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A rat histone H4 gene closely associated with the testis-specific H1t gene
Experimental
Cell Research 173 (1987) 534-545
A Rat Histone H4 Gene Closely Associated Testis-Specific HI t Gene
with the
SIDNEY GRIMES,*+ PAUL WEISZ-CARRING’IGN,” HENRY DAUM III, *V2 JOHN SMITH,? LINDA GREEN,S KENNETH WRIGHT,S GARY STEIN,* and JANET STEINS *Research Service (ISI), Veterans Administration Medical Center, Shreveport, Louisiana 71130: TDepartment of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 73130; fDepartment of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, Florida 32610; and ODepartment of Immunology and Medical Microbiology, University of Florida, College of Medicine, Gainesville, Florida 32610
A rat histone H4 gene closely associated with the testis-specific Hlt gene was isolated by screening the Sargent-Bonner rat genomic library using cloned human histone genes as probes. Both the H4 gene and the Hlt gene are located on a 7-kb EcoRI genomic DNA fragment. Although the deduced amino acid sequence of the rat H4 histone is identical to that of the sequence of human histone H4, the nucleotide sequence of the coding region differs significantly from the coding region of the human H4 gene. Moreover, the relative spacing between the 5’-consensus sequence elements is unique for an H4 gene. Slnuclease protection analyses reveal that both the H4 and Hlt mRNA species are present in a fraction of rat testis cells highly enriched in pachytene spermatocytes, while only the H4 mRNA species is present in a rat myeloma cell line (Y3-Agl.2.3). During a l-h hydroxyurea treatment of the Y3 cells, which produces a 99% inhibition of DNA synthesis, the level of this H4 mRNA drops by only 50%, indicating that the stability of this mRNA is only partially coupled with DNA synthesis. @ 1987 Academic press, IIIC.
Histone H4 plays a central role in nucleosome formation [l-2]. The highly conserved amino acid sequence of this histone reveals the essential nature of the sequence. On the other hand, the nucleotide sequence of histone H4 mRNA is not conserved to the same degree. For example, at least seven distinguishable histone H4 mRNA species are expressed in HeLa cells [3]. A summary of several amino acid and nucleotide sequences of H4 and the other histones has been published recently [4]. Mammalian germinal cell histone variants of all of the histones except histone H4 have been reported. A rat germinal variant Hlt is the only Hl variant which is synthesized in pachytene spermatocytes [5-6]. A testis-specific H2A variant TH2A [7] and a testis H2B variant TH2B [8] are also synthesized predominantly in pachytene spermatocytes, and a testis H3 variant TH3 191 and an H2A variant H2A.X [7] are synthesized in spermatogonia. These mammalian germinal cell ’ TO whom reprint requests should be addressed at Veterans Administration E. Stoner Ave., Shreveport, LA 71130. 2 Present address: FMC BioEroducts, 5 Maple Street, Rockland, ME. Copyright @ 1967 by Academic Press, Inc. AU rights of reproduction in any form reserved 0014-4827/87 $03.00
534
Medical Center, 510
Rat histone genes 535 histone variants are clearly products of unique genes which are differentially expressed during spermatogenesis. In the present paper we report the isolation and sequence analysis of an H4 gene located with 1300 base pairs of the rat Hlt gene. The data presented reveal that mRNA from this H4 gene and the closely associated Hlt gene are present in testis pachytene spermatocytes, and thus both the H4 gene and the Hlt gene appear to be expressed in pachytene spermatocytes contributing to the unique pattern of histones found in germinal cells. In addition, the H4 gene is expressed in rat myeloma cells, and the H4 mRNA is quite stable when the cells are treated with hydroxyurea at concentrations which effectively block DNA synthesis. MATERIALS
AND METHODS
Materials. [‘H]Thymidine was purchased from New England Nuclear. [a-‘*P]dCTP (600 Ci/mmol), [a-‘*P]dATP (600 Ci/mmol), and [Y-~*P]ATP (4000 Ci/mmol) were ordered from ICN. X-ray film was ordered from Eastman Kodak (XOmat XAR-5) and DuPont (Cronex 7) and hydroxyurea was ordered from Sigma. Restriction enzymes, plasmids, and Ml3 bacteriophage RF DNA cloning vectors were ordered from Bethesda Research Laboratories and IBI. Other enzymes were ordered from Boehringer-Mannheim Biochemicals. Cell culture and pulse labeling. Rat myeloma cells (Y3-Agl.2.3) obtained from the American Type Culture Collection were grown exponentially in RPM1 media. DNA synthesis inhibition by hydroxyurea was followed by measuring the incorporation of [‘Hlthymidine (5 @i/ml, 1x 106 cells/ml) into 10% (w/v) trichloroacetic acid-precipitable material in a 2-h pulse. Hydroxyurea was added and the cells were incubated for 1 h before the addition of [‘Hlthymidine. Screening a bacteriophage L library. A genomic library constructed using bacteriophage 1 strain Charon 4A and a partial EcoRI digest of liver DNA from a Sprague-Dawley rat [lo] was kindly supplied by Thomas Sargent. The technique of phage hybridization [ll] was performed utilizing human histone gene probes pF0108A (H4) [12] and pFNCl6A (Hl) [13] to screen the library. The probes were labeled by nick-translation with [32P]dCTP [14]. Hybridization was conducted in a solution composed of 4xSSPE (0.72 M NaCl, 0.04 M NaHrPO,, 4 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4), SxDenhardt’s ((0.04% (w/v) Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin (BSA)), 0.1% sodium dodecyl sulfate (SDS), and 0.1 mg/ml Escherichia coli DNA, and a stringent wash was conducted in a solution composed of 1x SSPE and 0.1% SDS. Both the hybridization and wash were conducted at 65°C when using the H4 probe and at 60°C when using the Hl probe. All experiments involving recombinant DNA techniques were performed within the NIH guidelines for research involving recombinant DNA. Isolation of DNA and RNA. Bacteriophage 1 was prepared from liquid culture [15, 161, and I DNA was extracted by adjusting the solution to 20 mM EDTA, 50 ug/ml proteinase K, and 0.5% SDS, followed by phenol extraction and dialysis against a solution containing 10 mM ‘Iris, pH 7.5, and 1 mM EDTA. Plasmid DNA, prepared by alkaline lysis [17], was purified by equilibrium density centrifugation for 16 h on CsCl-ethidium bromide gradients in a Beckman VTi65.2 rotor followed by treatment with T4 RNase and centrifugation through 1 M NaCl in an SWSO. 1 rotor. Total cellular RNA was isolated by the guanidinium isothiocyanate-cesium chloride method [18] from enriched rat testis germinal cell types prepared by centrifugal elutriation [19] and from rat myeloma Y3 cells. The cellular composition of the enriched germinal cell types has been reported [19]. The concentration of RNA in each sample was determined by measuring the optical density at 260 nm. Analysis of DNA and RNA. DNA restriction fragments were separated by electrophoresis through agarose gels in buffer composed of 90 mM Tris, 90 mM boric acid, 2 mM EDTA containing 0.5 &ml ethidium bromide. DNA was transferred to nitrocellulose by the Southern blotting technique 1201. Although phage hybridization using heterologous probes was conducted using aqueous conditions, hybridization to ‘*P-labeled DNA probes in Southern blot experiments was conducted in 50% formamide solution containing 5xSSC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.4), SxDenhardt’s (-BSA), 0.1% SDS, and 0.25 mg/ml E. coli DNA at 49°C with homologous probes 1211. Total cellular RNA was analyzed under denaturing conditions by electrophoresis in agarose gels containing formaldehyde [22]. The gels were soaked to remove formaldehyde, and RNA was trans-
536 Grimes et al. ferred to nitrocellulose by the Northern blotting technique [23], conducting hybridization in 50% formamide solution at 37°C as described for the Southern blotting technique with probes which were “P-labeled by nick-translation. A stringent wash was conducted in a solution of 0.1 x SSC and 0.1% SDS at 60°C. Cloning of DNA fragments. DNA fragments were isolated from low melting agarose gels (FMC BioProducts) by melting sections of agarose gels at 70°C in at least 4 vol of low salt buffer (0.1 M NaCl, 10 mM Tris, pH 7.5, 1 rr& EDTA) and purification of DNA by binding to an Elutip column (Cooper Biomedical Corp.), washing with low salt solution, and elution with 0.5 ml high salt solution (1 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA). The plasmid pPS3 was constructed from the 7-kb EcoRI genomic fragment containing both the Hlt and H4 genes. The plasmid pPS7 was constructed using the 1.5kb Sau3A fragment cut from pPS3 containing 197 bp of the 5’-coding region of H4 and an additional 1.3 kb of 5’-upstream noncoding region. The plasmid pHD1 was constructed using the 1.2kb PuuII-Hi&III fragment containing 320 bp of 5’-coding region of Hlt and 980 bp of 5’upstream noncoding region (tilled using the Klenow fragment of DNA polymerase I to form blunt ends). These plasmids were constructed to aid in the production of probes to be used in Southern blot and Northern blot analyses and in Sl-nuclease protection analyses. E. coli strain JM83 was transformed [24] using the recombinant plasmids and grown on L-Broth plates with ampicillin using 5-bromo-4-chloro-3indolyl-/I-u-galactoside (X-Gal) as color indicator. SI-m&ease protection analyses. Sl-nuclease protection analyses were performed to determine the cap sites of the mRNAs on the histone H4 and histone Hlt genes [25]. In these experiments the 1.5-kb Sau3A DNA insert from pPS7 (H4 probe) and a l.l-kb EcoRI-AL& insert fragment from pHD1 (Hl probe) were dephosphorylated and 5’-end-labeled with polynucleotide kinase and [y-‘*P]ATP. A sample containing approximately 10 ug of total cellular RNA from a specific testis cell type or from rat myeloma Y3 cells was mixed in hybridization buffer with the labeled probe, and the mixture was heated at 79°C to denature the DNA and hybridized for 3 h at 60°C for the H4 probe or 50°C for the Hl probe to allow formation of DNA-RNA hybrids. The mix was treated with Sl-nuclease to digest single-stranded nucleic acid and analyzed by polyacrylamide gel electrophoresis with a sequencing ladder and in separate experiments with “P-end-labeled DNA fragments of known length in adjacent lanes to determine the exact length of the protected DNA fragment. Sequence analysis of DNA. DNA fragments to be sequenced were cloned using bacteriophage Ml3 mp18 or mp19 [26] as vectors and the recombinants were used to transform E. cofi strain JMlOl [24]. The transformants were plated on YT plates containing X-Gal, clear plaques were selected, and DNA was prepared [24]. The Sanger dideoxy sequencing method [27] was used to sequence the cloned fragments. Autoradiograms were prepared using Dupont Cronex 7 film without an intensifying screen and analyzed with the aid of an IBI gel reader and the IBI/Pustell DNA sequence analysis system using an IBM-XT microcomputer. Further sequence analyses were accomplished using the Bionet resource of IntelliGenetics, Inc., Mountain View, California.
