Translation of maternal histone mRNAs in sea urchin embryos: a test of control by 5' cap methylation

Recent results have demonstrated the occurrence of mRNA cap methylation in the sea urchin embryo following fertilization. It has been suggested that this methylation event is responsible for the translational activation of maternal histone mRNAs in these embryos. We have used aphidicolin, an effective inhibitor of both DNA synthesis and cap methylation in cleavage stage sea urchin embryos, to examine the relationship between cap methylation and translation. At 5 micrograms/ml, a dose which rapidly abolishes DNA replication and blocks cleavage, we note no effect on recruitment or translation of maternal alpha-subtype histone mRNAs. This suggests that a postfertilization cap methylation event is not critical to the process of regulation of the translation of stored alpha-subtype histone mRNAs.     

Methylation changes of lysine 9 of histone H3 during preimplantation mouse development

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-m2K9) during pronucleus development, while the maternal genome stained intensively. Such H3-m2K9 asymmetry between the parental pronuclei was independent of HP1b localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-m2K9. A conspicuous increase in H3-m2K9 level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-m2K9 was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.     

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.     

Spatial distribution of messenger RNA in the cytoskeletal framework of ascidian eggs

Maternal poly(A)⁺RNA, histone mRNA, and actin mRNA exhibit unique spatial distributions in the different ooplasmic regions of ascidian eggs. These RNAs also appear to migrate with their respective ooplasms during the episode of extensive cytoplasmic rearrangement that occurs after fertilization, suggesting they are associated with a structural framework. The role of the cytoskeletal framework (CF) in determining the spatial distribution of maternal mRNA was tested by subjecting Triton X-100 extracted (Styela plicata) eggs and early embryos to in situ hybridization with poly(U) and cloned DNA probes. Grain counts indicated that substantial proportions of the egg poly(A)⁺RNA, histone mRNA, and actin mRNA were present in the CF and that there was no alteration in the extent of mRNA-CF interactions during the period between fertilization and the two-cell stage. Analysis of grain distributions indicated that poly(A)⁺RNA, histone mRNA, and actin mRNA were concentrated in the same regions of detergent-extracted eggs as they are in intact eggs. The proportions and spatial distribution of these RNAs in the CF were not affected when the actin cytoskeleton was destabilized by cytochalasin B or DNAse I. The data suggest that maternal mRNA is associated with the CF, that this association is responsible for mRNA rearrangement during ooplasmic segregation, and that mRNA-CF interactions are not dependent on the integrity of the actin cytoskeleton.     

Cytoplasmic 3'' poly(A) addition induces 5'' cap ribose methylation: implications for translational control of maternal mRNA.

During the early development of many animal species, the expression of new genetic information is governed by selective translation of stored maternal mRNAs. In many cases, this translational activation requires cytoplasmic poly(A) elongation. However, how this modification at the 3' end of an mRNA stimulates translation from the 5' end is unknown. Here we show that cytoplasmic polyadenylation stimulates cap ribose methylation during progesterone-induced oocyte maturation in Xenopus laevis. Translational recruitment of a chimeric reporter mRNA that is controlled by cytoplasmic polyadenylation coincides temporally with cap ribose methylation during this period. In addition, the inhibition of cap ribose methylation by S-isobutyladenosine significantly reduces translational activation of a reporter mRNA without affecting the increase of general protein synthesis or polyadenylation during maturation. These results provide evidence for a functional interaction between the termini of an mRNA molecule and suggest that 2'-O-ribose cap methylation mediates the translational recruitment of maternal mRNA.     

The spatial distribution of maternal mRNA is determined by a cortical cytoskeletal domain in Chaetopterus eggs

Messenger RNA molecules are localized in the cortical region of eggs and unevenly segregated to the embryonic cells during early development of the annelid Chaetopterus. The egg cortex is enriched in two organelles, ectoplasmic spherules and associated structures, which are similar in appearance to nuage. The physical basis of cortical mRNA localization was examined in stratified eggs and in eggs extracted with the nonionic detergent Nonidet P-40 (NP-40). The cortical organelles were displaced to the most centrifugal zone of stratified eggs. In situ hybridization with poly(U) or cloned DNA probes showed that a large proportion of the poly(A)+RNA, histone mRNA, and actin mRNA molecules was also displaced to the centrifugal zone. Extraction with NP-40 revealed a detergent-insoluble cytoskeletal domain (CD) in the egg cortex which contained the remnants of ectoplasmic spherules and nuage embedded in a fibrous network. Although most of the total protein and RNA was extracted by NP-40, a large proportion of the poly(A)+RNA, histone mRNA, and actin mRNA molecules was retained in the CD. In situ hybridization of stratified eggs extracted with NP-40 indicated that the CD, with its associated organelles and mRNA molecules, is displaced to the centrifugal zone as a unit. The results suggest that the tenacious association of mRNA molecules with the cortical CD may be responsible for maternal mRNA localization during early development.     

