Cleavage Stage Histone Research Articles

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five α-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.

A mammalian oocyte-specific linker histone gene H1oo: homology with the genes for the oocyte-specific cleavage stage histone (cs-H1) of sea urchin and the B4/H1M histone of the frog.

Oocytes and early embryos of multiple (non-mammalian) species lack the somatic form of the linker histone H1. To the best of our knowledge, a mammalian oocyte-specific linker (H1) histone(s) has not, as yet, been reported. We have uncovered the cDNA in question in the course of a differential screening (suppression subtractive hybridization (SSH)) project. Elucidation of the full-length sequence of this novel 1.2 kb cDNA led to the identification of a 912 bp open reading frame. The latter encoded a novel 34 kDa linker histone protein comprised of 304 amino acids, tentatively named H1oo. Amino acid BLAST analysis revealed that H1oo displayed the highest sequence homology to the oocyte-specific B4 histone of the frog, the respective central globular (putative DNA binding) domains displaying 54% identity. Substantial homology to the cs-H1 protein of the sea urchin oocyte was also apparent. While most oocytic mRNAs corresponding to somatic linker histones are not polyadenylated (and remain untranslated), the mRNAs of (non-mammalian) oocyte-specific linker histones and of mammalian H1oo, are polyadenylated, a process driven by the consensus signal sequence, AAUAAA, detected in the 3'-untranslated region of the H1oo cDNA. Our data suggest that the mouse oocyte-specific linker histone H1oo (1) constitutes a novel mammalian homolog of the oocyte-specific linker histone B4 of the frog and of the cs-H1 linker histone of the sea urchin; (2) is expressed as early as the GV (PI) stage oocyte, persisting into the MII stage oocyte, the oocytic polar bodies, and the two-cell embryo, extinction becoming apparent at the four- to eight-cell embryonic stage; and (3) may play a key role in the control of gene expression during oogenesis and early embryogenesis, presumably through the perturbation of chromatin structure.

Cytoplasm of sea urchin unfertilized eggs contains a nucleosome remodeling activity.

After fertilization the sea urchin sperm nucleus transforms into the male pronucleus which later fuses with the female pronucleus re-establishing the diploid genome of the embryo. This process requires remodeling of the sperm chromatin structure including the replacement of the sperm histones by maternally derived cleavage stage histone variants. In recent years, a group of protein complexes that promote chromatin-remodeling in an ATP-dependent manner have been described. To gain understanding into the molecular mechanisms operating during sea urchin male pronuclei formation, we analyzed whether chromatin-remodeling activity was present in unfertilized eggs as well as during early embryogenesis. We report that in the sea urchin Tetrapygus niger, protein extracts from the cytoplasm but not from the nucleus, of unfertilized eggs exhibit ATP-dependent nucleosome remodeling activity. This cytosolic activity was not found at early stages of sea urchin embryogenesis. In addition, by using polyclonal antibodies in Western blot analyses, we found that an ISWI-related protein is primarily localized in the cytoplasm of the sea urchin eggs. Interestingly, SWI2/SNF2-related proteins were not detected neither in the nucleus nor in the cytoplasm of unfertilized eggs. During embryogenesis, as transcriptional activity is increased an ISWI-related protein is found principally in the nuclear fraction. Together, our results indicate that the cytoplasm in sea urchin eggs contains an ATP-dependent chromatin-remodeling activity, which may include ISWI as a catalytic subunit.

Phosphorylation protects sperm-specific histones H1 and H2B from proteolysis after fertilization.

At intermediate stages of male pronucleus formation, sperm-derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A-H2B (SpH2A-SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP-ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine-protease (SpH-protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP-ribose) moiety of these proteins. Because of the selectivity displayed by the SpH-protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant alpha-subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH-protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling.

