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Psoralen crosslinking of active and inactive sea urchin histone and rRNA genes
Biochimica et Biophysica Acta 1397 Ž1998. 285–294
Psoralen crosslinking of active and inactive sea urchin histone and rRNA genes A. Jasinskas 1, N. Jasinskiene 1, J.P. Langmore
)
Biophysics Research DiÕision and Department of Biology, UniÕersity of Michigan, 930 N. UniÕersity, Ann Arbor, MI 48109-1055, USA Received 17 September 1997; revised 5 January 1998; accepted 5 January 1998
Abstract Chromatin structure is highly correlated with the transcriptional activity of specific genes. For example, it has been found that the regularity of nucleosome spacing is compromised when genes are transcribed. The rRNA genes from fungi, plants, and animals give distinctly bimodal distributions of psoralen crosslinking, which has led to the suggestion that these genes might be largely devoid of nucleosomes when transcriptionally active. We investigated the chromatin structure of the multicopy rRNA and histone genes during sea urchin early embryogenesis. The rRNA genes, which are weakly expressed, give a unimodal distribution of weak psoralen crosslinking, in contrast to the situation in all other organisms studied. The early histone genes were more accessible to psoralen crosslinking when active than inactive. The pattern of crosslinking suggests that these polII genes have a homogeneous structure and are still highly protected by nucleosomes when in the active conformation, unlike the situation in polI genes. q 1998 Elsevier Science B.V. Keywords: Psoralen; Crosslinking; rDNA; Histone gene; RNA polymerase II; Transcription
1. Introduction Regulation of transcription of eukaryotic singlecopy genes is achieved by control of the rate of initiation w1–4x. Transcription of multicopy genes can also be regulated by copy selection, which describes the selection of some copies of the genes for transcription, while the remainder are silent. The mechanism of copy selection is not known, but could be the result of limiting numbers of specific transcription factors or subtle differences in the structure of different gene copies. ) Corresponding author. Fax: q1-313-764-3323; E-mail: [email protected] 1 A permanent address is: Institute of Biochemistry, Mokslininku 12, 2600 Vilnius, Lithuania.
The most studied multicopy genes are the ribosomal RNA genes, which range from less than one hundred up to several thousand copies per genome in different organisms w5x. Although expression of eukaryotic genes is correlated with an ‘open’ chromatin structure, characterized by an indistinct micrococcal nuclease digestion pattern, this feature cannot be used to quantify the fraction of genes that are being transcribed, because the digestion patterns of mixtures of conformational states are difficult to interpret. For example, the micrococcal nuclease digestion patterns of active mouse rRNA genes are almost indistinguishable from those of inactive rRNA genes w6x. To overcome this problem, Sogo et al. w7x developed psoralen crosslinking to distinguish between active and inactive copies of the rRNA genes on the basis of differential protection of the DNA from psoralen
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 8 . 0 0 0 1 7 - 7
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crosslinking and the resulting differential electrophoretic mobility of the DNA. The active copies exist in a more ‘open’ conformation, which is more highly crosslinked and therefore retarded on agarose gels. The inactive copies are less highly crosslinked and therefore migrate faster. A distinctive characteristic of the active copies of the rRNA genes is that the level of crosslinking is similar to that of naked DNA. Conconi et al. w6x showed that in yeast, plants, and mice ribosomal genes were divided between active and inactive copies, and this copy-selection was maintained during the cell cycle, in dividing and inert tissues w8x. In every case the psoralen-accessible region was limited to the coding regions of the genes w6,7,9x. Changes in growth conditions leading to reduction in rRNA expression reduce the fraction of gene copies in the active state w6,9x. Thus it is well-documented that rRNA genes exhibit copyselection regulation in addition to or instead of control of the rate of initiation on the active copies of the gene, and that the active copies of these polI genes are not protected from psoralen crosslinking. Ribosomal genes and early histone genes are reiterated in the sea urchin genome several hundred times w10,11x, revealing an independent opportunity to test for regulation by copy selection. In the period between fertilization and the gastrula stage primary cell differentiation occurs, leading to dramatic alterations in length of cell division, to changes in the repertoire of gene expression, overall transcriptional activity, and chromatin structure w12–19x. During sea urchin early development rRNA synthesis drops from oogenesis to an early cleavage stage and is very low before the feeding larva stage Ž; 5% of maximal activity. w20x. This conclusion was made by measuring the absolute rates of rRNA synthesis w21–24x and by electron microscopy of ribosomal RNA genes as chromatin w25x. A much higher level of rRNA synthesis occurs at the pluteus stage, when the embryos increase mass w26x. In contrast, the transcriptional activity of early histone genes increases at early morula, reaching the highest transcriptional activity during early blastula Ž at 8–10 h of development in Strongylocentrotus purpuratus. and then drops again to a very low level at late blastula Žat 18 h of development. w27–29x. Chromatin structure is changing during this time as well. DNase I hypersensitive sites are detected on these genes in their active state
and disappear completely after gene inactivation w30– 32x. In the active state of the histone genes at 10 h of development the nucleosomes are distributed randomly, while at 18 h and 36 h of development when the early histone genes are transcriptionally inactive, they posses a typical nucleosomal repeat pattern w17,30–32x with positioned nucleosomes w33x. However, these studies did not address the question of the heterogeneity in histone gene chromatin structure. The weak bands seen in the higher molecular weight region on agarose gels could be due to a minor fraction of chromatin resembling inactive chromatin w34x. On the other hand the typical nucleosomal pattern obtained for inactive histone genes at 18 h and 36 h of development might mask a less distinct pattern arising from gene copies without periodic nucleosomes or depleted in number of nucleosomes, as would be expected if fractions of the gene copies were still in the active conformation. Thus, the indistinct micrococcal nuclease digestion pattern of active early histone gene chromatin does not exclude the possibilities that a fraction of the genes is inactive and has a typical nucleosomal repeat, or that a fraction of the genes is devoid of nucleosomes as in the case of the rRNA genes. We used psoralen crosslinking to analyze chromatin heterogeneity of ribosomal RNA genes and early histone genes of S. purpuratus during early development. Results showed that both gene families have a homogeneous chromatin structure in the inactive state and Žfor histone genes. a homogeneously ‘open’ structure in the active state, evidence against copy selection regulation of these genes during early embryogenesis. 2. Materials and methods 2.1. Sea urchin embryo cultures and nuclear isolation S. purpuratus were purchased from Marinas, Long Beach, CA. The collection of the gametes, their fertilization, growing of embryos and nuclei isolation were done essentially as described by Vincenz et al. w34x except that all nuclei isolation buffers contained 10 mM sodium butyrate to inhibit histone deacetylation w35x. For DNA labeling, embryos were grown continuously in the presence of w 3 H-methylxthymidine Ž1 m Cirml, Amersham.. Typical specific activ-
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ity was 10 5 dpmrm g of DNA. The nuclei were stored at a concentration of 1 mgrml in buffer A containing 15 mM HEPES Ž pH 7.5. , 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 0.1 mM PMSF, 5 mM iodoacetate, 6 mM leupeptin, 10 mM sodium butyrate and 50% glycerol. Protein and DNA analysis revealed no degradation during isolation and storage at y808C for more than a year. Nuclei from mouse erythroleukemic cells ŽMEL cells. were isolated by the method described by Langmore and Paulson w36x with some modifications. Briefly, collected cells were washed two times in PBS, then twice in 15 mM HEPES ŽpH 7.5., 60 mM KCl, 3 mM MgCl 2 , 1 mM PMSF, 1 mM leupeptin. Cells were washed in lysis buffer containing 15 mM HEPES, pH 7,5, 60 mM KCl, 15 mM NaCl, 3 mM MgCl 2 , 1 mM PMSF, 1 mM leupeptin, 0.1% digitonin repeatedly until all cells lysed. All solutions contained 10 mM sodium butyrate. Nuclei were used immediately or stored in 50% glycerol at y808C. Nuclei from the kangaroo rat liver were isolated by modified Hewish and Burgoyne method w37x as described by Makarov et al. w38x.