RESULTS Since several human histone genes have been isolated and sequenced, these served as probes for screening a rat genomic library. Duplicate filters were lifted from each plate containing E. coli infected by the bacteriophage library and one of each pair of filters was hybridized to 32P-labeled human H4 probe pF0108A in aqueous solution and the duplicate filter was hybridized to 32P-labeled human Hl probe pFNC16A. After three rounds of screening, two of the selected clones hybridized to both the HI and H4 probes. A third clone hybridized only to the H4 probe. Single and double restriction enzyme digestions were conducted utilizing DNA isolated from each of the three bacteriophage clones. DNA fragments were separated by electrophoresis on agarose gels and transferred to nitrocellulose by Southern blotting. Blots were initially probed separately with 32P-labeled pF0108A (H4) and pFNC16A (Hl). A partial restriction map of one of the clones containing genes for both histones H 1 and H4 is presented in Fig. 1 A. In one of
Rat histone genes
s
HSH
Hli SE
I IlJ IIll Hlt
B
E
E
HE
I I I, .II.
H
.I.
537
E
I
H4
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Fig. 1. (A) Restriction map of the genomic insert containing the Hlt and H4 histone genes. Single and double restriction digestions were conducted and the resulting DNA fragments were analyzed electrophoretically on agarose gels as described under Materials and Methods (E, EcoRI; H, Hi&III; Ps, PsrI; Pv, PuuII; and S, Sau3A). The orientation of each gene is designated by an arrow below the gene. (B) Detailed restriction map of the H4 gene with the sequencing strategy. Single and double restriction digestions were conducted and DNA fragments analyzed as described in (A). (H, HindIII). The heavy arrow designates the mRNA cap site. The light arrows designate the sequencing strategy. Note that the orientation of the H4 gene is reversed with respect to the orientation in (A).
the other two clones, which lacked a detectable Hl gene, the histone H4 gene was present on a 3200-bp Hi&III fragment, indicating that it is located in a genomic region significantly different from the one presented in Fig. 1B. Figure 1 A reveals the close proximity of the H4 gene to the Hl gene in the first of these genomic fragments. The Hl and H4 genes reside together on a 7-kb EcoRI fragment, separated by only about 1300 bp of DNA, and are oriented with their 3’-termini facing each other. DNA fragments containing all or parts of the genes and regions flanking the genes were cloned into the pUC series plasmid vectors to be used as hybridization probes and into Ml3 mp18 or mp19 for sequence analysis. The identity of the Hl gene as the Hlt gene was revealed by restriction mapping (Fig. 1 A) and sequencing (data not shown), and comparison of our data with the published restriction map and sequence of the rat Hlt gene [6]. Sequence analysis of the 440-bp Hind11 fragment (Fig. 2) reveals that this sequence contains all of, the coding region, about 90 bp of the flanking 3’noncoding region and 35 bp of the flanking 5’noncoding region of the H4 gene. Since a unique Sau3A site exists near the center of this gene, the 440-bp Hid11 fragment was cut with Sau3A to obtain two HimfIIESau3A fragments. These two smaller fragments were force cloned into mp18 which had been cut with Hi&III and BamHI and sequenced to complete the analysis of the entire 440-bp fragment. In order to obtain additional flanking 5’-noncoding sequence of this H4 gene, we isolated and sequenced a 236-bp HhaI fragment which overlaps the 5’Hi&III site (Fig. 1 B). Thus, the sequence was extended to 189 bp upstream from the start codon.
538
Grimes et al. -189 -130
CGCAMGAA
TAGCTGAGGT TTGCTAGCCA ATGAAAACAT TCAGATTGCA ATGACTCATC
CTTGTTGCCC CACCCCCTTT
-70 TT~CA~GCAGAAG -10 AUCAACC
CAAGGACCTG ATTCGAAAAC
CCCTCCTAAC
GCCTGTGGTC
GTCTATTTAA A$G"AGCTT
+ GCTTTCTTCC CTKACTTTT
ATG TCT GGR CGT GGT F&A GGT GGT AAA GGG CTT GGG AAA GGT kt Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly LYS Gly 10 1
43 GGC GCT AAG CGT CAT CGC AAA GTC CTG CGC GAC ARC ATC CAG GGC ATC ACA AAG Gly Ala Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile l$r Lys 20 97 CCC GCC ATC CGC CGC CTG GCC CGG CGC GGA GGA GTG AAG CGC ATC TCC GGC CTC Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser Gly Leu 40 151 ATC TAC GAG GIG ACC CGC GGT GTG CTG AAG GTG TTC CTG GAG AAC GTG ATC CGC Ile Tyr Glu Glu Thr Aq Gly Val Leu Lys 1'1 Phe Leu Glu Asn Val Ile Arg 50 205 6AC GCC GTC ACC TAC ACG GAG CAC GCC AAG CGC AAG ACG GTC ACT GCC ATG GAC Asp Ala ;;l Thr Tyr Thr Glu His Ala Lys Arg Lys ;hOr Val Thr Ala Met Asp 259 GTG GTC TAC GCG CTT AAA CGC CAG GGC CGC ACA CTC TAC GGC TTC GGT GGT TAA Val Val Tyr Ala ;a Lys Arg Gin Gly Arg Thr Leu Tyr Gly Phe Gly Gly l 100 313 ACTWTAATG
CGCTTTCCTT AGCTGTTGCA AAAGGCCCTT t
373 CATTWAAGG ,
GTTGTTCACT TITCGMMTT .
Fig. 2. Nucleic acid sequence of the rat histone H4 cloned into bacteriophage M13mp18 and M13mp19 and codon and the coding region is translated. Underlined arrow indicates the H4 mRNA cap site determined by
TTCAGGGCCA CCCACGWT I
gene. DNA fragments noted in Fig. 1B were sequenced. Numbering starts at the ATG start regions represent consensus sequences. The Sl-nuclease protection analysis.