Poly(ADP-ribosylation) protects maternally derived histones from proteolysis after fertilization

Fertilization in sea urchins is followed by the replacement of sperm-specific histones by cleavage-stage histone variants recruited from maternal stores. Such remodelling of zygote chromatin involves a cysteine proteinase that degrades the sperm-specific histones in a selective manner, leaving the maternal cleavage-stage histone variants intact. The mechanism that determines the selectivity of the sperm-histone-selective proteinase (SpH-proteinase) was analysed by focusing on the post-translational modification status of both sets of histones. It has previously been reported that only native cleavage-stage histones are poly(ADP-ribosylated), whereas the sperm-specific histones are not modified. To determine whether the poly(ADP-ribose) moiety afforded protection from degradation, the ADP-ribose polymers were removed from the cleavage-stage histones in vitro; these proteins were then assayed as potential substrates of the SpH-proteinase. Strikingly, the cleavage-stage histone variants were extensively degraded after the enzymic removal of their ADP-ribose moieties. In addition, the SpH cysteine proteinase was not inhibited by isolated poly(ADP-ribose) polymers. Consequently, only poly(ADP-ribosylated) cleavage-stage histone variants are protected from proteolysis. These results demonstrate a novel role for this type of post-translational modification, namely the protection of nuclear proteins against nuclear proteinases after fertilization.

Poly(ADP-ribosylation) protects maternally derived histones from proteolysis after fertilization

Fertilization in sea urchins is followed by the replacement of sperm-specific histones by cleavage-stage histone variants recruited from maternal stores. Such remodelling of zygote chromatin involves a cysteine proteinase that degrades the sperm-specific histones in a selective manner, leaving the maternal cleavage-stage histone variants intact. The mechanism that determines the selectivity of the sperm-histone-selective proteinase (SpH-proteinase) was analysed by focusing on the post-translational modification status of both sets of histones. It has previously been reported that only native cleavage-stage histones are poly(ADP-ribosylated), whereas the sperm-specific histones are not modified. To determine whether the poly(ADP-ribose) moiety afforded protection from degradation, the ADP-ribose polymers were removed from the cleavage-stage histones in vitro; these proteins were then assayed as potential substrates of the SpH-proteinase. Strikingly, the cleavage-stage histone variants were extensively degraded after the enzymic removal of their ADP-ribose moieties. In addition, the SpH cysteine proteinase was not inhibited by isolated poly(ADP-ribose) polymers. Consequently, only poly(ADP-ribosylated) cleavage-stage histone variants are protected from proteolysis. These results demonstrate a novel role for this type of post-translational modification, namely the protection of nuclear proteins against nuclear proteinases after fertilization.

Isolation and Amino Acid Sequence Analysis Reveal an Ancient Evolutionary Origin of the Cleavage Stage (CS) Histones of the Sea Urchin

The cleavage stage (CS) H1, H2A, and H2B histones of the sea urchin, which have previously been identified by their distinct electrophoretic mobility on Triton/acid/urea gels, are known to be maternally expressed during oogenesis and have been implicated in chromatin remodeling of the male pronucleus following fertilization. Here, we describe the isolation of these three CS histones by reverse-phase HPLC chromatography. Moreover, a novel CS H3 protein was identified by the same purification procedure. A low incorporation of radioactive amino acids into the CS histones during early development revealed that the bulk of these proteins in the blastula embryo are derived from the maternal pool of the egg. Amino acid analysis, together with the previously described electrophoretic mobilities, unequivocally identified the purified proteins as CS histones. Peptide sequence analysis confirmed the novel nature of the CS variants as they are distantly related to the early, late, and sperm histone subtypes of the sea urchin. The CS H1 protein displays highest sequence similarity with the H1M (B4) histone of Xenopus laevis, indicating that the frog H1M protein may be a vertebrate homologue of the CS H1 histone. These data suggest an ancient evolutionary origin and wide distribution of the CS histone variants.

The five cleavage-stage (CS) histones of the sea urchin are encoded by a maternally expressed family of replacement histone genes: functional equivalence of the CS H1 and frog H1M (B4) proteins.