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crococcal nuclease. DNA digestion and purification was essentially as described by Fronk et al. w32x. DNA fragments were separated on 1.2% agarose gels in 1 = TBE buffer. 2.4. DNA isolation, restriction and gel electrophoresis DNA was isolated as described by Vincenz et al. w34x. DNA concentration was monitored by scintillation counting. Restriction was performed as recommended by the manufacturer Ž Gibco BRL, Boehringer Mannheim. using 5–20 U of restriction enzymes per m g of crosslinked DNA for 3–6 h at 378C. Noncrosslinked control DNA was digested with 1 U of restriction enzyme per m g of DNA for 1 h at 378C. Electrophoresis of restriction fragments was performed in 30 cm-long 1.5% or 0.8% nondenaturing agarose gels in 1 = TAE standard buffer w39x at 80 V for 24 h with recirculation of the buffer. Electrophoresis of micrococcal nuclease digested DNA was performed in 1.2% agarose nondenaturing gels in 1 = TBE. 2.5. Transfer and hybridization
2.2. Psoralen photocrosslinking of nuclei Psoralen photocrosslinking of nuclei was performed essentially as described by Conconi et al. w8x. Nuclei in storage buffer were diluted twice with 50 mM NaCl, 50 mM Tris, pH 7.5, 5 mM MgCl 2 , 5 mM dithiothreitol, 6 mM leupeptin at the concentration of 2–4 A 260 . Photocrosslinking was done in open polypropylene dishes using a 450 W Conrad–Hanovia medium pressure UV lamp for different times on ice at a distance of ; 15 cm. The measured intensity was 50 Wrm2. The light was filtered to cut off wavelengths - 300 nm using Schott glass filter WG 320. One 20th volume of 4,5X ,8-trimethylpsoralen ŽSigma. stock solution in ethanol Ž200 m grml. was added at 20 min intervals during irradiation. After each addition of psoralen, the nuclei were preincubated in the dark for 5 min on ice before irradiation. 2.3. Micrococcal nuclease digestion Nuclei isolated from 10 and 36 h embryos were digested at a concentration of 50 m grml using mi-
Before blotting, the gels were irradiated on ice for 2 h with short wavelength Ž l s 254 nm. light to reverse the psoralen crosslinks, as described by Sogo et al. w7x. DNA was transferred to Zeta-Probe GT membrane ŽBio-Rad. using alkaline vacuum blotting ŽBio-Rad.. Hybridization probes used were pCO2A, containing the 6.5 kb sea urchin early histone gene repeat w40x and pSPTmr100, containing 6.6 kb mouse ribosomal gene sequences, subcloned from lgtWESmr100 w41x by A. Conconi. Probes were prepared using 50 ng of plasmid DNA by random priming ŽBoehringer Mannheim.. Specific activities were ) 10 8 cpmrm g of DNA. Prehybridization and hybridization were performed as recommended for Zeta-Probe GT membrane by the manufacturer Ž BioRad. with some modifications. Prehybridization was done for 15 min at 508C in 35% formamide, 0.12 M sodium phosphate, pH 7.2, 0.25 M NaCl, 7% SDS. Fresh solution and denatured radioactive probe was added, and the filters were hybridized overnight at 508C in a hybridization oven ŽHoefer.. Post-hybridization washes for histone genes were performed as
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described by Vincenz et al. w34x, for ribosomal genes last wash was done in 0.5 = SSC for 15 min at room temperature. Stripping of the probes was achieved by incubation of the filters twice for 20 min in a large volume of 0.5% SDS at 958C. Autoradiography was done using preflashed Kodak X-ray film or Molecular Dynamics image plates.
3. Results 3.1. Confirmation of rRNA and histone gene structure by micrococcal nuclease digestion In our experiments we used nuclei from 10 and 36 h embryos, which differ drastically in cell division times Ž2 h and 16 h, respectively. , and have different amounts of stored RNA and proteins. Moreover, these nuclei differ in chromatin condensation as revealed by electron microscopy, and by micrococcal nuclease and DNase I digestion w17,30–32,42x. To confirm that the nucleosomal structure of these genes in both developmental stages agreed with the known patterns of expression during development, we digested nuclei with micrococcal nuclease, separated DNA fragments on 1.2% agarose gels, transferred to Zeta-Probe membrane, and hybridized to probes specific to the rRNA or histone genes. To determine the structure of the rRNA genes, the filters were hybridized to the ribosomal gene specific probe pSPTmr100, which consists of 6.6 kb of transcriptionally active sequences from the mouse rRNA repeat including ; 90% of the 18S, the entire 5.8S, and ; 80% of the 28S rRNA genes. Because the sea urchin rRNA locus has not been mapped or completely sequenced, we cannot be certain which regions of the sea urchin rRNA genes hybridize to this probe. However, given the high sequence homology within the 18S genes Ž87% identity between mouse and sea urchin. and the small size of the sea urchin rRNA repeat Ž 10–12 kb. w43,44x we are confident that the probe substantially overlaps the coding sequences of the sea urchin repeat. Ethidium bromidestained gels Žnot shown. showed regular nucleosomal patterns that were similar for both stages of embryos. Fig. 1, lanes 1–4, shows Southern blots of the rRNA genes at 10 h and at 36 h of development, which have nucleosomal repeat ladders characteristic of inactive
Fig. 1. Micrococcal nuclease digestion of the sea urchin rRNA and early histone genes. Nuclei were isolated from 10 and 36 h embryos and digested with different amounts of micrococcal nuclease. Isolated DNA was fractionated on 1.2% agarose gels and Southern blots were hybridized to ribosomal gene specific probe pSPTmr100 Žlanes 1–4. or histone gene specific probe, pCO2A, Žlanes 5–8.. ŽLanes 1,5. Digested with 1 urml; Žlanes 2,6. 10 urml; Žlanes 3,7. 5 urml; Žlanes 4,8. 20 urml. ŽLane M. 123 bp repeat molecular weight marker. ŽLanes 1,2. rRNA genes from 10 h nuclei; Žlanes 3,4. rRNA genes from 36 h nuclei; Žlanes 5,6. histone genes from 10 h nuclei; Žlanes 7,8. histone genes from 36 h nuclei.
genes. The calculated nucleosome repeat length for the rDNA was the same as for total chromatin and corresponded to 212 " 4 bp, which is close to that described in the literature w17,45,46x. A different picture was revealed in the Southern blots of early histone genes Ž Fig. 1, lanes 5–8., which were probed with the plasmid pCO2A, consisting of the complete S. purpuratus early histone repeat with all five histone genes, which are independently transcribed w47,48x. The amount of spacer DNA that is transcribed is unknown, because the 3X
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end of the mature mRNA is apparently produced by post-transcriptional processing w49,50x. At 10 h of development Ž lanes 5,6. early histone genes gave weakened nucleosome bands with a shorter nucleosome repeat length, characteristic of active early histone gene chromatin w32,42x. This indistinct ladder is the same in the coding and non-coding regions, indicating that the majority of even the non-coding sequences have altered nucleosome structure w32x. At 36 h of development Žlanes 7,8. the histone gene sequences acquired a stronger nucleosomal ladder with repeat length characteristic of bulk embryo chromatin. The presence of a weak nucleosomal ladder at 10 h might be due to structure present at the transcriptionally active loci, or to a fraction of the copies of the histone genes remaining in an inactive conformation.