These results, summarized in Fig. 2, reveal the start and stop codons as well as the specific upstream elements described by Wells [4]. The underlined upstream sequence elements include a CAAT sequence at -68, GGTCC at -62, TATTTkAA at -47, and CTCA at -9. A region conspicuously absent is a purine-rich sequence sometimes present in H4 genes immediately upstream from the CAAT sequence [4]. The other very unusual aspect about this upstream region is the relative spacing between the sequence elements just described. Specifically, there are usually 15 to 58 bases between the ATG start codon and the CTCA consensus sequence, 15 to 22 bases between the TATAAATA sequence and the CTCA sequence, and 5 to 58 bases between the CAAT sequence and the GGTCC sequence [4], while in the H4 gene being described in this paper there are 5, 30, and 2 bases, respectively, between these elements. Consensus sequence elements downstream from the stop codon include GGCCCT’MTCAGGGCC at 342, ACCCA at 362, and CAAAGGGTTG at 377. The first of these directs the formation of a hairpin structure in the H4 mRNA, and this palindromic element and the contiguous ACCCA element mark the site of the H4 mRNA terminus [28]. Nothing unusual is apparent in these consensus sequence elements or in the relative spacing of these elements in the flanking 3’-noncoding region. The protein coding region, presented in Figure 2, reveals that this gene codes for a histone H4 which is identical in amino acid sequence to the consensus
Rat histone genes Y3
I
539
TESTIS
2
12345 28s 18s
H4mRNA
Fig. 3. Detection of histone H4 mRNA by Northern blot analysis. Total cellular RNA was prepared as described under Materials and Methods, and 20 ug of each RNA sample was electrophoresed on a denaturing agarose gel. The RNA was transferred to nitrocellulose and analyzed by the Northern blotting procedure using a [32P]-labeled 440-bp Hind11 fragment containing histone H4 as the probe. The RNA samples are as follows: RNA from rat myeloma Y3 cells, untreated control (I), and 1mM hydroxyurea treated (2); RNA from five enriched populations of adult rat testis germinal cells, the three fractions of greatest purity being late spermatids (steps 13-19) (I), early spermatids (steps 1-8) (3), and pachytene spermatocytes (5). The relative migration rates of 18 and 28 S rRNA are shown for size markers.
sequence for this histone [4]. The nucleotide sequence of the coding region of this gene is also highly conserved with 62 of 102 codons identical to those in the consensus nucleotide sequence of the H4 gene [4]. Of these 62 identical codons, 27 are highly conserved codons found in vertebrate histone H4 genes [4]. The nucleotide sequences of the flanking noncoding regions of this gene are much less conserved. In order to examine the expression of rat histone H4 genes, Northern blot analyses were conducted using total cellular RNA isolated from a rat myeloma cell line (Y3 Ag1.2.3) and a number of rat tissues including enriched fractions of rat testis germinal cells. The blots were probed with our rat histone H4 probe. Interestingly, we observed that the members of the H4 mRNA family detected in Y3 cells under our hybridization conditions are quite resistant to inhibition of DNA synthesis in contrast to the behavior of most histone mRNAs (Fig. 3A). Thus, 1 mM hydroxyurea treatment, which inhibits incorporation of [3H]thymidine into rat Y3 cells by more than 99% failed to significantly reduce the level of H4 mRNA to less than 50% of the control levels. Although the wash conditions used for our blots were quite stringent, the Northern blot method is limited in specificity for highly homologous mRNAs, and we are most likely measuring several histone H4 mRNA species. If this is the case, a significant amount of H4 mRNA is relatively stable when DNA synthesis in the Y3 cells is blocked by hydroxyurea. Furthermore, histone H4 mRNA was most abundant in a population of germinal cells enriched in rat testis pachytene spermatocytes in which there is no DNA replication (Fig. 3, lane 5). Much less histone H4 mRNA was detected in the other enriched populations of rat testis germinal cell types in this experiment which included early spermatids (steps 1-8) (Fig. 3 B, lane 3) and late spermatids (steps 13-19) (Fig. 3B, lane I). Sl-nuclease protection assays were conducted to provide evidence for the
540 Grimes et al. GlAT2C
-217
Fig. 4. Detection of histone H4 mRNA in rat myeloma Y3 cells by Sl-nuclease protection analysis. The 1.5kb Sau3A fragment which spans the 5’-half of the coding region and the flanking 5’-noncoding region of the H4 gene (Fig. 1 A) was used as an Sl-nuclease probe. Approximately 10 pg of total cellular RNA from untreated Y3 cells was used in the assay. A sequencing ladder derived from the 1.9-kb Hi&III fragment containing the 3’coding region of the Hl t gene and flanking the H4 gene (Fig. 1) is used as a size marker. The number of bases represented by the largest protected fragment is indicated. The sample in lanes marked 1 and 2 were treated with 100 and 400 units of Sl-nuclease, respectively.
presence of the specific H4 and Hlt mRNAs relevant to this study. The data presented in Fig. 4 indicate that mRNA encoded by this specific rat histone H4 gene is present in rat myeloma Y3 cells. The larger protected DNA fragment (lanes 1 and 2) marked in the figure is 217 bases in length based upon its electrophoretic mobility compared to sequencing ladders and compared to DNA fragments of known length run in parallel experiments, which indicates that adenine at position -16 (Fig. 2) represents the cap site of the H4 mRNA. A second protected fragment 200 bases in length presumably represents H4 mRNA species which protect the DNA probe only to the AUG codon. Note that the smaller protected fragment is shortened (compare lane 2 to lane I) when higher Sl-nuclease concentrations are used (100 units-lane I, 400 units-lane 2) while the larger fragment is not shortened, which suggests less-perfect base pairing for the smaller fragment. Treatment of the myeloma cells for 60 min with 10 rnM hydroxyurea (Fig. 5A, lane 5) leads to a 50% reduction in the level of the two histone H4 protected fragments compared to untreated controls (Fig. 5A, lane 6) as approximated from densitometric scans of autoradiograms . Additional S 1nuclease protection analyses revealed that both types of these histone H4 mRNA species are present and most abundant in pachytene spermatocytes (Fig. 5, lane 9), but a trace of these H4 mRNA species can be detected in populations of testis cells enriched in late spermatids and early spermatids (Fig. 5, lanes 7 and 8), presumably due to contamination of the purified cell types with other testis cell types. However, note in the testis cells that the H4 mRNA species which results in the protection
Rat histone genes
541
Fig. 5. (A) Histone mRNA in Y3 and testis germinal cells. Total cellular RNA (10 ug) from control rat Y3 cells (untreated), hydroxyurea-treated Y3 cells, or rat testis germinal cells was used for each Sl-nuclease protection assay. Y3 cells were treated with hydroxyurea as follows: control (no hydroxyurea) lanes I knes a and 6; 0.5 mM, lane 2; 1 ti, lane 3; 5 mM, lane 4; 10 n&f, lane 5. The last three lanes represent RNA from late spermatids (steps 13-19) (lane 7), early spermatids (steps l-8) (lane 8), and pachytene spermatocytes (lane 9). (A) H4 probe as in Fig. 4. (B) Hlt probe (see Materials and Methods).