The cleavage-stage (CS) histones of the sea urchin are known to be maternally expressed in the egg, have been implicated in chromatin remodeling of the male pronucleus following fertilization, and are the only histone variants present in embryonic chromatin up to the four-cell stage. With the help of partial peptide sequence information, we have isolated and identified CS H1, H2A, H2B, H3, and H4 cDNAs from egg poly(A)+ mRNA of the sea urchin Psammechinus miliaris. All five CS proteins correspond to replacement histone variants which are encoded by replication-independent genes containing introns, poly(A) addition signals, and long nontranslated sequences. Transcripts of the CS histone genes could be detected only during oogenesis and in development up to the early blastula stage. The CS proteins, with the exception of H4, are unique histones which are distantly related in sequence to the early, late, and sperm histone subtypes of the sea urchin. In contrast, the CS H1 protein displays highest sequence homology with the H1M (B4) histone of Xenopus laevis. Both H1 proteins are replacement histone variants with very similar developmental expression profiles in their respective species, thus indicating that the frog H1M (B4) gene is a vertebrate homolog of the CS H1 gene. These data furthermore suggest that the CS histones are of ancient evolutionary origin and may perform similar conserved functions during oogenesis and early development in different species.

Hybrid nucleoprotein particles containing a subset of male and female histone variants form during male pronucleus formation in sea urchins.

To determine the changes in chromatin organization during male pronucleus remodeling, we have compared the composition of nucleoprotein particles (NP-ps) resulting from digestion with endogenous nuclease (ENase) and with micrococcal nuclease (MNase). Whole nuclei were isolated from sea urchin gametes and zygotes containing partially decondensed (15 min postinsemination, p.i.) or a fully decondensed (40 min p.i.) male pronucleus and digested with nucleases. The NP-ps generated were analyzed in agarose gels, and their histone composition was determined. Sperm core histones (SpH) and cleavage stage (CS) variants were identified by Western immunoblots revealed with specific antibodies. A single NP-ps was generated after digestion of sperm nucleus with MNase, which migrated in agarose gels between DNA fragments of 1.78-1.26 Kb. Sperm chromatin remained undigested after incubation in ENases activating buffer, indicating that these nuclei do not contain ENases. One type of NP-ps was obtained by digestion of unfertilized egg nuclei, either with ENase or MNase; the NP-ps was located in the region of the agarose gel corresponding to DNA fragments of 3.4-1.95 Kb [Imschenetzky et al. (1989): Exp Cell Res 182:436-444]. When whole nuclei from zygotes containing the female pronucleus and a partially remodeled male pronucleus were digested with ENase, a single NP-ps was generated, which migrated between DNA fragments of 2.5-1.9 Kb. This particle contained only CS histone variants. Alternatively, when these nuclei were digested with MNase, two NP-ps were generated; the slower migrating NP-ps (s) was located in the same position of the agarose gel as those resulting from ENase digestion and the faster migrating NP-ps (f) migrated between DNA fragments of 1.95-1.26 Kb. It was found that NP-ps (s) contained only CS histone variants, whereas NP-ps (f) were formed by a subset of SpH and by CS histone variants. When nuclei from zygotes containing a fully decondensed male pronucleus were digested either with ENase or MNase, a single type of NP-ps was observed, which migrated in the same position as NP-ps (s) in agarose gels. This particle contained only CS histone variants. On the basis of the histone compositions and on electrophoretic similarities, it was concluded that NP-ps (s) originated from the female pronucleus and that NP-ps (f) were generated from the partially remodeled male pronucleus. Consequently, our results indicate that at an intermediate stage of male pronucleus remodeling the chromatin is formed by NP-ps containing a subset of both SpH and of CS histone variants, whereas at final stages of male pronucleus decondensation chromatin organization is similar to that of the female pronucleus.

Sea urchin zygote chromatin exhibit an unfolded nucleosomal array during the first S phase.