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development, differ in accessibility to many restriction enzymes. In order to probe chromatin structure of early histone genes in their active and inactive stages, we searched for enzymes that have single restriction sites in the early histone gene repeat and could efficiently restrict crosslinked DNA of both development stages. Ten and 36 h nuclei were crosslinked with trimethylpsoralen for different times as indicated in Fig. 2, the DNA was isolated, restricted, separated on nondenaturing agarose gels, UV irradiated, vacuum blotted, and hybridized to pCO2A DNA. HindIII and EcoRI Žnot shown. failed to significantly digest crosslinked DNA. Sal I did not completely digest the crosslinked DNA. XhoI and SacII gave similar results Žnot shown. . SacI efficiently digested crosslinked early histone genes as well as ribosomal genes Ž not shown. and was used in further experiments.
3.2. Restriction analysis of psoralen crosslinked nuclei
3.3. Psoralen photocrosslinking of rRNA genes
Initial experiments showed that psoralen-crosslinked DNA, isolated from nuclei at 10 and 36 h of
Psoralen crosslinking to purified DNA depends upon extent of UV exposure, DNA sequence, and
Fig. 2. Restriction analysis of psoralen crosslinked DNA, isolated from nuclei of 10 and 36 h sea urchin embryo developmental stages. Nuclei were photocrosslinked with trimethylpsoralen. DNA was isolated, hydrolyzed with restriction enzymes and fragments were separated on 0.8% agarose gel. After reversal of crosslinks, DNA was transferred to nylon filters and hybridized with early histone gene probe pCO2A. ŽLane M. lrHindIII molecular weight marker; Žlane C. control uncrosslinked DNA digested with HindIII. Restriction enzymes and development times indicated at the top. ŽOdd lanes. 25 min crosslinking; Ževen lanes. 1 h crosslinking.
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supercoil torsion w51x. In our hands, crosslinking naked rDNA and lambda DNA for increasing times causes progressive retardation of the DNA on agarose gels. This retardation is not easily saturated. For example, the 6.6 kb lrHindIII fragment is retarded 2.2, 3.3, 4.3, 5.4, and 6.4% for crosslinking times of 0.5, 1, 1.5, 2, and 2.5 h Ždata not shown. . In nuclei, psoralen should bind to DNA, RNA, proteins and membranes w51–53x. Thus, the extent of crosslinking of nuclear DNA depends on specific properties of the nuclei, such as amount of RNA and protein present. To eliminate the potential differences in psoralen crosslinking of DNA in nuclei at the two development stages, we determined the crosslinking conditions that gave the same electrophoretic retardation of the inactive rRNA genes at both 10 and 36 h of development. Fig. 3 shows the results of crosslinking sea urchin rRNA chromatin for different times, as probed with the mouse rRNA probe, pSPTmr100. One hour of crosslinking was insufficient to completely shift the mobility of DNA from both develop-
Fig. 3. Analysis of psoralen crosslinked sea urchin ribosomal genes. After nuclei were crosslinked with psoralen DNA was isolated, restricted with Sac I and fragments were separated on a 1.5% agarose gel. DNA transferred to nylon filters and analyzed with ribosome gene specific probe pSPTmr100. ŽLane M. l r HindIII molecular weight marker; Žlanes 1,4. control uncrosslinked DNA, isolated from 36 h nuclei and digested with SacI; Žlanes 2,3. DNA of 1 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlanes 5,6. DNA of 2 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlane 7. 1 h crosslinked purified DNA isolated from 36 h nuclei.
Table 1 Percentage electrophoretic retardation of crosslinked sea urchin rRNA genes relative to uncrosslinked DNA Crosslinking time Žh.
DNA Ž%.
10 h nuclei Ž%.
36 h nuclei Ž%.
1 2
4.8 6.8
3.5 3.4
2.3 3.4
mental stages Ž lanes 2,3.. Two hours of UV irradiation Ž lanes 5,6. and longer Žnot shown. saturated and equalized band mobility. We conclude that 2 h trimethylpsoralen photocrosslinking eliminates the intrinsic differences in the conditions of irradiation of the nuclei and creates saturation conditions for the nuclei of both development stages studied. After 2 h crosslinking, the 4.4 kb rDNA band Žwhich contains ) 90% of the sequences that hybridize with the mouse 18S and 28S probe. was retarded by 3.4%. In contrast to the situation found in other cells, there was no band splitting for sea urchin rRNA genes at either development stage. The two weak bands at higher molecule weights Ž which could reflect sequence overlap with the probe or sequence heterogeneity. were similarly retarded and remained monodisperse. The restricted DNA from 2-h crosslinked nuclei was retarded only half as much as from 2-h crosslinked naked DNA ŽTable 1.. Thus, the rDNA crosslinked in the nuclei was highly protected from the photoreaction, as expected for inactive chromatin. No bands or shoulders were detected that migrated with greater or less retardation. The simplest interpretation of this result is that all or most all of the copies of the sea urchin embryonic rRNA genes have a homogeneous, ‘closed’ chromatin structure. To check our ability to reproduce the splitting of active rDNA bands seen by Sogo et al., we used mouse erythroleukemic cell nuclei as a positive control for ribosomal gene band splitting. Fig. 4 shows that after psoralen crosslinking, the rRNA separates into two bands, representing about 60% and 40% of active and inactive rRNA gene copies, respectively, in the agreement with the literature results w6x. Splitting of the bands occurred after 1 h, as well as after 2 h of psoralen photocrosslinking, showing that the effect can be achieved over a range of crosslinking conditions. The active and inactive rRNA fragments were retarded by 6.2 and 1.6% ŽTable 2. , with the
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very weak doublet at 10 kb is retarded too much to represent the active component of the 7.5 kb fragment, and is probably due to sequence heterogeneity or incomplete restriction, however the lack of a restriction map for the kangaroo rat makes any conclusion about these minor bands difficult. Thus, the experimental conditions for psoralen photocrosslinking were appropriate for resolving the differences between active and inactive copies of mammalian rRNA genes. 3.4. Psoralen photocrosslinking of histone genes
Fig. 4. Investigation of chromatin structure of rRNA genes by psoralen photocrosslinking assay in MEL cells and kangaroo rat liver cells. Nuclei were photocrosslinked with trimethylpsoralen and isolated DNA was processed as in Fig. 3. Filters were hybridized with ribosomal gene specific probe pSPTmr100. EcoRI was used as restriction enzyme. ŽLane M. l r HindIII molecular weight marker; Žlane 1. control uncrosslinked DNA from MEL cells; Žlanes 2,3. DNA from MEL cell nuclei crosslinked for 1 and 2 h, respectively; Žlane 4. 1 h crosslinked purified MEL cell DNA; Žlane 5. control uncrosslinked DNA from kangaroo rat; Žlanes 6,7. DNA from rat liver nuclei crosslinked for 1 and 2 h, respectively. s And f represent bands of slow and fast moving ribosomal gene copies, respectively.
active copies behaving almost like naked DNA. In the case of kangaroo rat liver nuclei, a broad tail formed on the 7.5 kb major rRNA band, similar to the results from tomato leaves and cell culture w8x, which seem to have only minor active fractions. The
The filter from the experiment shown in Fig. 3 was stripped and rehybridized with the early histone gene probe, pCO2A, containing the entire 6.5 kb sea urchin early histone gene repeat. Fig. 5 shows that after 2 h of psoralen crosslinking active Žlane 5. and inactive Žlane 6. early histone gene sequences are retarded by 5.2 and 4.0%, respectively Ž Table 3. . The small difference in mobility is unlike the finding in mouse rRNA genes, which exhibited almost a 5% difference in mobility between the active and inactive copies. As in the case of the rRNA genes from sea urchin, even the inactive copies underwent appreciable crosslinking. The active histone genes were still substantially protected from psoralen crosslinking, relative to the active rRNA genes of mouse. The incomplete psoralen crosslinking is consistent with results in yeast showing that several pol II genes are partially protected from crosslinking w54,55x. The observed differences between the active and inactive stages for the early histone genes are consistent with increase in the nucleosome density or decrease in the accessibility of the nucleosomes when the genes are inactivated. Unlike the situation with active rDNA genes from other organisms, however, the DNA from crosslinked active histone genes was not split into two bands representative of active and
Table 2 Percentage electrophoretic retardation of the 4.4 kb band representative of the majority of the 18S and 28S crosslinked mouse rRNA genes relative to uncrosslinked DNA Crosslinking time Žh.