of the larger protected DNA fragment represents approximately 10 % of the total H4 mRNA which provides protection to the DNA probe. The same RNA samples used in Sl-nuclease protection analysis with the H4 probe in Fig. 5A were also used in Sl-nuclease protection analyses with the Hlt probe. The size of the protected fragment (marked in Fig. 5B) indicates that adenine at position 1 (according to the published numbering scheme for the Hlt gene [6]) represents the cap site of the Hlt mRNA. Furthermore, the results shown in Fig. 5B reveal that the Hlt mRNA is most abundant in pachytene spermatocytes (lane 9) with only trace amounts in the fractions of cells enriched in early spermatids (lane 7) or late spermatids (lane 8). No Hlt mRNA could be detected in any of the Y3 cell RNA samples including those treated with hydroxyurea (lanes l-6). Analysis of histone proteins from Y3 cells by electrophoresis on acid-urea gels containing Triton X-100 also failed to reveal histone Hlt (data not shown). DISCUSSION Cloned human histone genes have been used successfully to screen a rat genomic library. Two of the three isolated bacteriophage clones which contained a histone H4 gene also contained a histone Hl gene. Detailed studies of histone genes indicate that in many organisms the genes are clustered with each cluster containing one copy of each of the five histone genes [30-311. The clone examined in this paper contains both the rat testis-specific histone Hlt gene and a closely associated rat histone H4 gene. Whether this genomic fragment contains additional histone genes or whether it is part of a larger fragment containing additional histone genes is unkonwn at the present time. Since histone H4 is the most highly conserved of the five histones with respect to amino acid sequence and histone Hl is the least conserved, we used a slightly different screening strategy for each of these probes. When using the human H4 probe, hybridization was conducted at 65°C but when using the human Hl probe, hybridization was
542 Grimes et al. conducted at 60°C; the lower hybridization temperature was chosen to allow a greater degree of mismatching. Fortunately, this strategy was adequate for the successful identification of both rat H4 and HI histone genes. It is presumed that this strategy was necessary for this purpose, but we did not test other temperatures. Tissue-specific histone variants replace at least in part the usual somatic-type histone variants in germinal cells during spermatogenesis in many organisms [32-341. Rat histone Hl species are designated Hla, b, c, d, e, and t [51, where Hla, c, and t are prominent in the testis germinal cells and Hlb, d, and e are somatic variants. Hlt is the only Hl variant synthesized in pachytene spermatocytes [5]. The switch seen in histone Hl gene expression in germinal cells is also seen for histones H2A, H2B, and H3 where the rat testis-specific or testisenriched variants are designated H2A.X, TH2A, TH2B, and TH3 [7-91. Recently, the TH2B variant gene has been isolated and sequenced (personal communication from Dr. Chi-Born Chae). However, a unique mammalian germinal cell histone H4 has not been described, which may reflect the absolute requirement for the highly conserved amino acid sequence of this histone. Nevertheless, synthesis of histones H4 and Hlt has been demonstrated in premeiotic pachytene spermatocytes which are not active in DNA synthesis [35-361. Along these lines, it has been reported [36] that synthesis of histones H4 and Hlt occurs at significant levels in germinal cells even in the presence of hydroxyurea (50% of control levels for H4 and 90% of control levels for Hlt) which blocks testis DNA synthesis by 90% in those cells active in DNA synthesis and normally leads to a rapid turnover of histone mRNA [29, 371. Other related work [37] indicates that high concentrations of DNA synthesis inhibitors can have a significant inhibitory effect on histone gene transcription, which we presume may occur in our experiments as well. However, the level of transcription has not been determined in the experiments described in the present study. In any case the data clearly show that we have isolated the testis-specific Hlt and a closely associated H4 gene both of which are under the control of regulatory mechanisms which lead to the expression of these histone mRNAs in pachytene spermatocytes which are not active in DNA synthesis. Our restriction mapping, subcloning, and sequencing data reveal that the Hl gene presented in this report is in fact Hlt by comparison to the published restriction map and sequence [6]. The H4 gene closely associated with this Hlt gene appears to be functional in synthesis of H4 mRN,A in pachytene spermatocytes and in rat Y3 cells. We speculate that the H4 gene reported in this paper contributes to histone H4 synthesis in rat testis pachytene spermatocytes. The close association of this H4 gene with the testis Hlt suggests that these two genes may share some aspects of gene regulation perhaps by virtue of being part of the same chromosomal domain. The structural juxtaposition of the rat Hlt and H4 genes most closely resembles the genomic organization of some early sea urchin, some amphibian, and some chicken histone gene clusters [30]. At least one human Hl gene is located between two core histone genes [13], but in this case the core histone genes are H2B and H3. There is clearly a developmental
Rat histone genes
543
regulation of expression of histone genes in eukaryotes as demonstrated by the differential expression of sea urchin early, late, and testis sperm histone genes [301. The early Hl and core histone genes are clustered as are the early histone genes of many other organisms where fertilized eggs enter a period of extremely rapid cell division requiring large quantities of histones for chromatin assembly. In this case the coordinate expression of the clustered histone genes is facilitated by the close proximity of the genes to each other. By analogy we speculate that the close structural association of the rat Hlt and H4 genes facilitates the coordinate expression of these two genes in premeiotic pachytene spermatocytes. The biological significance of the testis histone variants is unknown [32], but the coordinate expression of testis-specific histone genes is widespread in eukaryotes, being seen in species as divergent as sea urchins, amphibians, rodents, and primates including humans [30-341. There is an obvious requirement for histone synthesis presumably for restructuring of the premiotic chromosomes which occurs in the absence of DNA synthesis in pachytene spermatocytes. In the rat this requirement is fulfilled at least in part by the coordinate expression of the testis-specific histone genes Hlt (THl), TH2A, TH2B, TH3, and the H4 gene closely associated with Hlt. It will be important to determine whether the other rat testis-specific core histone genes are also in close proximity to the Hlt and H4 genes. The 5’-flanking sequence elements of the H4 gene are unusual in that a consensus purine-rich sequence is absent altogether, that CAAT sequence is unusual, and the other consensus sequence elements have a unique relative spacing. The relative spacing of flanking 5’-promoter elements is critical for stereospecific alignment and cooperative association of transacting factors in eucaryotic genes [38]. Thus, the unusual 5’-noncoding region may play a key role in the expression of this histone H4 mRNA species which is quite stable in pachytene spermatocytes and perhaps in other cell types including rat myeloma Y3 cells treated with hydroxyurea. The Sl-nuclease protection data indicating only a 50 % drop in the level of histone H4 mRNA in rat myeloma cells treated with 10 mM hydroxyurea for 1 h and published data on Hlt [36] indicating continued synthesis during hydroxyurea treatment suggest that this rat histone H4 gene and the Hlt gene are similar in this respect to some cell cycle-independent histone genes. Cell cycle-dependent histone mRNA levels normally drop to low or undetectable levels within 15 to 30 min in cells treated with 1 mM hydroxyurea due to a greatly increased turnover rate of histone mRNA [29, 371. However, this particular histone H4 gene as well as the Hlt gene have no intervening sequences or polyadenylation signals, both of which are found in some basally expressed histone genes [39]. Although, the available evidence indicates that the histone H4 gene and the closely associated Hlt gene are both expressed in pachytene spermatocytes, it appears that this H4 gene is also expressed in rat myeloma cells. On the other hand, there appears to be a tissue-specific expression of the Hlt gene only in pachytene spermatocytes. Therefore, the H4 gene may contribute to the synthesis of H4 mRNA in cells which are active in DNA synthesis such as Y3 cells as 35-878342
544 Grimes et al. well as in those which are inactive in DNA synthesis such as rat testis pachytene spermatocytes. If this behavior can be confirmed in other cell types, then the expression of this gene in somatic cells may contribute to the H4 mRNA pool which is synthesized by cell cycle-independent or basally expressed histone genes [40]. We have also identified a second rat histone H4 gene in another bacteriophage L clone isolated from the same rat genomic library. The restriction map of this second H4 gene is quite different from the map of the H4 gene presented in this report. It will be important of compare the two H4 genes in greater detail and examine the pattern of expression of these two histone genes in various rat tissues and specific cell types including the germinal cells at different stages of maturation. We acknowledge the excellent technical assistance of Jeffrey Anderson, Pamela Smart, and Janice Yager. Some data analysis was conducted with the Bionet computer resource of IntelliGenetics, Inc., Mountain View, California supported by NIH Grant 1 U41 RR-0168503. This research was supported by the Medical Research Service of the Veterans Administration and by BRSG Grant 2 SO7 RR 0582206 (administered by LSU Medical Ctr., Shreveport) to S.R.G.
REFERENCES 1. Komberg, R. D. (1977) Annu. Rev. Biochem. 46, 931. 2. Wu, R. S., Panusy, H. T., Hatch, C. L., and Bonner, W. M. (1986) CRC Crit. Rev. Biochem. 20, 201. 3. Lichtler, A. C., Sierra, F., Clark, S., Wells, J. R. E., Stein, J., and Stem, G. S. (1982) Nature (London) 298, 195. 4. Wells, D. E. (1986) Nucleic Acids Res. 14, (Supplement) r119. 5. Bucci, L. R., Brock, W. A., and Meistrich, M. L. (1982) Exp. Cell Res. 140, 111. 6. Cole, K. D., Kandala, J. C., and Kistler, W. S. (1986) J. Biol. Chem. 261, 7178. 7. ‘Bostle-Weige, P. K., Meistrich, M. L., Brock, W. A., Nishioka, K., and Bremer, J. W. (1982) J. Biol. Chem. 257, 5560. 8. Branson, R. E., Grimes, S. R., Yonuschot, G., and Irvin, J. L. (1975) Arch. Biochem. Biophys. 168,403.
9. ‘Bustle-Weige, P. K., Meistrich, M. L., Brock, W. A., and Nishioka, K. (1984) J. Biol. Chem. 259, 8769. 10. Sargent, T. D., Wu, J.-R., Sala-‘Bepat, J. M., Wallace, R. B., Reyes, A. A., and Bonner, J. (1979) Proc. Natl. Acad. Sci. USA 76, 3256. 11. Benton, W. D., and Davis, R. W. (1977) Science l%, 180. 12. Sierra, F., Stein, G., and Stein, J. (1983) Nucleic Acids Res. 11, 7069. 13. Carozzi, N., Marashi, F., Plumb, M., Zimmerman, S., Zimmerman, A., Coles, L. S., Wells, J. R. E., Stein, G., and Stein, J. (1984) Science 22, 1115. 14. Rigby, R. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977) J. Mol. Biol. 113, 237. 15. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O’Connell, C., Quon, D., Sim, D. K., and Efstratiadis, A. (1978) Cell 15, 687. 16. Yamamoto, K. R., Alberts, B. M., Benzinger, R., Lawhome, L., and ‘Beiber, G. (1970) Virology 40,734. 17. Bimboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513. 18. Chirgwin, J. M., Pizybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294. 19. Meistrich, M. L., Longtin, J., Brock, W. A., Grimes, S. R., and Mace, M. L. (1981) Biol. Reprod. 25,1065. 20. Southern, E. (1975) J. Mol. Biol. 98, 503. 21. Casey, J., and Davidson, N. (1977) Nucleic Acids Res. 4, 1539. 22. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16, 4743. 23. Thomas, P. (1980) Proc. Natl. Acad. Sci. USA 77, 5201.
