol-acetic acid-fixed chromosomes from a variety of species. Liea and Hamkalo [6] and Kaelbling et &. [7] used ~fferent restriction endonucleases on chromosomes to produce different banding patterns according to the enzymes used. For example, HoeIII, AZd, and ZTinfI produced a C-type pattern whereas AuuII gave G-banded types of chromosomes [7]. Similar types of studies have been carried on human [8] and Indian Muntjac cells [9]. Recently, Ferraro and I’rantera [IO] combined both approaches, i.e., the use of DNase I and restriction endonucleases, to study the hu-

Edinburgh

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Scotbd,

United

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man ribosomal gene clusters. They found a positive correlation between ribosomal gene activity using silver staining, the presence of DNase I-sensitive domains, and cleavage by the restriction e~do~u~~@aseZ&XI. An approach similar to that of Ferraro and Pramera [lo] has been adopted to study ihe ~h~~rn~ti~ conformation of the ~ent~orneri~ C-band some 9 in human s~ermatoc~es~ A the secondary constriction of this ~br~rn~sQrn~ shows pronounced stretching [II]. revkusly we demcmstrated [XX?]that this region co isted solely of a simI3le repetitive DNA sequence and s~g~~ate~ that unciermethylation of the DNA sequences within the constitutive heterochromatin might be res~~~a~~~e for the stretching or unwinding of what is ~~~rn~ll~ a tightly condensed region of chromatin. e present report addresses this particular question.

Meiotti metaphases, Air-dried metaphases were prepared according to the technique of Evans et ai. 1131~ Conventional staining was carried out using Giemsa in Gurr’s huger. Mitotic metaphases. To obtain human lympbacyte cultures with undercondensed heterochromatin the procedure of S&mid et &. [l4] was carried out. Briefly, 1Q ml of peripheral blood from a healthy male was cultured for 72 h. Six hours before harvesting 5-azacytidine was added to a final concentration of 5 X 10e7 &liter and, 1 h before harvesting, colcemid was added to a fmal co~ce~trat~o~ of 0.1 ~&ml. The ceils were harvested and, after 15 min in monotonic KCh metaphase chromosome spreads were prepared by nxation using 31 rnethanol-acetic acid” Control cultures were set up in the same way witbout the 5’-azacytidine. In Situ

&Tick Translation

und Autoradiography

Nick translation was carried out basically as describedby Ke~era & uZ. [5]. Slides containing the fixed cells were incubated for 1Q min at l5’C with a 50-~1 solution of50 mMTris-HCl (pH 7.9), 5 m.MIVlgC18, 10 m&f 2-mercaptoethanol, 50 @g/ml bovine serum a!burnin, 10 U/ml DNA polymerase I, 1 ng/rnl pancreatic DNase I, and 4 j&f dATP, dCTP, and dGTP plus 0.3 PM [%]dTTP (57 Ci/rnmok Arnersbam Inc.). The reaction was terminated by washing (2X) in ice-cold 2X

204

SHOR

NOTE

SSCP, pH 7.40 [SSCP is 0.15 M NaCl, 0.015 M trisodium citrate, 1 rnM EDTA (disodium salt)] followed by dehydration through 70:90 and 100% absolute alcohol. After air drying, the slides were placed in ice-cold 5% TCA for 5 min and finally dehydrated through the alcohol series as before. Endonuclease

in Situ

Nick

Translation

The enzymes used were &&JI and its isoschizomer &aII. Slides containing the fixed cells were covered by 50 ~1 of the manufacturers recommended restriction enzyme buffer plus 20 U of either &J.spI or HpaII. Coverslips were sealed and the slides were incubated for 18 h at 37’C in a moist chamber. A limit digest was therefore obtained. Following this the slides were washed briefly in 10 miV Tris-HCl, pH 7.4, before undergoing the nick translation procedure as described above in the absence of the DNase I enzyme. Control slides for the DNase I in situ nick translation procedure were incubated as above in the absence of the enzyme DNase I. For the restriction endonuclease experiments control slides were incubated as described for the experimental slides in the absence of the restriction endonucleases. Autoradiographs were prepared using Ilford L4 liquid emulsion and the slides were exposed for Z-3 days at 4’C before developing in Kodak Dl9 developer followed by Giemsa staining. Grain

Counting

over Meiotic

and Mitotic

Metaphases

In the meiotic divisions only well-spread metaphases with clearly identifiable chromosomes were scored in the M2 divisions. Grains were scored only where the whole grain was within the heterochromatin domain. In many somatic cells chromosomes 1 and 16 could easily be identified with similar decondensed regions but only chromosome 9 was included in this study (they are, however, noted in the relevant figures). As a check to ensure that the correct chromosome was being identified the original stain was removed and the slides were restained through the film using methyl green and DAPI as the fluorochrome [ 151.

