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Site and stage specific action of endogenous nuclease and micrococcal nuclease on the histone genes of sea urchin embryos
DEVELOPMENTAL
BIOLOGY
(1986)
117,109-113
Site and Stage Specific Action of Endogenous Nuclease and Micrococcal Nuclease on the Histone Genes of Sea Urchin Embryos’ OLIN D. ANDERSON,' MEI-MINYU,~ Department Received
of Zoology, October
The early histone genes of sea urchin chromatin containing these genes was produces 1300-bp segments containing sites close to the cap site for Hl, H2A, are not expressed, do not display this contain histone genes when used with
University
of California,
II, 1985; accepted
in revised
AND Berkeley, form
FREDWILT~ California March
94720
31, 19%
embryos are expressed exclusively during cleavage stages of embryogenesis. The examined by nuclease sensitivity. An endogenous nuclease, active during cleavage, early histone genes. The cutting sites have been mapped; there are very sensitive H2B, and H4. Chromatin obtained from embryos of later stages, when the genes pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that nuclei from later stages, but not with nuclei from cleavage stages. Q 1986 Academic
Press, Inc.
INTRODUCTION
The sea urchin histone genes comprise a large and complex family of genes whose expression has been particularly well studied (reviewed by Maxson et al., 1983). During the first 24 hr of development, there is a sequential modulation in the activity of three main groups of histone genes. The “early” gene set is comprised of hundreds of tandem repeating units of the five histone genes, and is apparently active only from early cleavage until midblastula, with maximal transcription at the 12%cell stage (Maxson and Wilt, 1981; Mauron et al, 1982). These characteristics offer an attractive system for the study of gene activity and changes in chromatin structure. Bryan et al. (1983) found changes in sensitivity to micrococcal nuclease and DNase I at specific sequences in histone genes during early development of Psammechinus miliaris. Wu and Simpson (1985) also found DNase-hypersensitive sites just upstream from some histone genes in Strongylocentrotus purpuratus during a period of transcriptional activity. Spinelli et al. (1982) examined the histone DNA pattern of micrococcal nuclease-treated nuclei of sea urchin embryos; they report ’ Research was supported by research grants from the National Science Foundation and National Institutes of Health (to F.W.), a postdoctoral fellowship of the National Institutes of Health (to O.D.A.), and the visiting scholar’s program of the People’s Republic of China (to M.-M.Y.). ’ Present address: Western Regional Research Laboratories, U.S. Dept. Agriculture, Albany, Calif. 94706. 3 Present address: Dept. Biology, Peking University, Beijing, People’s Republic of China. 4 To whom reprint requests should be addressed.
a developmental change in nucleosomal packaging of the early histone repeats. We have examined the chromatin structure of the early histone genes during development of S. purpuratus, utilizing digestions with both micrococcal nuclease and an endogenous sea urchin nuclease activity. The endogenous nuclease activity is especially prominent in cleavage stage nuclei (100-200 cells), and it cuts the early histone repeats in the chromatin just upstream of each cap site. There is also a temporal relationship between the transcriptional activity of the early histone gene family and their association with nucleosomes. The results further establish that alterations in chromatin conformation accompany changes in expression of the early histone genes. METHODS
Embryos of locally collected S. prpuratus were raised at 15°C by conventional methods (Hinegardner, 1967), and nuclei were isolated by the methods of Hinegardner (1967) and Shaw et al. (1981). Nuclease digestions were carried out in 50 mMTris, pH 7.9, 80 mMNaC1, 1 mM CaC12,2 mM MgClz, 0.5 mM dithiothreitol, 250 mM sucrose. Isolation of DNA, its electrophoresis in agarose gels in Tris-acetate-EDTA, blotting, and detection with nick-translated probes were all carried out by standard methods (Maniatis et ab, 1982). Subclones of the early histone genes were isolated from clones pSpll’7 and pSplO2 obtained from Professor Laurence Kedes, whom we thank. A map of the early histone gene repeat showing the position of relevant restriction sites and the subcloned probes used in the experiments is shown in Fig. 1.