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24. Mandel, hi., and Higa, A. (1970) J. Mol. Biol. 53, 159. 25. Favaloro, J., ‘Beisman, R., and Kamen, R. (1980) In Methods in Enzymology (Grossman, L., and Moldave, K., Eds.), Vol. 65, p. 718, Academic Press, New York. 26. Messing, J. (1983) In Methods in Enzymology, (Wu, R., Grossman, L., Moldave, K., Eds.), Vol. 101, p. 20, Academic Press, New York. 27. Sanger, F., Nicklen, S., and Coulson, A. A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463. 28. Bimstiel, M. L., Busslinger, M., and Strub, K. (1985) Cell 41, 349. 29. Stein, G. S., Sierra, F., Stein, J. L., Plumb, M., Marashi, F., Carozzi, N., Prokopp, K., and Baumback, L. (1984) In Histone Genes (Stein, G. S., Stein, J. L., and Marzluff, W. F., Eds.), p. 397, Wiley, New York. 30. Maxson, R., Cohn, R., Kedes, L., and Mohun, T. (1983) Annu. Rev. Genet. 17, 239. 31. Connor, W., Mezquita, J., Winkfein, R. J., States, J. C., and Dixon, G. H. (1984) .I. Mol. Evol. 32. 33. 34. 35. 36. 37. 38.
20, 227. Grimes, S. R. (1986) Comp. Biochem.
Physiol. 83B, 495.
Lieber, T., Weisser, K., and Childs, G. (1986) Mol. Cell. Biol. 6, 2602. Zweidler, A. (1980) Dev. Biochem. 15,47. Brock, W. A., ‘Bostle, P. K., and Meistrich, M. L. (1980) Proc. Nail. Acad. Sci. USA 77, 371. Chiu, M. L., and Irvin, J. L. (1985) Arch. Biochem. Biophys. 236, 260. Sittman, D. B., Graves, R. A., and Marzluff, W. F. (1983) Proc. Natl. Acad. Sci. USA 80, 1849. Takahashi, K., Vigneron, M., Matthes, H., Wildeman, A., Zenke, M., and Chambon, P. (1986) Nature (London) 319, 121. 39. Wu, R. S., and Bonner, W. M. (1981) Cell 27, 321. 40. Wells, D., and Kedes, L. (1985) Proc. Natl. Acad. Sci. USA 82, 2834.
Received March 30,1987 Revised version received June 11, 1987
Printed in Sweden
Cell Research 173 (1987) 534-545
A Rat Histone H4 Gene Closely Associated Testis-Specific HI t Gene
with the
SIDNEY GRIMES,*+ PAUL WEISZ-CARRING’IGN,” HENRY DAUM III, *V2 JOHN SMITH,? LINDA GREEN,S KENNETH WRIGHT,S GARY STEIN,* and JANET STEINS *Research Service (ISI), Veterans Administration Medical Center, Shreveport, Louisiana 71130: TDepartment of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 73130; fDepartment of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, Florida 32610; and ODepartment of Immunology and Medical Microbiology, University of Florida, College of Medicine, Gainesville, Florida 32610
A rat histone H4 gene closely associated with the testis-specific Hlt gene was isolated by screening the Sargent-Bonner rat genomic library using cloned human histone genes as probes. Both the H4 gene and the Hlt gene are located on a 7-kb EcoRI genomic DNA fragment. Although the deduced amino acid sequence of the rat H4 histone is identical to that of the sequence of human histone H4, the nucleotide sequence of the coding region differs significantly from the coding region of the human H4 gene. Moreover, the relative spacing between the 5’-consensus sequence elements is unique for an H4 gene. Slnuclease protection analyses reveal that both the H4 and Hlt mRNA species are present in a fraction of rat testis cells highly enriched in pachytene spermatocytes, while only the H4 mRNA species is present in a rat myeloma cell line (Y3-Agl.2.3). During a l-h hydroxyurea treatment of the Y3 cells, which produces a 99% inhibition of DNA synthesis, the level of this H4 mRNA drops by only 50%, indicating that the stability of this mRNA is only partially coupled with DNA synthesis. @ 1987 Academic press, IIIC.
Histone H4 plays a central role in nucleosome formation [l-2]. The highly conserved amino acid sequence of this histone reveals the essential nature of the sequence. On the other hand, the nucleotide sequence of histone H4 mRNA is not conserved to the same degree. For example, at least seven distinguishable histone H4 mRNA species are expressed in HeLa cells [3]. A summary of several amino acid and nucleotide sequences of H4 and the other histones has been published recently [4]. Mammalian germinal cell histone variants of all of the histones except histone H4 have been reported. A rat germinal variant Hlt is the only Hl variant which is synthesized in pachytene spermatocytes [5-6]. A testis-specific H2A variant TH2A [7] and a testis H2B variant TH2B [8] are also synthesized predominantly in pachytene spermatocytes, and a testis H3 variant TH3 191 and an H2A variant H2A.X [7] are synthesized in spermatogonia. These mammalian germinal cell ’ TO whom reprint requests should be addressed at Veterans Administration E. Stoner Ave., Shreveport, LA 71130. 2 Present address: FMC BioEroducts, 5 Maple Street, Rockland, ME. Copyright @ 1967 by Academic Press, Inc. AU rights of reproduction in any form reserved 0014-4827/87 $03.00
534
Medical Center, 510
Rat histone genes 535 histone variants are clearly products of unique genes which are differentially expressed during spermatogenesis. In the present paper we report the isolation and sequence analysis of an H4 gene located with 1300 base pairs of the rat Hlt gene. The data presented reveal that mRNA from this H4 gene and the closely associated Hlt gene are present in testis pachytene spermatocytes, and thus both the H4 gene and the Hlt gene appear to be expressed in pachytene spermatocytes contributing to the unique pattern of histones found in germinal cells. In addition, the H4 gene is expressed in rat myeloma cells, and the H4 mRNA is quite stable when the cells are treated with hydroxyurea at concentrations which effectively block DNA synthesis. MATERIALS
AND METHODS
Materials. [‘H]Thymidine was purchased from New England Nuclear. [a-‘*P]dCTP (600 Ci/mmol), [a-‘*P]dATP (600 Ci/mmol), and [Y-~*P]ATP (4000 Ci/mmol) were ordered from ICN. X-ray film was ordered from Eastman Kodak (XOmat XAR-5) and DuPont (Cronex 7) and hydroxyurea was ordered from Sigma. Restriction enzymes, plasmids, and Ml3 bacteriophage RF DNA cloning vectors were ordered from Bethesda Research Laboratories and IBI. Other enzymes were ordered from Boehringer-Mannheim Biochemicals. Cell culture and pulse labeling. Rat myeloma cells (Y3-Agl.2.3) obtained from the American Type Culture Collection were grown exponentially in RPM1 media. DNA synthesis inhibition by hydroxyurea was followed by measuring the incorporation of [‘Hlthymidine (5 @i/ml, 1x 106 cells/ml) into 10% (w/v) trichloroacetic acid-precipitable material in a 2-h pulse. Hydroxyurea was added and the cells were incubated for 1 h before the addition of [‘Hlthymidine. Screening a bacteriophage L library. A genomic library constructed using bacteriophage 1 strain Charon 4A and a partial EcoRI digest of liver DNA from a Sprague-Dawley rat [lo] was kindly supplied by Thomas Sargent. The technique of phage hybridization [ll] was performed utilizing human histone gene probes pF0108A (H4) [12] and pFNCl6A (Hl) [13] to screen the library. The probes were labeled by nick-translation with [32P]dCTP [14]. Hybridization was conducted in a solution composed of 4xSSPE (0.72 M NaCl, 0.04 M NaHrPO,, 4 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4), SxDenhardt’s ((0.04% (w/v) Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin (BSA)), 0.1% sodium dodecyl sulfate (SDS), and 0.1 mg/ml Escherichia coli DNA, and a stringent wash was conducted in a solution composed of 1x SSPE and 0.1% SDS. Both the hybridization and wash were conducted at 65°C when using the H4 probe and at 60°C when using the Hl probe. All experiments involving recombinant DNA techniques were performed within the NIH guidelines for research involving recombinant DNA. Isolation of DNA and RNA. Bacteriophage 1 was prepared from liquid culture [15, 161, and I DNA was extracted by adjusting the solution to 20 mM EDTA, 50 ug/ml proteinase K, and 0.5% SDS, followed by phenol extraction and dialysis against a solution containing 10 mM ‘Iris, pH 7.5, and 1 mM EDTA. Plasmid DNA, prepared by alkaline lysis [17], was purified by equilibrium density centrifugation for 16 h on CsCl-ethidium bromide gradients in a Beckman VTi65.2 rotor followed by treatment with T4 RNase and centrifugation through 1 M NaCl in an SWSO. 1 rotor. Total cellular RNA was isolated by the guanidinium isothiocyanate-cesium chloride method [18] from enriched rat testis germinal cell types prepared by centrifugal elutriation [19] and from rat myeloma Y3 cells. The cellular composition of the enriched germinal cell types has been reported [19]. The concentration of RNA in each sample was determined by measuring the optical density at 260 nm. Analysis of DNA and RNA. DNA restriction fragments were separated by electrophoresis through agarose gels in buffer composed of 90 mM Tris, 90 mM boric acid, 2 mM EDTA containing 0.5 &ml ethidium bromide. DNA was transferred to nitrocellulose by the Southern blotting technique 1201. Although phage hybridization using heterologous probes was conducted using aqueous conditions, hybridization to ‘*P-labeled DNA probes in Southern blot experiments was conducted in 50% formamide solution containing 5xSSC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.4), SxDenhardt’s (-BSA), 0.1% SDS, and 0.25 mg/ml E. coli DNA at 49°C with homologous probes 1211. Total cellular RNA was analyzed under denaturing conditions by electrophoresis in agarose gels containing formaldehyde [22]. The gels were soaked to remove formaldehyde, and RNA was trans-
