Histone variants and chromatin structure during sea urchin development

At the late blastula stage of sea urchin development a changeover of histone synthesis and chromatin composition takes place. Synthesis of the early histone variants declines while another set, the late histone variants, begins to be detected. During subsequent development the late histones accumulate steadily. In the 9-day larva only late histone variants are detectable. Micrococcal nuclease acts differentially on early and late nuclei. There is a depressed release of acid-soluble DNA when chromatin containing the late histones is digested. Nucleosomal repeat lengths change systematically and in parallel with the changing histone composition. Blastula and preblastula chromatin have a significantly shorter major repeat length than does the chromatin of 9-, 11-, and 16-day larvae. Intermediate stages of development have chromatin with intermediate periodicities. These differences are observed when the determinations are made under denaturing conditions of electrophoresis. Repeat lengths were found to be independent of the extent of digestion at all stages examined except the pluteus, in which there is an increase of the apparent repeat length as digestion proceeds. Pancreatic DNase I digests nuclei from blastulae and 9-day larvae similarly. Changes in the histone composition of chromatin, in nuclease accessibility of chromatin, and in nucleosomal repeat length are all very closely correlated, implying that there are underlying causal relationships.     

Interaction of sperm histone variants and linker DNA during spermiogenesis in the sea urchin

Several physical properties of sea urchin spermatid chromatin, which contains phosphorylated Sp H1 and Sp H2B histone variants, and mature sperm chromatin, in which these histones are dephosphorylated, were compared. Density, thermal stability, average nucleosomal repeat length, and resistance to micrococcal nuclease digestion are all increased in mature sperm relative to spermatid chromatin. Since the chromatins are identical in histone variant subtypes, the altered physical properties are not a consequence of changes in histone primary structure during spermiogenesis. The data are interpreted to mean that dephosphorylation of the N-terminal regions of Sp H1 and Sp H2B in late spermatid nuclei permits strong ionic binding of these highly basic regions to the extended linker, stabilizing the highly condensed structure of sperm chromatin.     

The sea urchin (Sphaerechinus granularis) codes different H2B histones to assemble sperm and embryo chromatin.

Histones from sperm and gastrula nuclei of the sea urchin Sphaerechinus granularis were analyzed by three different electrophoretic methods, stained with specific color reactions and compared with one another. The histones of the two cell types showed identical properties in none of the three analytical methods employed but the greatest differences involved the H2B type of histones. H2B from embryo chromatin had properties similar to those of the corresponding molecule from calf thymus. In the sperm chromatin no such molecule was observed but two additional ones were found that differed from embryo H2B in size, charge and specific colour reactions. The differences were such that different genes may be postulated for the synthesis of the H2B histones found in sperm and in embryo.     

Phosphorylation of sea urchin sperm H1 and H2B histones precedes chromatin decondensation and H1 exchange during pronuclear formation

Immediately following fertilization in the sea urchin, sperm-specific histones Sp H1 and Sp H2B are phosphorylated. Then, in parallel with chromatin decondensation, nearly all phosphorylated Sp H1 is lost from the pronuclear chromatin, with the concurrent assimilation of the egg phosphoprotein CS H1. Chemical cleavage of in vivo labeled Sp H1 and Sp H2B shows that serine phosphorylation occurs in the unusually long N-terminal region of these proteins. These regions contain tandemly repeated tetra- and pentapeptide units each containing serine, proline, and two basic amino acids. It is proposed that sperm chromatin decondensation may require prior phosphorylation of these unusual N-terminal regions, whose function in the mature sperm may be to condense or stabilize its highly compact chromatin.     

Separation and properties of an H2B histone variant from the sperm chromatin of the sea urchin Sphaerechinus granularis

The purification and the physico-chemical characterization of one of the two H2B histone variants from the sperm of the sea urchin Sphaerechinus granularis are reported. The molecule shows, in addition to a distinctive molecular weight value, an amino acid composition different both from that of calf thymus H2B histone and from those of H2B histones from chromatin of sperm and embryos of other sea urchins. Circular dichroism and fluorescence data are discussed in comparison to those of calf thymus H2B.     

Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.     

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

Histone H2A.F/Z mRNA is stored in the egg cytoplasm and basally regulated in the sea urchin embryo.

The sea urchin H2A.F/Z histone is a member of a subclass of highly conserved H2A variants. Sequence analysis confirms that H2A.F/Z mRNA is polyadenylated. In situ hybridization studies demonstrate that maternal H2A.F/Z message is stored in the egg cytoplasm and present at equal levels in all cells of the mesenchyme blastula-stage embryo, suggesting that H2A.F/Z is not coordinately regulated with DNA synthesis. When blastula-stage embryos were exposed to DNA synthesis inhibitors, no effect on the steady-state level of H2A.F/Z mRNA was observed, while the level of late class H2B mRNA decreased substantially. These results provide evidence that the basal mode of regulation of this unusual histone variant is conserved evolutionarily.     

DNAase hydrolysis of chromatin DNA in intact sea urchin sperm cells. Effect of the ionic strength on the digestion parameters.

Chromatin DNA in intact functional Sphaerechinus granularis sperm cells has been digested with micrococcal nuclease at three different ionic strength conditions. The results show a highly-flexible chromatin organization similar to that found in sperm heads. The rate of digestion, the limit value of acid-soluble material and the fragmentation pattern show that the sensitivity of nucleosome and internucleosome DNA regions to nuclease hydrolysis depends on a delicate balance of polar and non polar interactions. At low ionic strength, both nucleosome and internucleosome regions are rapidly and completely hydrolysed at the same time and a transient subunit fragment of 120 b.p. average length is formed. At high ionic strength, internucleosome regions are preferentially hydrolysed; there is a limit digest value and a stable subunit fragment of 140 b.p. average length is formed. A supernucleosome organization in the high ionic strength environment of the sperm cells is suggested by the transient preferential formation of heptamers of nucleosome DNA fragments.     

Induction of cross-fertilization between sea urchin eggs and starfish sperm by polyethylene glycol treatment

Cross-fertilization between sea urchin eggs (Strongylocentrotus nudus) and starfish sperm (Asterina pectinifera) was induced by treatment with polyethylene glycol (PEG). Without treatment with PEG, the denuded egg surface (jelly coat- and vitelline coat-free) engulfed the head of acrosome-reacted sperm; however, sperm penetration did not occur [Kyozuka and Osanai, 1988]. When these eggs were exposed briefly to PEG (molecular weight 3,000) in seawater, the sperm entered the egg by membrane fusion. Cortical granules were discharged, and embryogenesis began following sperm penetration. PEG did not induce parthenogenesis in Strongylocentrotus eggs. Egg activation is thus closely linked with gamete membrane fusion.     

Histone crosstalk directed by H2B ubiquitination is required for chromatin boundary integrity

Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3' boundary of a heterochromatin region located upstream of the chicken β-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5' boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.     

Interaction of histone H1 from sea urchin sperm with superhelical and relaxed DNA

Complexes of histone H1 from sea urchin sperm (H1S) and calf thymus (H1T) with superhelical DNA I and relaxed circular DNA II have been analyzed by analytical sedimentation. Similar to H1T, the highly basic and relatively arginine-rich histone H1S preferentially interacts with DNA I compared to DNA II under competition conditions. However, H1S induces a stronger aggregation of bothforms of DNA than H1T. Below 0.05 M NaCl, the soluble complexes formed by both histones have similar properties, but aggregation proceeds in a different manner: H1S induces a stronger aggregation of DNA II as compared to DNA I, whereas H1T fails to aggregate DNA I.The results are explained on the basis of differences in amino acid sequence and structure of the two histones and related to the special chromatin condensing ability of histone H1S.     

The primary structure of histone H2A from the sperm cell of the sea urchin Parechinus angulosus

The 125 residues of the histone H2A from the sperm cells of the sea urchin Parechinus angulosus have been positioned. The N terminus is blocked by an acetyl group. Compared to the bovine histone, the sea urchin protein differs in 14 positions.