109
OOlZ-1606/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
110
DEVELOPMENTAL BIOLOGY
-
VOLUME 117,1986
TRANSCRIPTION
2 R llR2i-l
21
4
R
i-lml
IH2bl
I 8
2
I
l-m
44
2 t
Hl
1 I
!
II
2 I
PROBE 2
R
H
Hh
B
4
I
PROBE 1
FIG. 1. A diagram of the major early histone gene repeat of S. ~~~puratus is redrawn from the data of Kedes (cf. Maxson et al., 1983). Restriction endonuclease sites are designated: R, EcoRI; B, BamHl; H, HaeIII; Hh, Hha. The probes 1 and 2 used in this study are shown by the thick black lines. Probe 2 extends from the indicated Hh to R sites, but since the samples are digested with BamHl only the portion of the probe from Hh to B is effective in mapping. The arrows show the preferred cutting sites of endogenous nuciease derived from data in Fig. 4. The numbers 1 and 2 on the arrows indicate the use of probes 1 and 2.
RESULTS
AND
DISCUSSION
If the chromatin bearing the early histone genes has somewhat different structures at various times during development, this might be revealed by the sensitivity to digestion by DNAses. Accordingly, nuclei were prepared from lOO-200-cell cleavage stage embryos, a time
B
0
I
4
15
4063
0
I
4
15
40
63
FIG. 2. Nuclei were isolated from embryos possessing about 160 cells/ embryo. The isolated nuclei were suspended in MN buffer and incubated at 26OC, and aliquots were removed at selected times as shown at the bottom of the figure. DNA was purified, subjected to electrophoresis on an agarose gel, and then stained with ethidium bromide (A). The DNA was blotted to nitrocellulose and hybridized with radioactive histone gene DNA with coding sequences and spacers for H2B, H4,and Hl (B).
when the early histone genes are actively transcribed (Maxson and Wilt, 1981), and the DNA was fractionated by electrophoresis. The DNA had been digested during the incubation by endogenous nucleases (Fig. 2A). The DNA was blotted to nitrocellulose and hybridized with a nick-translated early histone gene probe. Figure 2B shows prominent regions of hybridization arranged as rungs on a ladder. The higher rungs progressively disappear as the bulk DNA is degraded. The bands containing early histone genes appear to have a regular spacing pattern (Fig. 2B, 1310 bp). The estimates of spacing are necessarily inexact because the width of the histone gene fragments is sufficient to suggest there is some heterogeneity in the precise cutting sites. In addition, the histone repeats can be of varying lengths in different individuals (Overton and Weinberg, 19’78).Nonetheless, the data support the idea of a site or region specific nicking of early histone genes by an endogenous nuclease in cleavage nuclei. The distribution of about 1300 bases is approximately the distance of the different coding regions of the five histone genes from one another in the major early histone gene repeat suggesting a region of sensitivity associated with each gene of the repeat. To determine if such nicks in the chromatin existed in the living embryo, in situ, DNA was prepared from intact embryos without nuclear isolation and compared to DNA isolated from nuclei from the same culture and stage. Some degree of DNA nicking occurs during the isolation of both lOO-200-cell cleavage stage (Fig. 3) and 500-cell mesenchyme blastula stage nuclei (data not shown). The DNA isolated from lOO-200~cell nuclei displays the typical “ladder” array of histone gene sequences (Fig. 3B2). Nuclei from mesenchyme blastulae and gastrulae show limited degradation, and the DNA does not show a prominent “ladder” (data not shown).