536 Grimes et al. ferred to nitrocellulose by the Northern blotting technique [23], conducting hybridization in 50% formamide solution at 37°C as described for the Southern blotting technique with probes which were “P-labeled by nick-translation. A stringent wash was conducted in a solution of 0.1 x SSC and 0.1% SDS at 60°C. Cloning of DNA fragments. DNA fragments were isolated from low melting agarose gels (FMC BioProducts) by melting sections of agarose gels at 70°C in at least 4 vol of low salt buffer (0.1 M NaCl, 10 mM Tris, pH 7.5, 1 rr& EDTA) and purification of DNA by binding to an Elutip column (Cooper Biomedical Corp.), washing with low salt solution, and elution with 0.5 ml high salt solution (1 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA). The plasmid pPS3 was constructed from the 7-kb EcoRI genomic fragment containing both the Hlt and H4 genes. The plasmid pPS7 was constructed using the 1.5kb Sau3A fragment cut from pPS3 containing 197 bp of the 5’-coding region of H4 and an additional 1.3 kb of 5’-upstream noncoding region. The plasmid pHD1 was constructed using the 1.2kb PuuII-Hi&III fragment containing 320 bp of 5’-coding region of Hlt and 980 bp of 5’upstream noncoding region (tilled using the Klenow fragment of DNA polymerase I to form blunt ends). These plasmids were constructed to aid in the production of probes to be used in Southern blot and Northern blot analyses and in Sl-nuclease protection analyses. E. coli strain JM83 was transformed [24] using the recombinant plasmids and grown on L-Broth plates with ampicillin using 5-bromo-4-chloro-3indolyl-/I-u-galactoside (X-Gal) as color indicator. SI-m&ease protection analyses. Sl-nuclease protection analyses were performed to determine the cap sites of the mRNAs on the histone H4 and histone Hlt genes [25]. In these experiments the 1.5-kb Sau3A DNA insert from pPS7 (H4 probe) and a l.l-kb EcoRI-AL& insert fragment from pHD1 (Hl probe) were dephosphorylated and 5’-end-labeled with polynucleotide kinase and [y-‘*P]ATP. A sample containing approximately 10 ug of total cellular RNA from a specific testis cell type or from rat myeloma Y3 cells was mixed in hybridization buffer with the labeled probe, and the mixture was heated at 79°C to denature the DNA and hybridized for 3 h at 60°C for the H4 probe or 50°C for the Hl probe to allow formation of DNA-RNA hybrids. The mix was treated with Sl-nuclease to digest single-stranded nucleic acid and analyzed by polyacrylamide gel electrophoresis with a sequencing ladder and in separate experiments with “P-end-labeled DNA fragments of known length in adjacent lanes to determine the exact length of the protected DNA fragment. Sequence analysis of DNA. DNA fragments to be sequenced were cloned using bacteriophage Ml3 mp18 or mp19 [26] as vectors and the recombinants were used to transform E. cofi strain JMlOl [24]. The transformants were plated on YT plates containing X-Gal, clear plaques were selected, and DNA was prepared [24]. The Sanger dideoxy sequencing method [27] was used to sequence the cloned fragments. Autoradiograms were prepared using Dupont Cronex 7 film without an intensifying screen and analyzed with the aid of an IBI gel reader and the IBI/Pustell DNA sequence analysis system using an IBM-XT microcomputer. Further sequence analyses were accomplished using the Bionet resource of IntelliGenetics, Inc., Mountain View, California.
RESULTS Since several human histone genes have been isolated and sequenced, these served as probes for screening a rat genomic library. Duplicate filters were lifted from each plate containing E. coli infected by the bacteriophage library and one of each pair of filters was hybridized to 32P-labeled human H4 probe pF0108A in aqueous solution and the duplicate filter was hybridized to 32P-labeled human Hl probe pFNC16A. After three rounds of screening, two of the selected clones hybridized to both the HI and H4 probes. A third clone hybridized only to the H4 probe. Single and double restriction enzyme digestions were conducted utilizing DNA isolated from each of the three bacteriophage clones. DNA fragments were separated by electrophoresis on agarose gels and transferred to nitrocellulose by Southern blotting. Blots were initially probed separately with 32P-labeled pF0108A (H4) and pFNC16A (Hl). A partial restriction map of one of the clones containing genes for both histones H 1 and H4 is presented in Fig. 1 A. In one of
Rat histone genes
s
HSH
Hli SE
I IlJ IIll Hlt
B
E
E
HE
I I I, .II.
H
.I.
537
E
I
H4
<
Fig. 1. (A) Restriction map of the genomic insert containing the Hlt and H4 histone genes. Single and double restriction digestions were conducted and the resulting DNA fragments were analyzed electrophoretically on agarose gels as described under Materials and Methods (E, EcoRI; H, Hi&III; Ps, PsrI; Pv, PuuII; and S, Sau3A). The orientation of each gene is designated by an arrow below the gene. (B) Detailed restriction map of the H4 gene with the sequencing strategy. Single and double restriction digestions were conducted and DNA fragments analyzed as described in (A). (H, HindIII). The heavy arrow designates the mRNA cap site. The light arrows designate the sequencing strategy. Note that the orientation of the H4 gene is reversed with respect to the orientation in (A).
the other two clones, which lacked a detectable Hl gene, the histone H4 gene was present on a 3200-bp Hi&III fragment, indicating that it is located in a genomic region significantly different from the one presented in Fig. 1B. Figure 1 A reveals the close proximity of the H4 gene to the Hl gene in the first of these genomic fragments. The Hl and H4 genes reside together on a 7-kb EcoRI fragment, separated by only about 1300 bp of DNA, and are oriented with their 3’-termini facing each other. DNA fragments containing all or parts of the genes and regions flanking the genes were cloned into the pUC series plasmid vectors to be used as hybridization probes and into Ml3 mp18 or mp19 for sequence analysis. The identity of the Hl gene as the Hlt gene was revealed by restriction mapping (Fig. 1 A) and sequencing (data not shown), and comparison of our data with the published restriction map and sequence of the rat Hlt gene [6]. Sequence analysis of the 440-bp Hind11 fragment (Fig. 2) reveals that this sequence contains all of, the coding region, about 90 bp of the flanking 3’noncoding region and 35 bp of the flanking 5’noncoding region of the H4 gene. Since a unique Sau3A site exists near the center of this gene, the 440-bp Hid11 fragment was cut with Sau3A to obtain two HimfIIESau3A fragments. These two smaller fragments were force cloned into mp18 which had been cut with Hi&III and BamHI and sequenced to complete the analysis of the entire 440-bp fragment. In order to obtain additional flanking 5’-noncoding sequence of this H4 gene, we isolated and sequenced a 236-bp HhaI fragment which overlaps the 5’Hi&III site (Fig. 1 B). Thus, the sequence was extended to 189 bp upstream from the start codon.