DNA Ž%.
Active component Ž%.
Inactive component Ž%.
1 2
4.3 6.0)
4.7 6.2
1.6 1.6
)Extrapolated from results with sea urchin DNA.
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4. Discussion
Fig. 5. Trimethylpsoralen photocrosslinking of sea urchin early histone genes. The filter in Fig. 3 was stripped and rehybridized with early histone gene probe pCO2A. ŽLane M. l r HindIII marker; Žlanes 1,4. control uncrosslinked DNA, isolated from 36 h nuclei and digested with SacI; Žlanes 2,3. DNA of 1 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlanes 5,6. DNA of 2 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlane 7. 1 h crosslinked purified DNA isolated from 36 h nuclei. The active early histone gene repeat is crosslinked to a greater extent than the inactive repeat even after a saturation dose of crosslinking. Psoralen crosslinking does not resolve either the active or inactive genes into separate bands.
inactive chromatin conformations. The simplest interpretation of this result is that most of the copies of the genes have the same chromatin structure. Despite the similar migration of the active and inactive genes, from the intensity profiles from the PhosphorImager data we estimate that as little as 10% of an inactive conformation would have given rise to a detectable shoulder on the bands in Fig. 5, lanes 2 and 5. We interpret this data as evidence that almost all copies of these genes are present in the ‘open’ state, suggesting that copy selection is not used in the regulation of the histone genes at 10 h of development. Table 3 Percentage electrophoretic retardation of crosslinked sea urchin histone genes relative to uncrosslinked DNA Crosslinking time Žh.
DNA Ž%.
10 h nuclei Ž%.
36 h nuclei Ž%.
1 2
6.0 8.6
4.9 5.2
2.5 4.0
Under saturating conditions, psoralen makes covalent linkages in DNA that is not protected by nucleosome core particles. DNA in an open chromatin conformation or free from proteins is more frequently crosslinked. The frequency of crosslinking can be assayed on an agarose gels. Conconi et al. w6x showed that crosslinking of rRNA genes gave a bimodal distribution of products, a slowly migrating transcriptionally active fraction and a rapidly migrating inactive fraction. Similar heterogeneity in rDNA structure was found in different organisms and in different physiological states w6,8,9x. In contrast, psoralen crosslinking of single copy pol II genes in yeast cells have shown a homogeneous, protected structure in both the active and inactive transcriptional states w54,55x. We have investigated the heterogeneity of chromatin structure of ribosomal RNA genes during sea urchin early embryogenesis using psoralen photocrosslinking to electrophoretically separate the active and inactive copies of the genes. We performed the experiments on nuclei isolated in the presence of n-butyrate, which should eliminate any potential effects of histone deacetylation upon psoralen crosslinking. Sea urchin rRNA genes have similar DNA organization as in some other higher eukaryotes. The length of repeat unit, containing 18S and 26S RNA genes, is 10–12 kb w43,44x. The units are clustered and repeated about 50 times w20x, and are transcriptionally inactive at cleavage and gastrula stages. Large amounts of rRNA are stored in the eggs and immediately utilized after fertilization to support rapid cell division w24,56,57x. By using micrococcal nuclease we showed that the chromatin in the coding region of these genes has a canonical, organized nucleosomal organization at 10 and 36 h of developmental, consistent with the observed low transcription rates of about 0.4 transcripts per minute w20x. Psoralen crosslinking showed that the sea urchin rRNA genes were uniformly crosslinked at 10 and 36 h of development, as if the coding regions were in a single conformation. The simplest interpretation of this result is that most of the gene copies were in the inactive conformation, consistent with the results of micrococcal nuclease digestion. However, the retardation of the sea urchin rRNA genes was substantially greater than
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that found for inactive mouse rRNA genes. Perhaps this effect is due to the fact that the rRNA genes are active earlier as well as later in development and therefore might carry some of the properties of active chromatin. Alternatively, the micrococcal nuclease patterns and psoralen crosslinking might reflect different aspects of chromatin structure, which might only sometimes be correlated with transcriptional activity. For example, psoralen crosslinking might be promoted by hyperacetylation of the histones, which could sometimes be correlated with transcriptional activity, as in studies of rat rRNA genes w58x, but not correlated with transcriptional activity and nucleosome positioning in sea urchin embryos. Another possibility is that the longer length of the linker DNA between nucleosome cores could lead to appreciable exposure of the DNA to psoralen even when fully occupied by nucleosomes. The sea urchin early histone genes are repeated about 500 times, comprise about 0.15% of the sea urchin genome, and are transcribed by RNA polymerase II at a moderate rate of 0.5–1 transcripts per gene per minute Žassuming all copies are active. w20x. This rate is comparable to other polII genes, substantially greater than the average rate of 0.2 transcripts per minute in the embryos, but less than the maximal rate for pol II genes in S. purpuratus w59x. The highest transcriptional activity of the early histone genes is during the early blastula stage. The transcripts are barely detected later at gastrula. The genes have positioned nucleosomes in their inactive state and a random distribution of nucleosomes in their active state w33x. Our analysis of psoralen-crosslinked early histone genes found a small, but easily detectable difference in mobility of the histone genes when they were active and inactive, but did not detect any splitting of the histone band at either stage of transcriptional activity. The extent of crosslinking of the active histone genes is considerably less than for naked DNA, suggesting that even in the active conformation there is appreciable protection from nucleosomes. This is in contrast to the rRNA genes in mouse and yeast, where the active genes were as highly crosslinked as naked DNA. However, unlike previous psoralen results from pol II genes in yeast, the histone genes in the active state were crosslinked significantly more frequently than those in the inactive state. Thus the level of exposure of active histone
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genes to psoralen was intermediate between that for multicopy polI genes and single copy pol II genes that have been studied previously.
5. Conclusion Even this moderate difference between the crosslinking of active and inactive histone genes should have revealed the use of copy selection in the regulation of early histone genes, yet we did not detect a second peak or shoulder representing nucleosome-free active, or highly-protected inactive conformations. The fact that only a single band was found after psoralen crosslinking suggests that the sea urchin has met the needs for histone mRNA by regulating the rate of transcription of a large number of genes, rather than regulating the fraction of genes that are active. The difference found between the behavior of rRNA genes in other systems Ž which have heterogeneous structure., and the early histone genes Ž which have homogeneous, more protected, structure. suggests that there might be a difference in the role of chromatin structure in regulation of pol I and pol II transcription of multicopy genes. The observed differences might be correlated with possible differences that nucleosome displacement, acetylation, or supercoiling play in transcription of polI and pol II genes w58,60x.
Acknowledgements We are very thankful to Dr. A. Conconi for a gift of the rRNA gene plasmid pSRTmr100, Dr. Weinberg for early histone gene plasmid pCO2A, and S. Lejnine for the gift of kangaroo rat liver nuclei. This research was supported by NSF MCB9514196.