ANDERSON, 1
2
Yu,
AND
WILT
3
Nucleuses and Histonc Germ
111
ences between the DNA obtained from the different embryo cultures. A diagram of the results is shown in Fig. 1. The other probe (2) allows mapping in the opposite orientation from probe 1 by using BamHI to restrict the DNA resulting from the endogenous nuclease digests. The result of mapping the same two nuclear digests with this probe is shown in Fig. 4B. Again, a sharp pattern of bands occurs, particularly just upstream of the H2A, Hl, and H4 genes (Fig. 1). Thus, there are nuclease sensitive regions upstream from H2a, H2b, H4, and Hl genes (the H3 gene was not mapped with these probes), with slight differences between the two cultures. This same kind of mapping experiment has been carried out on several additional cultures with similar results. We conclude that in nuclei of cleavage stages there are regions near the cap site of each of the early histone genes
I
II
II
I
B FIG. 3. Cleavage stage embryos were used to prepare nuclei, and DNA was extracted from an aliquot without further manipulation. Some embryos were also used to prepare total DNA. An aliquot of the nuclei was also subjected to digestion with micrococcal nuclease (see Fig. 5). Samples of total embryo DNA (l), nuclear DNA (Z), and micrococcal nuclease-digested nuclear DNA (3) were subjected to electrophoresis and stained (A) then blotted and probed with the entire early histone gene repeat (B).
Chromatin of mesenchyme blastula and gastrula nuclei does not undergo a progressive degradation during incubation in vitro (up to 4 hr; not shown). The apparent regular nicking of the DNA in cleavage stage nuclei suggested it might be possible to map the approximate sites susceptible to nuclease action. Accordingly, small probes were used for the indirect mapping procedure (Wu, 1980) (see Fig. 1). Figure 4 shows such an experiment from embryos from two different females in which the DNA from nuclei was allowed to undergo endogenous digestion and was then digested with EcoRI (A) prior to electrophoresis and blotting. The presence of bands show that preferred cutting sites have been utilized by the endogenous nuclease. In this experiment cutting occurs in regions just upstream from the H4- and HBb-coding sequences, with slight differ-
A FIG. 4. Nuclei were isolated from 2). The nuclear DNA was digested BamHl (panel B). Blotted DNA was A) or 2 (panel B) from two different
B cleavage stage embryos (see Fig. with either EcoRI (pane1 A) or hybridized with probes 1 (panel females (I, II).
112
DEVELOPMENTAL
that are especially nucleases. The Presence
sensitive
BIOLOGY
to the action of endogenous
of Nucleosomes
in Early
Histone
Genes
The presence and distribution of nucleosomes in the early histone gene tandem repeat was examined by micrococcal nuclease treatment of nuclei isolated from lOO200-cell cleavage and mesenchyme blastula stage embryos. In Fig. 5A the time course of digestion of mesenchyme blastula nuclei displays the characteristic nucleosome repeat “ladder” in an ethidium bromidestained gel, with a repeat spacing of approximately 220 bp. When the blot of this gel is probed with the sea urchin early histone genes, the same apparent pattern of 220-bp repeats is evident (Fig. 5B). Thus, in mesenthyme blastula embryos, a stage when the early histone genes are not transcribed, probably most of the early histone gene repeat family is packaged into nucleosomes similar to total DNA. The same type of analysis using cleavage nuclei is complicated by the activity of endogenous nuclease (s) described in the previous section. Since the level of endogenous nuclease varies considerably in different cultures of embryos, experiments were carried out on several batches of eggs, adding enough micrococcal nuclease to generate rapidly the typical nucleosomal ladder. The results from one experiment where the endogenous activity was relatively low is shown in Fig. 3. There is no evidence of packaging the early histone repeats into a regular nucleosomal array (Fig. 3B3) even though the ladder produced from bulk DNA shows prominent nu-
M
0
1
2
4.5
8
16 31
FIG. 5. Nuclei were isolated from the mesenchyme shows the time course of digestion from a gel stained repeat (panel B).