538
Grimes et al. -189 -130
CGCAMGAA
TAGCTGAGGT TTGCTAGCCA ATGAAAACAT TCAGATTGCA ATGACTCATC
CTTGTTGCCC CACCCCCTTT
-70 TT~CA~GCAGAAG -10 AUCAACC
CAAGGACCTG ATTCGAAAAC
CCCTCCTAAC
GCCTGTGGTC
GTCTATTTAA A$G"AGCTT
+ GCTTTCTTCC CTKACTTTT
ATG TCT GGR CGT GGT F&A GGT GGT AAA GGG CTT GGG AAA GGT kt Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly LYS Gly 10 1
43 GGC GCT AAG CGT CAT CGC AAA GTC CTG CGC GAC ARC ATC CAG GGC ATC ACA AAG Gly Ala Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile l$r Lys 20 97 CCC GCC ATC CGC CGC CTG GCC CGG CGC GGA GGA GTG AAG CGC ATC TCC GGC CTC Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser Gly Leu 40 151 ATC TAC GAG GIG ACC CGC GGT GTG CTG AAG GTG TTC CTG GAG AAC GTG ATC CGC Ile Tyr Glu Glu Thr Aq Gly Val Leu Lys 1'1 Phe Leu Glu Asn Val Ile Arg 50 205 6AC GCC GTC ACC TAC ACG GAG CAC GCC AAG CGC AAG ACG GTC ACT GCC ATG GAC Asp Ala ;;l Thr Tyr Thr Glu His Ala Lys Arg Lys ;hOr Val Thr Ala Met Asp 259 GTG GTC TAC GCG CTT AAA CGC CAG GGC CGC ACA CTC TAC GGC TTC GGT GGT TAA Val Val Tyr Ala ;a Lys Arg Gin Gly Arg Thr Leu Tyr Gly Phe Gly Gly l 100 313 ACTWTAATG
CGCTTTCCTT AGCTGTTGCA AAAGGCCCTT t
373 CATTWAAGG ,
GTTGTTCACT TITCGMMTT .
Fig. 2. Nucleic acid sequence of the rat histone H4 cloned into bacteriophage M13mp18 and M13mp19 and codon and the coding region is translated. Underlined arrow indicates the H4 mRNA cap site determined by
TTCAGGGCCA CCCACGWT I
gene. DNA fragments noted in Fig. 1B were sequenced. Numbering starts at the ATG start regions represent consensus sequences. The Sl-nuclease protection analysis.
These results, summarized in Fig. 2, reveal the start and stop codons as well as the specific upstream elements described by Wells [4]. The underlined upstream sequence elements include a CAAT sequence at -68, GGTCC at -62, TATTTkAA at -47, and CTCA at -9. A region conspicuously absent is a purine-rich sequence sometimes present in H4 genes immediately upstream from the CAAT sequence [4]. The other very unusual aspect about this upstream region is the relative spacing between the sequence elements just described. Specifically, there are usually 15 to 58 bases between the ATG start codon and the CTCA consensus sequence, 15 to 22 bases between the TATAAATA sequence and the CTCA sequence, and 5 to 58 bases between the CAAT sequence and the GGTCC sequence [4], while in the H4 gene being described in this paper there are 5, 30, and 2 bases, respectively, between these elements. Consensus sequence elements downstream from the stop codon include GGCCCT’MTCAGGGCC at 342, ACCCA at 362, and CAAAGGGTTG at 377. The first of these directs the formation of a hairpin structure in the H4 mRNA, and this palindromic element and the contiguous ACCCA element mark the site of the H4 mRNA terminus [28]. Nothing unusual is apparent in these consensus sequence elements or in the relative spacing of these elements in the flanking 3’-noncoding region. The protein coding region, presented in Figure 2, reveals that this gene codes for a histone H4 which is identical in amino acid sequence to the consensus
Rat histone genes Y3
I
539
TESTIS
2
12345 28s 18s
H4mRNA
Fig. 3. Detection of histone H4 mRNA by Northern blot analysis. Total cellular RNA was prepared as described under Materials and Methods, and 20 ug of each RNA sample was electrophoresed on a denaturing agarose gel. The RNA was transferred to nitrocellulose and analyzed by the Northern blotting procedure using a [32P]-labeled 440-bp Hind11 fragment containing histone H4 as the probe. The RNA samples are as follows: RNA from rat myeloma Y3 cells, untreated control (I), and 1mM hydroxyurea treated (2); RNA from five enriched populations of adult rat testis germinal cells, the three fractions of greatest purity being late spermatids (steps 13-19) (I), early spermatids (steps 1-8) (3), and pachytene spermatocytes (5). The relative migration rates of 18 and 28 S rRNA are shown for size markers.
sequence for this histone [4]. The nucleotide sequence of the coding region of this gene is also highly conserved with 62 of 102 codons identical to those in the consensus nucleotide sequence of the H4 gene [4]. Of these 62 identical codons, 27 are highly conserved codons found in vertebrate histone H4 genes [4]. The nucleotide sequences of the flanking noncoding regions of this gene are much less conserved. In order to examine the expression of rat histone H4 genes, Northern blot analyses were conducted using total cellular RNA isolated from a rat myeloma cell line (Y3 Ag1.2.3) and a number of rat tissues including enriched fractions of rat testis germinal cells. The blots were probed with our rat histone H4 probe. Interestingly, we observed that the members of the H4 mRNA family detected in Y3 cells under our hybridization conditions are quite resistant to inhibition of DNA synthesis in contrast to the behavior of most histone mRNAs (Fig. 3A). Thus, 1 mM hydroxyurea treatment, which inhibits incorporation of [3H]thymidine into rat Y3 cells by more than 99% failed to significantly reduce the level of H4 mRNA to less than 50% of the control levels. Although the wash conditions used for our blots were quite stringent, the Northern blot method is limited in specificity for highly homologous mRNAs, and we are most likely measuring several histone H4 mRNA species. If this is the case, a significant amount of H4 mRNA is relatively stable when DNA synthesis in the Y3 cells is blocked by hydroxyurea. Furthermore, histone H4 mRNA was most abundant in a population of germinal cells enriched in rat testis pachytene spermatocytes in which there is no DNA replication (Fig. 3, lane 5). Much less histone H4 mRNA was detected in the other enriched populations of rat testis germinal cell types in this experiment which included early spermatids (steps 1-8) (Fig. 3 B, lane 3) and late spermatids (steps 13-19) (Fig. 3B, lane I). Sl-nuclease protection assays were conducted to provide evidence for the
540 Grimes et al. GlAT2C
-217
Fig. 4. Detection of histone H4 mRNA in rat myeloma Y3 cells by Sl-nuclease protection analysis. The 1.5kb Sau3A fragment which spans the 5’-half of the coding region and the flanking 5’-noncoding region of the H4 gene (Fig. 1 A) was used as an Sl-nuclease probe. Approximately 10 pg of total cellular RNA from untreated Y3 cells was used in the assay. A sequencing ladder derived from the 1.9-kb Hi&III fragment containing the 3’coding region of the Hl t gene and flanking the H4 gene (Fig. 1) is used as a size marker. The number of bases represented by the largest protected fragment is indicated. The sample in lanes marked 1 and 2 were treated with 100 and 400 units of Sl-nuclease, respectively.
presence of the specific H4 and Hlt mRNAs relevant to this study. The data presented in Fig. 4 indicate that mRNA encoded by this specific rat histone H4 gene is present in rat myeloma Y3 cells. The larger protected DNA fragment (lanes 1 and 2) marked in the figure is 217 bases in length based upon its electrophoretic mobility compared to sequencing ladders and compared to DNA fragments of known length run in parallel experiments, which indicates that adenine at position -16 (Fig. 2) represents the cap site of the H4 mRNA. A second protected fragment 200 bases in length presumably represents H4 mRNA species which protect the DNA probe only to the AUG codon. Note that the smaller protected fragment is shortened (compare lane 2 to lane I) when higher Sl-nuclease concentrations are used (100 units-lane I, 400 units-lane 2) while the larger fragment is not shortened, which suggests less-perfect base pairing for the smaller fragment. Treatment of the myeloma cells for 60 min with 10 rnM hydroxyurea (Fig. 5A, lane 5) leads to a 50% reduction in the level of the two histone H4 protected fragments compared to untreated controls (Fig. 5A, lane 6) as approximated from densitometric scans of autoradiograms . Additional S 1nuclease protection analyses revealed that both types of these histone H4 mRNA species are present and most abundant in pachytene spermatocytes (Fig. 5, lane 9), but a trace of these H4 mRNA species can be detected in populations of testis cells enriched in late spermatids and early spermatids (Fig. 5, lanes 7 and 8), presumably due to contamination of the purified cell types with other testis cell types. However, note in the testis cells that the H4 mRNA species which results in the protection
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Fig. 5. (A) Histone mRNA in Y3 and testis germinal cells. Total cellular RNA (10 ug) from control rat Y3 cells (untreated), hydroxyurea-treated Y3 cells, or rat testis germinal cells was used for each Sl-nuclease protection assay. Y3 cells were treated with hydroxyurea as follows: control (no hydroxyurea) lanes I knes a and 6; 0.5 mM, lane 2; 1 ti, lane 3; 5 mM, lane 4; 10 n&f, lane 5. The last three lanes represent RNA from late spermatids (steps 13-19) (lane 7), early spermatids (steps l-8) (lane 8), and pachytene spermatocytes (lane 9). (A) H4 probe as in Fig. 4. (B) Hlt probe (see Materials and Methods).