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Psoralen crosslinking of active and inactive sea urchin histone and rRNA genes A. Jasinskas 1, N. Jasinskiene 1, J.P. Langmore
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Biophysics Research DiÕision and Department of Biology, UniÕersity of Michigan, 930 N. UniÕersity, Ann Arbor, MI 48109-1055, USA Received 17 September 1997; revised 5 January 1998; accepted 5 January 1998
Abstract Chromatin structure is highly correlated with the transcriptional activity of specific genes. For example, it has been found that the regularity of nucleosome spacing is compromised when genes are transcribed. The rRNA genes from fungi, plants, and animals give distinctly bimodal distributions of psoralen crosslinking, which has led to the suggestion that these genes might be largely devoid of nucleosomes when transcriptionally active. We investigated the chromatin structure of the multicopy rRNA and histone genes during sea urchin early embryogenesis. The rRNA genes, which are weakly expressed, give a unimodal distribution of weak psoralen crosslinking, in contrast to the situation in all other organisms studied. The early histone genes were more accessible to psoralen crosslinking when active than inactive. The pattern of crosslinking suggests that these polII genes have a homogeneous structure and are still highly protected by nucleosomes when in the active conformation, unlike the situation in polI genes. q 1998 Elsevier Science B.V. Keywords: Psoralen; Crosslinking; rDNA; Histone gene; RNA polymerase II; Transcription
1. Introduction Regulation of transcription of eukaryotic singlecopy genes is achieved by control of the rate of initiation w1–4x. Transcription of multicopy genes can also be regulated by copy selection, which describes the selection of some copies of the genes for transcription, while the remainder are silent. The mechanism of copy selection is not known, but could be the result of limiting numbers of specific transcription factors or subtle differences in the structure of different gene copies. ) Corresponding author. Fax: q1-313-764-3323; E-mail: [email protected] 1 A permanent address is: Institute of Biochemistry, Mokslininku 12, 2600 Vilnius, Lithuania.
The most studied multicopy genes are the ribosomal RNA genes, which range from less than one hundred up to several thousand copies per genome in different organisms w5x. Although expression of eukaryotic genes is correlated with an ‘open’ chromatin structure, characterized by an indistinct micrococcal nuclease digestion pattern, this feature cannot be used to quantify the fraction of genes that are being transcribed, because the digestion patterns of mixtures of conformational states are difficult to interpret. For example, the micrococcal nuclease digestion patterns of active mouse rRNA genes are almost indistinguishable from those of inactive rRNA genes w6x. To overcome this problem, Sogo et al. w7x developed psoralen crosslinking to distinguish between active and inactive copies of the rRNA genes on the basis of differential protection of the DNA from psoralen
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 8 . 0 0 0 1 7 - 7
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crosslinking and the resulting differential electrophoretic mobility of the DNA. The active copies exist in a more ‘open’ conformation, which is more highly crosslinked and therefore retarded on agarose gels. The inactive copies are less highly crosslinked and therefore migrate faster. A distinctive characteristic of the active copies of the rRNA genes is that the level of crosslinking is similar to that of naked DNA. Conconi et al. w6x showed that in yeast, plants, and mice ribosomal genes were divided between active and inactive copies, and this copy-selection was maintained during the cell cycle, in dividing and inert tissues w8x. In every case the psoralen-accessible region was limited to the coding regions of the genes w6,7,9x. Changes in growth conditions leading to reduction in rRNA expression reduce the fraction of gene copies in the active state w6,9x. Thus it is well-documented that rRNA genes exhibit copyselection regulation in addition to or instead of control of the rate of initiation on the active copies of the gene, and that the active copies of these polI genes are not protected from psoralen crosslinking. Ribosomal genes and early histone genes are reiterated in the sea urchin genome several hundred times w10,11x, revealing an independent opportunity to test for regulation by copy selection. In the period between fertilization and the gastrula stage primary cell differentiation occurs, leading to dramatic alterations in length of cell division, to changes in the repertoire of gene expression, overall transcriptional activity, and chromatin structure w12–19x. During sea urchin early development rRNA synthesis drops from oogenesis to an early cleavage stage and is very low before the feeding larva stage Ž; 5% of maximal activity. w20x. This conclusion was made by measuring the absolute rates of rRNA synthesis w21–24x and by electron microscopy of ribosomal RNA genes as chromatin w25x. A much higher level of rRNA synthesis occurs at the pluteus stage, when the embryos increase mass w26x. In contrast, the transcriptional activity of early histone genes increases at early morula, reaching the highest transcriptional activity during early blastula Ž at 8–10 h of development in Strongylocentrotus purpuratus. and then drops again to a very low level at late blastula Žat 18 h of development. w27–29x. Chromatin structure is changing during this time as well. DNase I hypersensitive sites are detected on these genes in their active state
and disappear completely after gene inactivation w30– 32x. In the active state of the histone genes at 10 h of development the nucleosomes are distributed randomly, while at 18 h and 36 h of development when the early histone genes are transcriptionally inactive, they posses a typical nucleosomal repeat pattern w17,30–32x with positioned nucleosomes w33x. However, these studies did not address the question of the heterogeneity in histone gene chromatin structure. The weak bands seen in the higher molecular weight region on agarose gels could be due to a minor fraction of chromatin resembling inactive chromatin w34x. On the other hand the typical nucleosomal pattern obtained for inactive histone genes at 18 h and 36 h of development might mask a less distinct pattern arising from gene copies without periodic nucleosomes or depleted in number of nucleosomes, as would be expected if fractions of the gene copies were still in the active conformation. Thus, the indistinct micrococcal nuclease digestion pattern of active early histone gene chromatin does not exclude the possibilities that a fraction of the genes is inactive and has a typical nucleosomal repeat, or that a fraction of the genes is devoid of nucleosomes as in the case of the rRNA genes. We used psoralen crosslinking to analyze chromatin heterogeneity of ribosomal RNA genes and early histone genes of S. purpuratus during early development. Results showed that both gene families have a homogeneous chromatin structure in the inactive state and Žfor histone genes. a homogeneously ‘open’ structure in the active state, evidence against copy selection regulation of these genes during early embryogenesis. 2. Materials and methods 2.1. Sea urchin embryo cultures and nuclear isolation S. purpuratus were purchased from Marinas, Long Beach, CA. The collection of the gametes, their fertilization, growing of embryos and nuclei isolation were done essentially as described by Vincenz et al. w34x except that all nuclei isolation buffers contained 10 mM sodium butyrate to inhibit histone deacetylation w35x. For DNA labeling, embryos were grown continuously in the presence of w 3 H-methylxthymidine Ž1 m Cirml, Amersham.. Typical specific activ-
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ity was 10 5 dpmrm g of DNA. The nuclei were stored at a concentration of 1 mgrml in buffer A containing 15 mM HEPES Ž pH 7.5. , 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 0.1 mM PMSF, 5 mM iodoacetate, 6 mM leupeptin, 10 mM sodium butyrate and 50% glycerol. Protein and DNA analysis revealed no degradation during isolation and storage at y808C for more than a year. Nuclei from mouse erythroleukemic cells ŽMEL cells. were isolated by the method described by Langmore and Paulson w36x with some modifications. Briefly, collected cells were washed two times in PBS, then twice in 15 mM HEPES ŽpH 7.5., 60 mM KCl, 3 mM MgCl 2 , 1 mM PMSF, 1 mM leupeptin. Cells were washed in lysis buffer containing 15 mM HEPES, pH 7,5, 60 mM KCl, 15 mM NaCl, 3 mM MgCl 2 , 1 mM PMSF, 1 mM leupeptin, 0.1% digitonin repeatedly until all cells lysed. All solutions contained 10 mM sodium butyrate. Nuclei were used immediately or stored in 50% glycerol at y808C. Nuclei from the kangaroo rat liver were isolated by modified Hewish and Burgoyne method w37x as described by Makarov et al. w38x.