54
81
blastula stage with ethidium
VOLUME
117, 1986
cleosomes (Fig. 3A3). We conclude that the preponderance of active early histone genes are not adorned with nucleosomes during cleavage. This is consistent with the examination of chromatin from cleavage cells by electron microscopy (Busby and Bakken, 1980), and with the findings of Spinelli et al. (1982), but Bryan et al. (1983) stated that during cleavage most histone coding sequences are in micrococcal nuclease resistant structures, a conclusion different from our findings. This may be due to differences in species used or precise methods of analysis. Finally, it is interesting to inquire if nucleosomes associated with early histone gene repeats in gastrula embryos are specifically associated with any particular sequences of the repeat, a phenomenon termed “phasing.” In principle, nucleosome phasing might be assessed by digesting the DNA produced by micrococcal nuclease digestion of chromatin with EcoRI, followed by electrophoresis, blotting, and probing with probe 1. If nucleosomes are phased, a series of bands defined by the EcoRI site and the sites of micrococcal nuclease action will be generated. Such experiments were conducted and a ladder of bands spaced 200-300 bp apart was observed (data not shown). However, control experiments with purified cloned histone DNA revealed micrococcal nuclease had marked sequence specificity (cf. Reeves, 1984). The pattern of bands generated by micrococcal nuclease and EcoRI with purified histone DNA is very similar to the pattern produced with chromatin (data not shown). One likely interpretation is that nucleosomes are not phased, i.e., are arranged randomly over the repeat, which has the effect of exposing all portions of the repeat to enzyme
M
0
I
2
4.5
and digested up to 81 min, bromide. The blotted DNA
8
16
31
54
81
with 2 U/ml of micrococcal was probed with the entire
nuclease. Panel A early histone gene
ANDERSON,
Yu,
AND
WILT
attack with similar probability. We find no evidence using micrococcal nuclease for phasing of histone gene nucleosomes. In conclusion, when early histone genes are actively transcribed there are regions of marked nuclease sensitivity in the regions immediately upstream from the cap sites of the coding sequences. There is no apparent nucleosomal organization of these actively transcribed genes. As transcription ceases, the chromatin of the early histone genes becomes organized into conventional nucleosomes, and the core particles are probably randomly positioned with respect to particular DNA sequences. There is little or no site specificity of endogenous nuclease at these later stages. It is likely the cessation of transcription of the early histone genes and the changes in chromatin structure are causally linked.
Nucleases
BRYAN, P. N., OLAH, J., and BIRNSTIEL, M. L. (1983). Major changes in the 5’ and 3’ chromatin structure of sea urchin histone genes accompany their activation and inactivation in development. Cell 33, 843848. B~JSBY,S., and BAKKEN, A. H. (1980). Transcription in developing sea urchins: Electron microscopic analysis of cleavage, gastrula and prism stages. Chromosma 79, 85-104. HINEGARDNER, R. T. (1967). In “Methods in Developmental Biology”
Histone
113
Genes
(F. H. Wilt and N. K. Wessells, eds.), pp. 139-155. Crowell, New York. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular cloning.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. MAURON, A., KEDES, L. H., HOUGH-EVANS, B., and DAVIDSON, L. H. (1982). Accumulation of individual histone mRNA’s during embryogenesis of the sea urchin Strongylocentrotus purpuratus. Dev. Biol. 94,425-434.
MAXSON, R. E., and WILT, F. H. (1981). The rate of synthesis of histone mRNA during the development of sea urchin embryos. Dev. Biol. 83,380-386. MAXSON, R. E., COHN, R., KEDES, L., and MOHUN, T. (1983). Expression and organization of the histone genes. Annu. Rev. Genet. 17, 239277. OVERTON, G. C., and WEINBERG, E. S. (1978). Length and sequence heterogeneity of the histone gene repeat unit of the sea urchin S, purpuratus.
Cell
14,247-257.
REEVES, R. (1984). Transcriptionally phys.
REFERENCES
and
active chromatin. Biochim.
Biro
Acta 782,343-393.