Multiple methylated cap sequences in adenovirus type 2 early mRNA.

The methylated constituents of early adenovirus 2 mRNA were studied. RNA was isolated from polyribosomes of cells double labeled with [methyl-3H]methionine and 32PO4 from 2 to 7 g postinfection in the presence of cycloheximide. Cycloheximide ensures that methylation and processing are performed by preexisting host cell enzymes. RNA was fractionated into polyadenylic [poly(A)]+ and poly(A)- molecules using poly(U)-Sepharose, and undergraded virus-specific RNA was isolated by hybridization to viral DNA in 50% formamide at 37 degrees C. Viral mRNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea. Two 3H-labeled RNase T2-resistant oligonucleotide fractions with charges between -5 and -6 were obtained, consistent with two classes of 5' terminal methyl "cap" structures, m7G(5')ppp(5')NmpNp (cap 1) and m7G(5')ppp(5')NmNmpNp (cap 2) (Nm is a ribose 2'-O-methylation). The putative cap 1 contains all the methylated constituents of cap 1 plus Cm. The molar ratios of m7G to 2'-O-methylnucleosides is about 1.0 for cap 1 and 0.5 for cap 2, consistent with the proposed cap structures. Most significant, compositional analysis indicates four different cap 1 structures and at least three different cap 2 structures. Thus there is a minimum of seven early viral mRNA species with different cap structures, unless each type of mRNA can have more than one 5' terminus. In addition to methylated caps, early mRNA contains internal base methylations, exclusively as m6A, as shown by analyses of the mononucleotide (-2 charge) fraction. m6A was present in the ratio of 1 mol of m6Ap per 450 nucleotides. Thus viral mRNA molecules contain two to three internal m6A residues per methyl cap, since there is on the average 1 cap per 1,250 nucleotides.     

Hypomethylation of paternal DNA in the late mouse zygote is not essential for development

Global demethylation of DNA which marks the onset of development occurs asynchronously in the mouse; paternal DNA is demethylated at the the zygote stage, whereas maternal DNA is demethylated later in development. The biological function of such asymmetry and its underlying mechanisms are currently unknown. To test the hypothesis that the early demethylation of male DNA may be associated with protamine-histone exchange, we ,used round spermatids, whose DNA is still associated with histones, for artificial fertilization (round spermatid injection or ROSI), and compared the level of methylation of metaphase chromosomes in the resulting zygotes with the level of methylation in zygotes obtained after fertilization using mature sperm heads (intracytoplasmic sperm injection or ICSI). In contrast to ICSI-derived zygotes, ROSI-derived zygotes possessed only slightly demethylated paternal DNA. Both types of zygotes developed to term with similar rates which shows that hypomethylation of paternal DNA at the zygotic metaphase is not essential for full development in mice. Incorporation of exogenously expressed histone H2BYFP into paternal pronuclei was significantly higher in ICSI-derived zygotes than in ROSI-derived zygotes. Surprisingly, in the latter the incorporation of histone H2BYFP into the paternal pronucleus was still significantly higher than into the maternal pronucleus, suggesting that some exchange of chromatin-associated proteins occurs not only after ICSI but also after ROSI. This may explain why after ROSI, some transient demethylation of paternal DNA occurs early after fertilization, thus providing support for the hypothesis regarding the link between paternal DNA demethylation and protamine/histone exchange.     

Epigenetic discrimination by mouse metaphase II oocytes mediates asymmetric chromatin remodeling independently of meiotic exit

In mammalian fertilization, paternal chromatin is exhaustively remodeled, yet the maternal contribution to this process is unknown. To address this, we prevented the induction of meiotic exit by spermatozoa and examined sperm chromatin remodeling in metaphase II (mII) oocytes. Methylation of paternal H3-K4 and H3-K9 remained low, unlike maternal H3, although paternal H3-K4 methylation increased in zygotes. Thus, mII cytoplasm can sustain epigenetic asymmetry in a cell-cycle dependent manner. Paternal genomic DNA underwent oocyte-mediated cytosine demethylation and acquired maternally-derived K12-acetylated H4 (AcH4-K12) independently of microtubule assembly and maternal chromatin. AcH4-K12 persisted without typical maturation-associated deacetylation, irrespective of paternal pan-genomic cytosine methylation. Contrastingly, somatic cell nuclei underwent rapid H4 deacetylation; sperm and somatic chromatin exhibited asymmetric AcH4-K12 dynamics simultaneously within the same mII oocyte. Inhibition of somatic histone deacetylation revealed endogenous histone acetyl transferase activity. Oocytes thus specify the histone acetylation status of given nuclei by differentially targeting histone deacetylase and acetyl transferase activities. Asymmetric H4 acetylation during and immediately after fertilization was dispensable for development when both parental chromatin sets were hyperacetylated. These studies delineate non-zygotic chromatin remodeling and suggest a powerful model with which to study de novo genomic reprogramming.     

Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchinStrongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.     

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.