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crococcal nuclease. DNA digestion and purification was essentially as described by Fronk et al. w32x. DNA fragments were separated on 1.2% agarose gels in 1 = TBE buffer. 2.4. DNA isolation, restriction and gel electrophoresis DNA was isolated as described by Vincenz et al. w34x. DNA concentration was monitored by scintillation counting. Restriction was performed as recommended by the manufacturer Ž Gibco BRL, Boehringer Mannheim. using 5–20 U of restriction enzymes per m g of crosslinked DNA for 3–6 h at 378C. Noncrosslinked control DNA was digested with 1 U of restriction enzyme per m g of DNA for 1 h at 378C. Electrophoresis of restriction fragments was performed in 30 cm-long 1.5% or 0.8% nondenaturing agarose gels in 1 = TAE standard buffer w39x at 80 V for 24 h with recirculation of the buffer. Electrophoresis of micrococcal nuclease digested DNA was performed in 1.2% agarose nondenaturing gels in 1 = TBE. 2.5. Transfer and hybridization
2.2. Psoralen photocrosslinking of nuclei Psoralen photocrosslinking of nuclei was performed essentially as described by Conconi et al. w8x. Nuclei in storage buffer were diluted twice with 50 mM NaCl, 50 mM Tris, pH 7.5, 5 mM MgCl 2 , 5 mM dithiothreitol, 6 mM leupeptin at the concentration of 2–4 A 260 . Photocrosslinking was done in open polypropylene dishes using a 450 W Conrad–Hanovia medium pressure UV lamp for different times on ice at a distance of ; 15 cm. The measured intensity was 50 Wrm2. The light was filtered to cut off wavelengths - 300 nm using Schott glass filter WG 320. One 20th volume of 4,5X ,8-trimethylpsoralen ŽSigma. stock solution in ethanol Ž200 m grml. was added at 20 min intervals during irradiation. After each addition of psoralen, the nuclei were preincubated in the dark for 5 min on ice before irradiation. 2.3. Micrococcal nuclease digestion Nuclei isolated from 10 and 36 h embryos were digested at a concentration of 50 m grml using mi-
Before blotting, the gels were irradiated on ice for 2 h with short wavelength Ž l s 254 nm. light to reverse the psoralen crosslinks, as described by Sogo et al. w7x. DNA was transferred to Zeta-Probe GT membrane ŽBio-Rad. using alkaline vacuum blotting ŽBio-Rad.. Hybridization probes used were pCO2A, containing the 6.5 kb sea urchin early histone gene repeat w40x and pSPTmr100, containing 6.6 kb mouse ribosomal gene sequences, subcloned from lgtWESmr100 w41x by A. Conconi. Probes were prepared using 50 ng of plasmid DNA by random priming ŽBoehringer Mannheim.. Specific activities were ) 10 8 cpmrm g of DNA. Prehybridization and hybridization were performed as recommended for Zeta-Probe GT membrane by the manufacturer Ž BioRad. with some modifications. Prehybridization was done for 15 min at 508C in 35% formamide, 0.12 M sodium phosphate, pH 7.2, 0.25 M NaCl, 7% SDS. Fresh solution and denatured radioactive probe was added, and the filters were hybridized overnight at 508C in a hybridization oven ŽHoefer.. Post-hybridization washes for histone genes were performed as
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described by Vincenz et al. w34x, for ribosomal genes last wash was done in 0.5 = SSC for 15 min at room temperature. Stripping of the probes was achieved by incubation of the filters twice for 20 min in a large volume of 0.5% SDS at 958C. Autoradiography was done using preflashed Kodak X-ray film or Molecular Dynamics image plates.
3. Results 3.1. Confirmation of rRNA and histone gene structure by micrococcal nuclease digestion In our experiments we used nuclei from 10 and 36 h embryos, which differ drastically in cell division times Ž2 h and 16 h, respectively. , and have different amounts of stored RNA and proteins. Moreover, these nuclei differ in chromatin condensation as revealed by electron microscopy, and by micrococcal nuclease and DNase I digestion w17,30–32,42x. To confirm that the nucleosomal structure of these genes in both developmental stages agreed with the known patterns of expression during development, we digested nuclei with micrococcal nuclease, separated DNA fragments on 1.2% agarose gels, transferred to Zeta-Probe membrane, and hybridized to probes specific to the rRNA or histone genes. To determine the structure of the rRNA genes, the filters were hybridized to the ribosomal gene specific probe pSPTmr100, which consists of 6.6 kb of transcriptionally active sequences from the mouse rRNA repeat including ; 90% of the 18S, the entire 5.8S, and ; 80% of the 28S rRNA genes. Because the sea urchin rRNA locus has not been mapped or completely sequenced, we cannot be certain which regions of the sea urchin rRNA genes hybridize to this probe. However, given the high sequence homology within the 18S genes Ž87% identity between mouse and sea urchin. and the small size of the sea urchin rRNA repeat Ž 10–12 kb. w43,44x we are confident that the probe substantially overlaps the coding sequences of the sea urchin repeat. Ethidium bromidestained gels Žnot shown. showed regular nucleosomal patterns that were similar for both stages of embryos. Fig. 1, lanes 1–4, shows Southern blots of the rRNA genes at 10 h and at 36 h of development, which have nucleosomal repeat ladders characteristic of inactive
Fig. 1. Micrococcal nuclease digestion of the sea urchin rRNA and early histone genes. Nuclei were isolated from 10 and 36 h embryos and digested with different amounts of micrococcal nuclease. Isolated DNA was fractionated on 1.2% agarose gels and Southern blots were hybridized to ribosomal gene specific probe pSPTmr100 Žlanes 1–4. or histone gene specific probe, pCO2A, Žlanes 5–8.. ŽLanes 1,5. Digested with 1 urml; Žlanes 2,6. 10 urml; Žlanes 3,7. 5 urml; Žlanes 4,8. 20 urml. ŽLane M. 123 bp repeat molecular weight marker. ŽLanes 1,2. rRNA genes from 10 h nuclei; Žlanes 3,4. rRNA genes from 36 h nuclei; Žlanes 5,6. histone genes from 10 h nuclei; Žlanes 7,8. histone genes from 36 h nuclei.
genes. The calculated nucleosome repeat length for the rDNA was the same as for total chromatin and corresponded to 212 " 4 bp, which is close to that described in the literature w17,45,46x. A different picture was revealed in the Southern blots of early histone genes Ž Fig. 1, lanes 5–8., which were probed with the plasmid pCO2A, consisting of the complete S. purpuratus early histone repeat with all five histone genes, which are independently transcribed w47,48x. The amount of spacer DNA that is transcribed is unknown, because the 3X
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end of the mature mRNA is apparently produced by post-transcriptional processing w49,50x. At 10 h of development Ž lanes 5,6. early histone genes gave weakened nucleosome bands with a shorter nucleosome repeat length, characteristic of active early histone gene chromatin w32,42x. This indistinct ladder is the same in the coding and non-coding regions, indicating that the majority of even the non-coding sequences have altered nucleosome structure w32x. At 36 h of development Žlanes 7,8. the histone gene sequences acquired a stronger nucleosomal ladder with repeat length characteristic of bulk embryo chromatin. The presence of a weak nucleosomal ladder at 10 h might be due to structure present at the transcriptionally active loci, or to a fraction of the copies of the histone genes remaining in an inactive conformation.