SHAW, B. R., COGNETTI, G., SHOLES, W. N., and RICHARDS, R. G. (1981). Shift in nucleosome populations during embryogenesis: Microheterogeneity in nucleosomes during development of the sea urchin embryo. Biochemistry 20,4971-4978. SPINELLI, G., ALBANESE, I., ANELLO, L., CIACUO, M., and DILIEGRO, I. (1982). Chromatin structure of histone genes in sea urchin sperms and embryos. Nucleic Acids Res. 10, 7977-7991. Wrr, C. (1980). The 5’ends of Drosophila heat shock genes in chromatin are hypersensitive to DNAse I. Nature (Low&m) 286,854-860. Wu, T.-C., and SIMPSON,R. T. (1985). Transient alterations of the chromatin structure of sea urchin early histone genes during embryogenesis. Nucleic Acid. Res. 13, 6185-6197.
BIOLOGY
(1986)
117,109-113
Site and Stage Specific Action of Endogenous Nuclease and Micrococcal Nuclease on the Histone Genes of Sea Urchin Embryos’ OLIN D. ANDERSON,' MEI-MINYU,~ Department Received
of Zoology, October
The early histone genes of sea urchin chromatin containing these genes was produces 1300-bp segments containing sites close to the cap site for Hl, H2A, are not expressed, do not display this contain histone genes when used with
University
of California,
II, 1985; accepted
in revised
AND Berkeley, form
FREDWILT~ California March
94720
31, 19%
embryos are expressed exclusively during cleavage stages of embryogenesis. The examined by nuclease sensitivity. An endogenous nuclease, active during cleavage, early histone genes. The cutting sites have been mapped; there are very sensitive H2B, and H4. Chromatin obtained from embryos of later stages, when the genes pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that nuclei from later stages, but not with nuclei from cleavage stages. Q 1986 Academic
Press, Inc.
INTRODUCTION
The sea urchin histone genes comprise a large and complex family of genes whose expression has been particularly well studied (reviewed by Maxson et al., 1983). During the first 24 hr of development, there is a sequential modulation in the activity of three main groups of histone genes. The “early” gene set is comprised of hundreds of tandem repeating units of the five histone genes, and is apparently active only from early cleavage until midblastula, with maximal transcription at the 12%cell stage (Maxson and Wilt, 1981; Mauron et al, 1982). These characteristics offer an attractive system for the study of gene activity and changes in chromatin structure. Bryan et al. (1983) found changes in sensitivity to micrococcal nuclease and DNase I at specific sequences in histone genes during early development of Psammechinus miliaris. Wu and Simpson (1985) also found DNase-hypersensitive sites just upstream from some histone genes in Strongylocentrotus purpuratus during a period of transcriptional activity. Spinelli et al. (1982) examined the histone DNA pattern of micrococcal nuclease-treated nuclei of sea urchin embryos; they report ’ Research was supported by research grants from the National Science Foundation and National Institutes of Health (to F.W.), a postdoctoral fellowship of the National Institutes of Health (to O.D.A.), and the visiting scholar’s program of the People’s Republic of China (to M.-M.Y.). ’ Present address: Western Regional Research Laboratories, U.S. Dept. Agriculture, Albany, Calif. 94706. 3 Present address: Dept. Biology, Peking University, Beijing, People’s Republic of China. 4 To whom reprint requests should be addressed.