RESULTS

Human chromosome 9 (large arrow in Fig. la) can be readily identified at metaphase M2 during male meiosis when the centromeric heterochromatic domain is extended (small arrow in Fig. la). A similar but less pronounced extension is also often observed at the Ml stage of spermatogenesis [12]. Figure lb illustrates the effect of growing peripheral blood in the presence of 5’-azacytidine (see Materials and Methods). In this example one chromosome 9 shows pronounced extension; the other is not so extended (large arrows in Fig. lb). In this figure the chromosomes have been stained with methyl green/DAPI [15] and the small arrows show the centromeric domains of chromosomes 1 and 16, the Y chromosome, and two unidentified acrocentric chromosomes. Control cultures do not demonstrate this phenomenom (Fig. lc). Figure 2a shows the results obtained when fixed M2 metaphases were digested with the endonuclease HpuII followed by incorporation of nucleoside triphosphates including tritium-labeled thymidine triphosphate as described (see Materials and Methods). The bivalent chromosome is labeled (large arrows in Fig. 2a). The centromeric heterochromatic domain is decondensed,

FIG. 1. (a) A C-banded M2 division during spermatogenesis with the large arrow showing human chromosome 9. The small arrow shows the decondensed centromeric chromatin. (b) Somatic metaphase from a 5’-azacytidine-treated cell stained with methyl green/ DAPI. The large arrows identify chromosome 9, and the small arrows show that chromosomes 1,15, and 16 and the Y chromosome can also be identified with this stain. (c) Control culture as in (b) without the addition of 5’-azacytidine.

showing pronounced stretching (small arrows in Fig. 2a). Grains can clearly be seen in both chromosome arms and in the decondensed centromeric heterochromatin. The chromosomes are stained with Geimsa. Fig-

SHORT

205

NOTE

in Table 1. Included in this table are the results obtaine ase I2 from both mitotic and meiotic material kng omoHpaII, and kl.spli to introduce nicks into the somal DNA~ With the meiotic material, the ratio of grains in the extended region within the centromeric domain to that in the rest of chrome sions) was significantly greater wit with MspI (t = 2.89; P = 0.02). DNase I as above, it was apparent at the MspI ratio of that in the cbromograins within the extended region some arms was also significantly greater than that found for DNase I (t = 2.4; P = OB2). The proportion of silver grains in the extended region to that in the cb mosome arms formed the decreasing sequence Hpe MspI-DNase I. The same order was f~un matic metaphases although in this instance the diEerences between the three treatments was not statistically significant.

Chromosome 9 is the only bivalent in man which its centro onstrates pronounced stretching within chromatin at metaphase although the sequences in this domain are not unique to this chromosome ]12? 161. The use of MspI and II combined with t tion reaction show that the repetitive present in the decondensed centromeric chromatic are undermethylated in comparison to the DNA in the chromosome arms. It has been shown that [IO] transcriptionally active human ribosoma hypomethylated and thus accessible the E&XXII endormclease enzyme. HQW bility is also correlated with increa nicking with DNase I. In the present case it is clear that the hypomethylated DNA sequences present in the condensed centromeric chromatin, although access to both MapI and &&I, remain relatively inaccessible FIG. 2. (a) M2 divisions labeled using I@aII to induce chromosomal nicks. Chromosomes 9 are shown (large arrows) with the decondensed chromatin (small arrow). Note the grains within the decondensed chromatin (b) A somatic metaphase showing decondensed centromeric cbromatin of chromosome 1 (large arrow) and chromosome 9 (small arrow). Nicks were introduced with A4spI. Mitotic

ure 2b is an example of a metaphase from cultured peripheral blood cells grown in the presence of 5’-azacytidine followed by digestion with the restriction endonuclease MspI and labeling as described for Fig. 2a. Both chromosomes 1 and 9 have pronounced decondensation of their centromeric chromatin (large and small arrows, respectively, in Fig. 2b). The label is apparent in both these regions as well as in the chromosome arms. The results from these experiments are summarized

Control DNasel

HpCLU iv&I Meiotic

Control DNasel

Hpd lapI Note. A = No. of grains “stretched” heterocbromatin

A

B

c = BfA

D (=SE

2 251 172 324

Q 41 41 71

0 Q.163 0.238 0.219

Q 0.023 O.Q32 0.022

3 174 214 415

0 18 .55 73

0 0.130 Q.254 Q. 176

0 0.023 0.029 0.018

on chromosome 9; B = No. of grains of chromosome 9.

in

C)

the

206

SHORT

to DNase I (see column C, Table 1). The implication is that these DNase I-insensitive regions are transcriptionally inactive. In contrast to the meiotic results, growing lymphocytes in the presence of 5’-azacytidine apparently leads to an undermethylation of the DNA sequences in both the decondensed centromeric chromatin and the DNA in the euchromatic arms of chromosome 9. Surprisingly, the decondensed region of this chromosome was sensitive to DNase I digestion. Chromatin configuration does appear to play an important role in the accessibility or otherwise of the underlying DNA in chromatin to nicking by DNase I. For example, the C-band heterochromatin of the X chromosome of Microtus agrestis [4] was found to be DNase I sensitive irrespective of the transcriptional activity of the X chromosome under study, and the heterochromatic region on the long arm of the human Y chromosome, a region thought to be devoid of active genes but enriched in simple repetitive DNA sequences [ 17,18], was also found to be sensitive to DNase I. Using 5’-aza-2’-deoxycytidine Michalowsky and Jones [19] have shown that extensive demethylation of chromatin does lead to an alteration in chromatin configuration with the consequence that the demethylated chromatin is accessible to digestion with DNase I. They also showed that under these conditions DNase I accessibility does not necessarily mean that the chromatin is transcriptionally active. It is interesting to note that a similar phenomenon of heterochromatic undercondensation has been observed in chorionic villus samples and in human sperm genomes [20, 211. It is known that, in Drosophila, gene inactivation can occur by the “spreading effect” caused by the near proximity of large blocks of heterochromatin. Perhaps the observation that undercondensation of large heterochromatic blocks at specific times during meiosis and early embryo development implies the activation of genes which under normal circumstances are inactivated by the suppressive effect exerted by the nor-

NOTE

mally highly condensed these chromosomes.

heterochromatic

blocks

on

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