To investigate changes in chromatin organization associated with DNA replication during the first stages of development of the sea urchin Tetrapygus niger, we compared micrococcal nuclease (MNase) digestion patterns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs. We observed that the majority of DNA fragments derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nuclei were larger (1,500 to 410 bp). This result indicates that in zygotes, where active DNA replication is occurring, the major chromatin fraction is represented as unfolded nucleosomes. In contrast, in unfertilized eggs chromatin appears to be organized into polynucleosomes. To determine if the unfolded structure of nucleosomes observed during S phase is related to the level of poly (ADP-ribosylation) of cleavage stage (CS) histone variants, zygotes were treated with 20 mM 3-Amino Benzamide (3 ABA) during the interval between 3 and 30 min post-insemination (p.i.). This treatment with 3 ABA decreases the poly (ADP-ribosylation) of CS histone variants and inhibits the first S phase in zygotes [Imschenetzky et al. (1991): J Cell Biochem 46:234-241; Imschenetzky et al. (1993): J Cell Biochem 51:198-205]. When the MNase digested patterns of chromatin from these 3 ABA treated and control zygotes were compared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly(ADP-ribose) synthetase with 3 ABA. This result indicates that the unfolded nucleosomal structure, particular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly(ADP-ribosylation) of CS histone variants.

Poly(ADP‐ribosylation) of atypical CS histone variants is required for the progression of S phase in early embryos of sea urchins

The patterns of poly(ADP-ribosylation) in vivo of CS (cleavage stage) histone variants were compared in sea urchin zygotes at the entrance and the exit of S1 and S2 in the initial developmental cell cycles. This post-translational modification was detected by Western immunoblots with rabbit sera anti-poly(ADP-ribose) that was principally reactive against ADP-ribose polymers and slightly against ADP-ribose oligomers. The effect of 3 aminobenzamide (3-ABA), an inhibitor of the poly(ADP-ribose) synthetase, on S phase progression was determined in vivo by measuring the incorporation of 3H thymidine into DNA. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) in a cell cycle dependent manner. A significantly positive reaction of several CS variants with sera anti-poly(ADP-ribose) was found at the entrance into S phase, which decreases after its completion. The incubation of zygotes in 3-ABA inhibited the poly(ADP-ribosylation) of CS variants and prevented both the progression of the first S phase and the first cleavage division. These observations suggest that the poly(ADP-ribosylation) of atypical CS histone variants is relevant for initiation of sea urchin development and is required for embryonic DNA replication.

Phosphorylation of sea urchin histone CS H2A

Phosphorylation of cleavage stage (CS) histones was studied during the first cell cycle in male pronuclei of the sea urchin. Histone CS H2A rapidly incorporated 32PO 4 during the replication period, but not before. Peptide mapping and amino acid analysis of radiolabelled CS H2A showed that phosphorylation occurred mainly on serine residues located in the C-terminal region of the molecule. When DNA replication was inhibited with aphidicolin both CS H2A and CS H2B accumulated in male pronuclei at the same rate as in the control culture, whereas accumulation of H3 and H4 histones was reduced. Incorporation of 32PO 4 by CS H2A doubled when DNA synthesis was inhibited with aphidicolin. Thus phosphorylation of CS H2A was correlated with transport of CS histones from the egg storage pool to the male pronucleus, but not with chromatin synthesis, indicating that this event precedes nucleosome formation. A role for phosphorylation and dephosphorylation of the CS H2A C-terminal region in modulating transport of stored CS histone dimers and their assembly into nucleosomes is discussed.

Translational regulation of histone synthesis in the sea urchin strongylocentrotus purpuratus.

The pattern and schedule of histone synthesis in unfertilized eggs and early embryos of the sea urchin Strongylocentrotus purpuratus were studied using two-dimensional gel electrophoresis. After fertilization there is an abrupt change in the pattern of histone variant synthesis. Although both cleavage-stage (CS) variants. However, after fertilization, both CS and alpha messages are translated. Since alpha histone mRNA isolated from unfertilized eggs can be translated in vitro, the synthesis of alpha histone subtypes appears to be under translational control. Although the synthesis of alpha subtypes is shown here to occur before the second S phase after fertilization, little or no alpha histone is incorporated into chromatin at this time. Thus, early chromatin is composed predominantly of CS variants probably recruited for the most part from the large pool of CS histones stored in the unfertilized egg.