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development, differ in accessibility to many restriction enzymes. In order to probe chromatin structure of early histone genes in their active and inactive stages, we searched for enzymes that have single restriction sites in the early histone gene repeat and could efficiently restrict crosslinked DNA of both development stages. Ten and 36 h nuclei were crosslinked with trimethylpsoralen for different times as indicated in Fig. 2, the DNA was isolated, restricted, separated on nondenaturing agarose gels, UV irradiated, vacuum blotted, and hybridized to pCO2A DNA. HindIII and EcoRI Žnot shown. failed to significantly digest crosslinked DNA. Sal I did not completely digest the crosslinked DNA. XhoI and SacII gave similar results Žnot shown. . SacI efficiently digested crosslinked early histone genes as well as ribosomal genes Ž not shown. and was used in further experiments.
3.2. Restriction analysis of psoralen crosslinked nuclei
3.3. Psoralen photocrosslinking of rRNA genes
Initial experiments showed that psoralen-crosslinked DNA, isolated from nuclei at 10 and 36 h of
Psoralen crosslinking to purified DNA depends upon extent of UV exposure, DNA sequence, and
Fig. 2. Restriction analysis of psoralen crosslinked DNA, isolated from nuclei of 10 and 36 h sea urchin embryo developmental stages. Nuclei were photocrosslinked with trimethylpsoralen. DNA was isolated, hydrolyzed with restriction enzymes and fragments were separated on 0.8% agarose gel. After reversal of crosslinks, DNA was transferred to nylon filters and hybridized with early histone gene probe pCO2A. ŽLane M. lrHindIII molecular weight marker; Žlane C. control uncrosslinked DNA digested with HindIII. Restriction enzymes and development times indicated at the top. ŽOdd lanes. 25 min crosslinking; Ževen lanes. 1 h crosslinking.
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supercoil torsion w51x. In our hands, crosslinking naked rDNA and lambda DNA for increasing times causes progressive retardation of the DNA on agarose gels. This retardation is not easily saturated. For example, the 6.6 kb lrHindIII fragment is retarded 2.2, 3.3, 4.3, 5.4, and 6.4% for crosslinking times of 0.5, 1, 1.5, 2, and 2.5 h Ždata not shown. . In nuclei, psoralen should bind to DNA, RNA, proteins and membranes w51–53x. Thus, the extent of crosslinking of nuclear DNA depends on specific properties of the nuclei, such as amount of RNA and protein present. To eliminate the potential differences in psoralen crosslinking of DNA in nuclei at the two development stages, we determined the crosslinking conditions that gave the same electrophoretic retardation of the inactive rRNA genes at both 10 and 36 h of development. Fig. 3 shows the results of crosslinking sea urchin rRNA chromatin for different times, as probed with the mouse rRNA probe, pSPTmr100. One hour of crosslinking was insufficient to completely shift the mobility of DNA from both develop-
Fig. 3. Analysis of psoralen crosslinked sea urchin ribosomal genes. After nuclei were crosslinked with psoralen DNA was isolated, restricted with Sac I and fragments were separated on a 1.5% agarose gel. DNA transferred to nylon filters and analyzed with ribosome gene specific probe pSPTmr100. ŽLane M. l r HindIII molecular weight marker; Žlanes 1,4. control uncrosslinked DNA, isolated from 36 h nuclei and digested with SacI; Žlanes 2,3. DNA of 1 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlanes 5,6. DNA of 2 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlane 7. 1 h crosslinked purified DNA isolated from 36 h nuclei.
Table 1 Percentage electrophoretic retardation of crosslinked sea urchin rRNA genes relative to uncrosslinked DNA Crosslinking time Žh.
DNA Ž%.
10 h nuclei Ž%.
36 h nuclei Ž%.
1 2
4.8 6.8
3.5 3.4
2.3 3.4
mental stages Ž lanes 2,3.. Two hours of UV irradiation Ž lanes 5,6. and longer Žnot shown. saturated and equalized band mobility. We conclude that 2 h trimethylpsoralen photocrosslinking eliminates the intrinsic differences in the conditions of irradiation of the nuclei and creates saturation conditions for the nuclei of both development stages studied. After 2 h crosslinking, the 4.4 kb rDNA band Žwhich contains ) 90% of the sequences that hybridize with the mouse 18S and 28S probe. was retarded by 3.4%. In contrast to the situation found in other cells, there was no band splitting for sea urchin rRNA genes at either development stage. The two weak bands at higher molecule weights Ž which could reflect sequence overlap with the probe or sequence heterogeneity. were similarly retarded and remained monodisperse. The restricted DNA from 2-h crosslinked nuclei was retarded only half as much as from 2-h crosslinked naked DNA ŽTable 1.. Thus, the rDNA crosslinked in the nuclei was highly protected from the photoreaction, as expected for inactive chromatin. No bands or shoulders were detected that migrated with greater or less retardation. The simplest interpretation of this result is that all or most all of the copies of the sea urchin embryonic rRNA genes have a homogeneous, ‘closed’ chromatin structure. To check our ability to reproduce the splitting of active rDNA bands seen by Sogo et al., we used mouse erythroleukemic cell nuclei as a positive control for ribosomal gene band splitting. Fig. 4 shows that after psoralen crosslinking, the rRNA separates into two bands, representing about 60% and 40% of active and inactive rRNA gene copies, respectively, in the agreement with the literature results w6x. Splitting of the bands occurred after 1 h, as well as after 2 h of psoralen photocrosslinking, showing that the effect can be achieved over a range of crosslinking conditions. The active and inactive rRNA fragments were retarded by 6.2 and 1.6% ŽTable 2. , with the
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very weak doublet at 10 kb is retarded too much to represent the active component of the 7.5 kb fragment, and is probably due to sequence heterogeneity or incomplete restriction, however the lack of a restriction map for the kangaroo rat makes any conclusion about these minor bands difficult. Thus, the experimental conditions for psoralen photocrosslinking were appropriate for resolving the differences between active and inactive copies of mammalian rRNA genes. 3.4. Psoralen photocrosslinking of histone genes
Fig. 4. Investigation of chromatin structure of rRNA genes by psoralen photocrosslinking assay in MEL cells and kangaroo rat liver cells. Nuclei were photocrosslinked with trimethylpsoralen and isolated DNA was processed as in Fig. 3. Filters were hybridized with ribosomal gene specific probe pSPTmr100. EcoRI was used as restriction enzyme. ŽLane M. l r HindIII molecular weight marker; Žlane 1. control uncrosslinked DNA from MEL cells; Žlanes 2,3. DNA from MEL cell nuclei crosslinked for 1 and 2 h, respectively; Žlane 4. 1 h crosslinked purified MEL cell DNA; Žlane 5. control uncrosslinked DNA from kangaroo rat; Žlanes 6,7. DNA from rat liver nuclei crosslinked for 1 and 2 h, respectively. s And f represent bands of slow and fast moving ribosomal gene copies, respectively.
active copies behaving almost like naked DNA. In the case of kangaroo rat liver nuclei, a broad tail formed on the 7.5 kb major rRNA band, similar to the results from tomato leaves and cell culture w8x, which seem to have only minor active fractions. The
The filter from the experiment shown in Fig. 3 was stripped and rehybridized with the early histone gene probe, pCO2A, containing the entire 6.5 kb sea urchin early histone gene repeat. Fig. 5 shows that after 2 h of psoralen crosslinking active Žlane 5. and inactive Žlane 6. early histone gene sequences are retarded by 5.2 and 4.0%, respectively Ž Table 3. . The small difference in mobility is unlike the finding in mouse rRNA genes, which exhibited almost a 5% difference in mobility between the active and inactive copies. As in the case of the rRNA genes from sea urchin, even the inactive copies underwent appreciable crosslinking. The active histone genes were still substantially protected from psoralen crosslinking, relative to the active rRNA genes of mouse. The incomplete psoralen crosslinking is consistent with results in yeast showing that several pol II genes are partially protected from crosslinking w54,55x. The observed differences between the active and inactive stages for the early histone genes are consistent with increase in the nucleosome density or decrease in the accessibility of the nucleosomes when the genes are inactivated. Unlike the situation with active rDNA genes from other organisms, however, the DNA from crosslinked active histone genes was not split into two bands representative of active and
Table 2 Percentage electrophoretic retardation of the 4.4 kb band representative of the majority of the 18S and 28S crosslinked mouse rRNA genes relative to uncrosslinked DNA Crosslinking time Žh.