a developmental change in nucleosomal packaging of the early histone repeats. We have examined the chromatin structure of the early histone genes during development of S. purpuratus, utilizing digestions with both micrococcal nuclease and an endogenous sea urchin nuclease activity. The endogenous nuclease activity is especially prominent in cleavage stage nuclei (100-200 cells), and it cuts the early histone repeats in the chromatin just upstream of each cap site. There is also a temporal relationship between the transcriptional activity of the early histone gene family and their association with nucleosomes. The results further establish that alterations in chromatin conformation accompany changes in expression of the early histone genes. METHODS
Embryos of locally collected S. prpuratus were raised at 15°C by conventional methods (Hinegardner, 1967), and nuclei were isolated by the methods of Hinegardner (1967) and Shaw et al. (1981). Nuclease digestions were carried out in 50 mMTris, pH 7.9, 80 mMNaC1, 1 mM CaC12,2 mM MgClz, 0.5 mM dithiothreitol, 250 mM sucrose. Isolation of DNA, its electrophoresis in agarose gels in Tris-acetate-EDTA, blotting, and detection with nick-translated probes were all carried out by standard methods (Maniatis et ab, 1982). Subclones of the early histone genes were isolated from clones pSpll’7 and pSplO2 obtained from Professor Laurence Kedes, whom we thank. A map of the early histone gene repeat showing the position of relevant restriction sites and the subcloned probes used in the experiments is shown in Fig. 1.
109
OOlZ-1606/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
110
DEVELOPMENTAL BIOLOGY
-
VOLUME 117,1986
TRANSCRIPTION
2 R llR2i-l
21
4
R
i-lml
IH2bl
I 8
2
I
l-m
44
2 t
Hl
1 I
!
II
2 I
PROBE 2
R
H
Hh
B
4
I
PROBE 1
FIG. 1. A diagram of the major early histone gene repeat of S. ~~~puratus is redrawn from the data of Kedes (cf. Maxson et al., 1983). Restriction endonuclease sites are designated: R, EcoRI; B, BamHl; H, HaeIII; Hh, Hha. The probes 1 and 2 used in this study are shown by the thick black lines. Probe 2 extends from the indicated Hh to R sites, but since the samples are digested with BamHl only the portion of the probe from Hh to B is effective in mapping. The arrows show the preferred cutting sites of endogenous nuciease derived from data in Fig. 4. The numbers 1 and 2 on the arrows indicate the use of probes 1 and 2.
RESULTS
AND
DISCUSSION
If the chromatin bearing the early histone genes has somewhat different structures at various times during development, this might be revealed by the sensitivity to digestion by DNAses. Accordingly, nuclei were prepared from lOO-200-cell cleavage stage embryos, a time
B
0
I
4
15
4063
0
I
4
15
40
63
FIG. 2. Nuclei were isolated from embryos possessing about 160 cells/ embryo. The isolated nuclei were suspended in MN buffer and incubated at 26OC, and aliquots were removed at selected times as shown at the bottom of the figure. DNA was purified, subjected to electrophoresis on an agarose gel, and then stained with ethidium bromide (A). The DNA was blotted to nitrocellulose and hybridized with radioactive histone gene DNA with coding sequences and spacers for H2B, H4,and Hl (B).
when the early histone genes are actively transcribed (Maxson and Wilt, 1981), and the DNA was fractionated by electrophoresis. The DNA had been digested during the incubation by endogenous nucleases (Fig. 2A). The DNA was blotted to nitrocellulose and hybridized with a nick-translated early histone gene probe. Figure 2B shows prominent regions of hybridization arranged as rungs on a ladder. The higher rungs progressively disappear as the bulk DNA is degraded. The bands containing early histone genes appear to have a regular spacing pattern (Fig. 2B, 1310 bp). The estimates of spacing are necessarily inexact because the width of the histone gene fragments is sufficient to suggest there is some heterogeneity in the precise cutting sites. In addition, the histone repeats can be of varying lengths in different individuals (Overton and Weinberg, 19’78).Nonetheless, the data support the idea of a site or region specific nicking of early histone genes by an endogenous nuclease in cleavage nuclei. The distribution of about 1300 bases is approximately the distance of the different coding regions of the five histone genes from one another in the major early histone gene repeat suggesting a region of sensitivity associated with each gene of the repeat. To determine if such nicks in the chromatin existed in the living embryo, in situ, DNA was prepared from intact embryos without nuclear isolation and compared to DNA isolated from nuclei from the same culture and stage. Some degree of DNA nicking occurs during the isolation of both lOO-200-cell cleavage stage (Fig. 3) and 500-cell mesenchyme blastula stage nuclei (data not shown). The DNA isolated from lOO-200~cell nuclei displays the typical “ladder” array of histone gene sequences (Fig. 3B2). Nuclei from mesenchyme blastulae and gastrulae show limited degradation, and the DNA does not show a prominent “ladder” (data not shown).