Electrophoretic analysis of the stored histone pool in unfertilized sea urchin eggs: quantification and identification by antibody binding.

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.

Phosphorylation of cleavage stage histone H1 in mitotic and prematurely condensed chromosomes

The cleavage stage variant of histone H1 (CS H1) in early sea urchin embryos is highly phosphorylated. As is the case for H1s from other cell types, CS H1 reaches its highest degree of phosphorylation during mitosis. Cs H1 from male chromosomes forced to prematurely condense and from mitotic chromosomes are phosphorylated to a similar extent. Despite the correlation between the degree of H1 phosphorylation and chromosome condensation, the two events can be separated. In the presence of emetine, a protein synthesis inhibitor, CS H1 is assembled onto the male pronuclear chromatin from a histone pool stored in the egg. Phosphorylation proceeds to a normal extent, but chromosome condensation is blocked. The data suggest that if the high degree of phosphorylation of H1 characteristic of mitotic chromosomes is required, it is not by itself sufficient, for chromosome condensation.

Alterations in chromatin structure during early sea urchin embryogenesis.

Sea urchin sperm before fertilization possess the longest nucleosome repeat length yet determined for any chromatin. By the time the fertilized egg gives rise to a blastula or gastrula embryo, the chromatin has a considerably shorter repeat length and, in addition, a sequence of different histone variants of H1, H2A, and H2B has appeared. We have investigated the relationship between these variations in histone composition and concomitant alterations in chromatin structure during the earliest stages of embryogenesis in two species of sea urchin. In contrast to the long repeat distance in sperm, chromatin loaded with cleavage stage histones has a much smaller repeat. Later stages containing predominantly alpha histones display an intermediate spacing. More detailed analysis of the events in the first cell cycle was carried out with polyspermically fertilized eggs. During the first 30 min after fertilization, in which sperm-specific H1 is completely replaced by cleavage-stage H1, the male pronuclear repeat remains unchanged. The decrease toward the repeat length of cleavage stages begins at about the time of DNA synthesis. Higher degrees of polyspermy extend the length of the cell cycle, including the duration of S phase and the length of time to reach the first chromosome condensation. At these higher degrees of polyspermy, the decrease in repeat length is also slowed. We conclude that the adjustment of the arrangement of nucleosomes in embryonic chromatin from that found in sperm can occur within the first cell cycle and that its timing is cell-cycle dependent. The adjustment is separable from a corresponding change in H1 composition.

Transitions in histone variants of the male pronucleus following fertilization and evidence for a maternal store of cleavage-stage histones in the sea urchin egg

A system has been developed for the biochemical study of male pronuclear histones following fertilization. Pronuclei are isolated in high yield and purity from polyspermically fertilized sea urchin eggs. Acid extracts of the pronuclei or chromatin are analyzed by two-dimensional gel electrophoresis. During male pronuclear decondensation, sperm-specific (Sp) H1 is completely replaced by the cleavage-stage (CS) H1 variant and two sperm H2B species are more gradually replaced by two closely migrating species (O, P) perhaps modifications of the Sp2Bs. Histones CS2A and CS2B begin to accumulate on the male chromatin predominantly during DNA synthesis, as does a subspecies of H3 (3″). By the time of chromosome condensation, the histone complement is chiefly CS1; CS2A and Sp2A; CS2B and O, P; three H3s and two H4s. Controls indicate that these patterns are not artifacts of cytoplasmic or maternal pronuclear contamination or modification of sperm histones during isolation. The histone transitions occur even if protein synthesis is inhibited, suggesting a maternal pool of CS histones. Such a store can be demonstrated directly in acid extracts of whole eggs. At least 30 haploid equivalents of CS histones are stored per egg. No α, β, γ, or δ variants appear on the chromatin in the first cycle. This system should allow, for the first time, detailed biochemical study of male pronuclear chromatin.