of the larger protected DNA fragment represents approximately 10 % of the total H4 mRNA which provides protection to the DNA probe. The same RNA samples used in Sl-nuclease protection analysis with the H4 probe in Fig. 5A were also used in Sl-nuclease protection analyses with the Hlt probe. The size of the protected fragment (marked in Fig. 5B) indicates that adenine at position 1 (according to the published numbering scheme for the Hlt gene [6]) represents the cap site of the Hlt mRNA. Furthermore, the results shown in Fig. 5B reveal that the Hlt mRNA is most abundant in pachytene spermatocytes (lane 9) with only trace amounts in the fractions of cells enriched in early spermatids (lane 7) or late spermatids (lane 8). No Hlt mRNA could be detected in any of the Y3 cell RNA samples including those treated with hydroxyurea (lanes l-6). Analysis of histone proteins from Y3 cells by electrophoresis on acid-urea gels containing Triton X-100 also failed to reveal histone Hlt (data not shown). DISCUSSION Cloned human histone genes have been used successfully to screen a rat genomic library. Two of the three isolated bacteriophage clones which contained a histone H4 gene also contained a histone Hl gene. Detailed studies of histone genes indicate that in many organisms the genes are clustered with each cluster containing one copy of each of the five histone genes [30-311. The clone examined in this paper contains both the rat testis-specific histone Hlt gene and a closely associated rat histone H4 gene. Whether this genomic fragment contains additional histone genes or whether it is part of a larger fragment containing additional histone genes is unkonwn at the present time. Since histone H4 is the most highly conserved of the five histones with respect to amino acid sequence and histone Hl is the least conserved, we used a slightly different screening strategy for each of these probes. When using the human H4 probe, hybridization was conducted at 65°C but when using the human Hl probe, hybridization was
542 Grimes et al. conducted at 60°C; the lower hybridization temperature was chosen to allow a greater degree of mismatching. Fortunately, this strategy was adequate for the successful identification of both rat H4 and HI histone genes. It is presumed that this strategy was necessary for this purpose, but we did not test other temperatures. Tissue-specific histone variants replace at least in part the usual somatic-type histone variants in germinal cells during spermatogenesis in many organisms [32-341. Rat histone Hl species are designated Hla, b, c, d, e, and t [51, where Hla, c, and t are prominent in the testis germinal cells and Hlb, d, and e are somatic variants. Hlt is the only Hl variant synthesized in pachytene spermatocytes [5]. The switch seen in histone Hl gene expression in germinal cells is also seen for histones H2A, H2B, and H3 where the rat testis-specific or testisenriched variants are designated H2A.X, TH2A, TH2B, and TH3 [7-91. Recently, the TH2B variant gene has been isolated and sequenced (personal communication from Dr. Chi-Born Chae). However, a unique mammalian germinal cell histone H4 has not been described, which may reflect the absolute requirement for the highly conserved amino acid sequence of this histone. Nevertheless, synthesis of histones H4 and Hlt has been demonstrated in premeiotic pachytene spermatocytes which are not active in DNA synthesis [35-361. Along these lines, it has been reported [36] that synthesis of histones H4 and Hlt occurs at significant levels in germinal cells even in the presence of hydroxyurea (50% of control levels for H4 and 90% of control levels for Hlt) which blocks testis DNA synthesis by 90% in those cells active in DNA synthesis and normally leads to a rapid turnover of histone mRNA [29, 371. Other related work [37] indicates that high concentrations of DNA synthesis inhibitors can have a significant inhibitory effect on histone gene transcription, which we presume may occur in our experiments as well. However, the level of transcription has not been determined in the experiments described in the present study. In any case the data clearly show that we have isolated the testis-specific Hlt and a closely associated H4 gene both of which are under the control of regulatory mechanisms which lead to the expression of these histone mRNAs in pachytene spermatocytes which are not active in DNA synthesis. Our restriction mapping, subcloning, and sequencing data reveal that the Hl gene presented in this report is in fact Hlt by comparison to the published restriction map and sequence [6]. The H4 gene closely associated with this Hlt gene appears to be functional in synthesis of H4 mRN,A in pachytene spermatocytes and in rat Y3 cells. We speculate that the H4 gene reported in this paper contributes to histone H4 synthesis in rat testis pachytene spermatocytes. The close association of this H4 gene with the testis Hlt suggests that these two genes may share some aspects of gene regulation perhaps by virtue of being part of the same chromosomal domain. The structural juxtaposition of the rat Hlt and H4 genes most closely resembles the genomic organization of some early sea urchin, some amphibian, and some chicken histone gene clusters [30]. At least one human Hl gene is located between two core histone genes [13], but in this case the core histone genes are H2B and H3. There is clearly a developmental
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regulation of expression of histone genes in eukaryotes as demonstrated by the differential expression of sea urchin early, late, and testis sperm histone genes [301. The early Hl and core histone genes are clustered as are the early histone genes of many other organisms where fertilized eggs enter a period of extremely rapid cell division requiring large quantities of histones for chromatin assembly. In this case the coordinate expression of the clustered histone genes is facilitated by the close proximity of the genes to each other. By analogy we speculate that the close structural association of the rat Hlt and H4 genes facilitates the coordinate expression of these two genes in premeiotic pachytene spermatocytes. The biological significance of the testis histone variants is unknown [32], but the coordinate expression of testis-specific histone genes is widespread in eukaryotes, being seen in species as divergent as sea urchins, amphibians, rodents, and primates including humans [30-341. There is an obvious requirement for histone synthesis presumably for restructuring of the premiotic chromosomes which occurs in the absence of DNA synthesis in pachytene spermatocytes. In the rat this requirement is fulfilled at least in part by the coordinate expression of the testis-specific histone genes Hlt (THl), TH2A, TH2B, TH3, and the H4 gene closely associated with Hlt. It will be important to determine whether the other rat testis-specific core histone genes are also in close proximity to the Hlt and H4 genes. The 5’-flanking sequence elements of the H4 gene are unusual in that a consensus purine-rich sequence is absent altogether, that CAAT sequence is unusual, and the other consensus sequence elements have a unique relative spacing. The relative spacing of flanking 5’-promoter elements is critical for stereospecific alignment and cooperative association of transacting factors in eucaryotic genes [38]. Thus, the unusual 5’-noncoding region may play a key role in the expression of this histone H4 mRNA species which is quite stable in pachytene spermatocytes and perhaps in other cell types including rat myeloma Y3 cells treated with hydroxyurea. The Sl-nuclease protection data indicating only a 50 % drop in the level of histone H4 mRNA in rat myeloma cells treated with 10 mM hydroxyurea for 1 h and published data on Hlt [36] indicating continued synthesis during hydroxyurea treatment suggest that this rat histone H4 gene and the Hlt gene are similar in this respect to some cell cycle-independent histone genes. Cell cycle-dependent histone mRNA levels normally drop to low or undetectable levels within 15 to 30 min in cells treated with 1 mM hydroxyurea due to a greatly increased turnover rate of histone mRNA [29, 371. However, this particular histone H4 gene as well as the Hlt gene have no intervening sequences or polyadenylation signals, both of which are found in some basally expressed histone genes [39]. Although, the available evidence indicates that the histone H4 gene and the closely associated Hlt gene are both expressed in pachytene spermatocytes, it appears that this H4 gene is also expressed in rat myeloma cells. On the other hand, there appears to be a tissue-specific expression of the Hlt gene only in pachytene spermatocytes. Therefore, the H4 gene may contribute to the synthesis of H4 mRNA in cells which are active in DNA synthesis such as Y3 cells as 35-878342
544 Grimes et al. well as in those which are inactive in DNA synthesis such as rat testis pachytene spermatocytes. If this behavior can be confirmed in other cell types, then the expression of this gene in somatic cells may contribute to the H4 mRNA pool which is synthesized by cell cycle-independent or basally expressed histone genes [40]. We have also identified a second rat histone H4 gene in another bacteriophage L clone isolated from the same rat genomic library. The restriction map of this second H4 gene is quite different from the map of the H4 gene presented in this report. It will be important of compare the two H4 genes in greater detail and examine the pattern of expression of these two histone genes in various rat tissues and specific cell types including the germinal cells at different stages of maturation. We acknowledge the excellent technical assistance of Jeffrey Anderson, Pamela Smart, and Janice Yager. Some data analysis was conducted with the Bionet computer resource of IntelliGenetics, Inc., Mountain View, California supported by NIH Grant 1 U41 RR-0168503. This research was supported by the Medical Research Service of the Veterans Administration and by BRSG Grant 2 SO7 RR 0582206 (administered by LSU Medical Ctr., Shreveport) to S.R.G.
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Received March 30,1987 Revised version received June 11, 1987
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