ANDERSON, 1
2
Yu,
AND
WILT
3
Nucleuses and Histonc Germ
111
ences between the DNA obtained from the different embryo cultures. A diagram of the results is shown in Fig. 1. The other probe (2) allows mapping in the opposite orientation from probe 1 by using BamHI to restrict the DNA resulting from the endogenous nuclease digests. The result of mapping the same two nuclear digests with this probe is shown in Fig. 4B. Again, a sharp pattern of bands occurs, particularly just upstream of the H2A, Hl, and H4 genes (Fig. 1). Thus, there are nuclease sensitive regions upstream from H2a, H2b, H4, and Hl genes (the H3 gene was not mapped with these probes), with slight differences between the two cultures. This same kind of mapping experiment has been carried out on several additional cultures with similar results. We conclude that in nuclei of cleavage stages there are regions near the cap site of each of the early histone genes
I
II
II
I
B FIG. 3. Cleavage stage embryos were used to prepare nuclei, and DNA was extracted from an aliquot without further manipulation. Some embryos were also used to prepare total DNA. An aliquot of the nuclei was also subjected to digestion with micrococcal nuclease (see Fig. 5). Samples of total embryo DNA (l), nuclear DNA (Z), and micrococcal nuclease-digested nuclear DNA (3) were subjected to electrophoresis and stained (A) then blotted and probed with the entire early histone gene repeat (B).
Chromatin of mesenchyme blastula and gastrula nuclei does not undergo a progressive degradation during incubation in vitro (up to 4 hr; not shown). The apparent regular nicking of the DNA in cleavage stage nuclei suggested it might be possible to map the approximate sites susceptible to nuclease action. Accordingly, small probes were used for the indirect mapping procedure (Wu, 1980) (see Fig. 1). Figure 4 shows such an experiment from embryos from two different females in which the DNA from nuclei was allowed to undergo endogenous digestion and was then digested with EcoRI (A) prior to electrophoresis and blotting. The presence of bands show that preferred cutting sites have been utilized by the endogenous nuclease. In this experiment cutting occurs in regions just upstream from the H4- and HBb-coding sequences, with slight differ-
A FIG. 4. Nuclei were isolated from 2). The nuclear DNA was digested BamHl (panel B). Blotted DNA was A) or 2 (panel B) from two different
B cleavage stage embryos (see Fig. with either EcoRI (pane1 A) or hybridized with probes 1 (panel females (I, II).
112
DEVELOPMENTAL
that are especially nucleases. The Presence
sensitive
BIOLOGY
to the action of endogenous
of Nucleosomes
in Early
Histone
Genes
The presence and distribution of nucleosomes in the early histone gene tandem repeat was examined by micrococcal nuclease treatment of nuclei isolated from lOO200-cell cleavage and mesenchyme blastula stage embryos. In Fig. 5A the time course of digestion of mesenchyme blastula nuclei displays the characteristic nucleosome repeat “ladder” in an ethidium bromidestained gel, with a repeat spacing of approximately 220 bp. When the blot of this gel is probed with the sea urchin early histone genes, the same apparent pattern of 220-bp repeats is evident (Fig. 5B). Thus, in mesenthyme blastula embryos, a stage when the early histone genes are not transcribed, probably most of the early histone gene repeat family is packaged into nucleosomes similar to total DNA. The same type of analysis using cleavage nuclei is complicated by the activity of endogenous nuclease (s) described in the previous section. Since the level of endogenous nuclease varies considerably in different cultures of embryos, experiments were carried out on several batches of eggs, adding enough micrococcal nuclease to generate rapidly the typical nucleosomal ladder. The results from one experiment where the endogenous activity was relatively low is shown in Fig. 3. There is no evidence of packaging the early histone repeats into a regular nucleosomal array (Fig. 3B3) even though the ladder produced from bulk DNA shows prominent nu-
M
0
1
2
4.5
8
16 31
FIG. 5. Nuclei were isolated from the mesenchyme shows the time course of digestion from a gel stained repeat (panel B).