DNA Ž%.
Active component Ž%.
Inactive component Ž%.
1 2
4.3 6.0)
4.7 6.2
1.6 1.6
)Extrapolated from results with sea urchin DNA.
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4. Discussion
Fig. 5. Trimethylpsoralen photocrosslinking of sea urchin early histone genes. The filter in Fig. 3 was stripped and rehybridized with early histone gene probe pCO2A. ŽLane M. l r HindIII marker; Žlanes 1,4. control uncrosslinked DNA, isolated from 36 h nuclei and digested with SacI; Žlanes 2,3. DNA of 1 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlanes 5,6. DNA of 2 h crosslinked nuclei from 10 and 36 h embryos, respectively; Žlane 7. 1 h crosslinked purified DNA isolated from 36 h nuclei. The active early histone gene repeat is crosslinked to a greater extent than the inactive repeat even after a saturation dose of crosslinking. Psoralen crosslinking does not resolve either the active or inactive genes into separate bands.
inactive chromatin conformations. The simplest interpretation of this result is that most of the copies of the genes have the same chromatin structure. Despite the similar migration of the active and inactive genes, from the intensity profiles from the PhosphorImager data we estimate that as little as 10% of an inactive conformation would have given rise to a detectable shoulder on the bands in Fig. 5, lanes 2 and 5. We interpret this data as evidence that almost all copies of these genes are present in the ‘open’ state, suggesting that copy selection is not used in the regulation of the histone genes at 10 h of development. Table 3 Percentage electrophoretic retardation of crosslinked sea urchin histone genes relative to uncrosslinked DNA Crosslinking time Žh.
DNA Ž%.
10 h nuclei Ž%.
36 h nuclei Ž%.
1 2
6.0 8.6
4.9 5.2
2.5 4.0
Under saturating conditions, psoralen makes covalent linkages in DNA that is not protected by nucleosome core particles. DNA in an open chromatin conformation or free from proteins is more frequently crosslinked. The frequency of crosslinking can be assayed on an agarose gels. Conconi et al. w6x showed that crosslinking of rRNA genes gave a bimodal distribution of products, a slowly migrating transcriptionally active fraction and a rapidly migrating inactive fraction. Similar heterogeneity in rDNA structure was found in different organisms and in different physiological states w6,8,9x. In contrast, psoralen crosslinking of single copy pol II genes in yeast cells have shown a homogeneous, protected structure in both the active and inactive transcriptional states w54,55x. We have investigated the heterogeneity of chromatin structure of ribosomal RNA genes during sea urchin early embryogenesis using psoralen photocrosslinking to electrophoretically separate the active and inactive copies of the genes. We performed the experiments on nuclei isolated in the presence of n-butyrate, which should eliminate any potential effects of histone deacetylation upon psoralen crosslinking. Sea urchin rRNA genes have similar DNA organization as in some other higher eukaryotes. The length of repeat unit, containing 18S and 26S RNA genes, is 10–12 kb w43,44x. The units are clustered and repeated about 50 times w20x, and are transcriptionally inactive at cleavage and gastrula stages. Large amounts of rRNA are stored in the eggs and immediately utilized after fertilization to support rapid cell division w24,56,57x. By using micrococcal nuclease we showed that the chromatin in the coding region of these genes has a canonical, organized nucleosomal organization at 10 and 36 h of developmental, consistent with the observed low transcription rates of about 0.4 transcripts per minute w20x. Psoralen crosslinking showed that the sea urchin rRNA genes were uniformly crosslinked at 10 and 36 h of development, as if the coding regions were in a single conformation. The simplest interpretation of this result is that most of the gene copies were in the inactive conformation, consistent with the results of micrococcal nuclease digestion. However, the retardation of the sea urchin rRNA genes was substantially greater than
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that found for inactive mouse rRNA genes. Perhaps this effect is due to the fact that the rRNA genes are active earlier as well as later in development and therefore might carry some of the properties of active chromatin. Alternatively, the micrococcal nuclease patterns and psoralen crosslinking might reflect different aspects of chromatin structure, which might only sometimes be correlated with transcriptional activity. For example, psoralen crosslinking might be promoted by hyperacetylation of the histones, which could sometimes be correlated with transcriptional activity, as in studies of rat rRNA genes w58x, but not correlated with transcriptional activity and nucleosome positioning in sea urchin embryos. Another possibility is that the longer length of the linker DNA between nucleosome cores could lead to appreciable exposure of the DNA to psoralen even when fully occupied by nucleosomes. The sea urchin early histone genes are repeated about 500 times, comprise about 0.15% of the sea urchin genome, and are transcribed by RNA polymerase II at a moderate rate of 0.5–1 transcripts per gene per minute Žassuming all copies are active. w20x. This rate is comparable to other polII genes, substantially greater than the average rate of 0.2 transcripts per minute in the embryos, but less than the maximal rate for pol II genes in S. purpuratus w59x. The highest transcriptional activity of the early histone genes is during the early blastula stage. The transcripts are barely detected later at gastrula. The genes have positioned nucleosomes in their inactive state and a random distribution of nucleosomes in their active state w33x. Our analysis of psoralen-crosslinked early histone genes found a small, but easily detectable difference in mobility of the histone genes when they were active and inactive, but did not detect any splitting of the histone band at either stage of transcriptional activity. The extent of crosslinking of the active histone genes is considerably less than for naked DNA, suggesting that even in the active conformation there is appreciable protection from nucleosomes. This is in contrast to the rRNA genes in mouse and yeast, where the active genes were as highly crosslinked as naked DNA. However, unlike previous psoralen results from pol II genes in yeast, the histone genes in the active state were crosslinked significantly more frequently than those in the inactive state. Thus the level of exposure of active histone
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genes to psoralen was intermediate between that for multicopy polI genes and single copy pol II genes that have been studied previously.
5. Conclusion Even this moderate difference between the crosslinking of active and inactive histone genes should have revealed the use of copy selection in the regulation of early histone genes, yet we did not detect a second peak or shoulder representing nucleosome-free active, or highly-protected inactive conformations. The fact that only a single band was found after psoralen crosslinking suggests that the sea urchin has met the needs for histone mRNA by regulating the rate of transcription of a large number of genes, rather than regulating the fraction of genes that are active. The difference found between the behavior of rRNA genes in other systems Ž which have heterogeneous structure., and the early histone genes Ž which have homogeneous, more protected, structure. suggests that there might be a difference in the role of chromatin structure in regulation of pol I and pol II transcription of multicopy genes. The observed differences might be correlated with possible differences that nucleosome displacement, acetylation, or supercoiling play in transcription of polI and pol II genes w58,60x.
Acknowledgements We are very thankful to Dr. A. Conconi for a gift of the rRNA gene plasmid pSRTmr100, Dr. Weinberg for early histone gene plasmid pCO2A, and S. Lejnine for the gift of kangaroo rat liver nuclei. This research was supported by NSF MCB9514196.
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