54
81
blastula stage with ethidium
VOLUME
117, 1986
cleosomes (Fig. 3A3). We conclude that the preponderance of active early histone genes are not adorned with nucleosomes during cleavage. This is consistent with the examination of chromatin from cleavage cells by electron microscopy (Busby and Bakken, 1980), and with the findings of Spinelli et al. (1982), but Bryan et al. (1983) stated that during cleavage most histone coding sequences are in micrococcal nuclease resistant structures, a conclusion different from our findings. This may be due to differences in species used or precise methods of analysis. Finally, it is interesting to inquire if nucleosomes associated with early histone gene repeats in gastrula embryos are specifically associated with any particular sequences of the repeat, a phenomenon termed “phasing.” In principle, nucleosome phasing might be assessed by digesting the DNA produced by micrococcal nuclease digestion of chromatin with EcoRI, followed by electrophoresis, blotting, and probing with probe 1. If nucleosomes are phased, a series of bands defined by the EcoRI site and the sites of micrococcal nuclease action will be generated. Such experiments were conducted and a ladder of bands spaced 200-300 bp apart was observed (data not shown). However, control experiments with purified cloned histone DNA revealed micrococcal nuclease had marked sequence specificity (cf. Reeves, 1984). The pattern of bands generated by micrococcal nuclease and EcoRI with purified histone DNA is very similar to the pattern produced with chromatin (data not shown). One likely interpretation is that nucleosomes are not phased, i.e., are arranged randomly over the repeat, which has the effect of exposing all portions of the repeat to enzyme
M
0
I
2
4.5
and digested up to 81 min, bromide. The blotted DNA
8
16
31
54
81
with 2 U/ml of micrococcal was probed with the entire
nuclease. Panel A early histone gene
ANDERSON,
Yu,
AND
WILT
attack with similar probability. We find no evidence using micrococcal nuclease for phasing of histone gene nucleosomes. In conclusion, when early histone genes are actively transcribed there are regions of marked nuclease sensitivity in the regions immediately upstream from the cap sites of the coding sequences. There is no apparent nucleosomal organization of these actively transcribed genes. As transcription ceases, the chromatin of the early histone genes becomes organized into conventional nucleosomes, and the core particles are probably randomly positioned with respect to particular DNA sequences. There is little or no site specificity of endogenous nuclease at these later stages. It is likely the cessation of transcription of the early histone genes and the changes in chromatin structure are causally linked.
Nucleases
BRYAN, P. N., OLAH, J., and BIRNSTIEL, M. L. (1983). Major changes in the 5’ and 3’ chromatin structure of sea urchin histone genes accompany their activation and inactivation in development. Cell 33, 843848. B~JSBY,S., and BAKKEN, A. H. (1980). Transcription in developing sea urchins: Electron microscopic analysis of cleavage, gastrula and prism stages. Chromosma 79, 85-104. HINEGARDNER, R. T. (1967). In “Methods in Developmental Biology”
Histone
113
Genes
(F. H. Wilt and N. K. Wessells, eds.), pp. 139-155. Crowell, New York. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular cloning.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. MAURON, A., KEDES, L. H., HOUGH-EVANS, B., and DAVIDSON, L. H. (1982). Accumulation of individual histone mRNA’s during embryogenesis of the sea urchin Strongylocentrotus purpuratus. Dev. Biol. 94,425-434.
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