A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1

Using circular dichroism to probe the extent of DNA condensation in chromatin, we have demonstrated that a major nucleolar protein, nucleolin can decondense chromatin. By means of various binding assays we show that nucleolin has a strong affinity for histone H1 and that the phosphorylated N-terminal domain, rich in lengthy stretches of acidic amino acids, is responsible for this ionic interaction. Additional experiments clearly demonstrate that nucleolin is unable to act as a nucleosome core assembly or disassembly factor and hence has little affinity for the core histone octamer. We propose that this nucleolar protein induces chromatin decondensation by binding to histone H1, and that nucleolin can therefore be regarded as a protein of the high-mobility-group type.     

Germ cell sex prior to meiosis in the rainbow trout

Germ cells make two major decisions when they move from an indeterminate state to their final stage of gamete production. One decision is sexual commitment for sperm or egg production, and the other is to maintain mitotic division or entry into meiosis. It is unclear whether the two decisions are made as a single event or separate events, because there has been no evidence for the presence of germ cell sex prior to meiosis. Here we report direct evidence in the fish rainbow trout that gonia have distinct sexuality. We show that dazl expression occurs in both male and female gonia but exhibits differential intracellular distribution. More strikingly, we show that boule is highly expressed in male gonia but absent in female gonia. Therefore, mitotic gonia possess sex, sperm/egg decision and mitosis/meiosis decision are two independent events, and sperm/egg decision precedes mitosis/meiosis decision in rainbow trout, making this organism a unique vertebrate model for mechanistic understanding of germ cell sex differentiation and relationship between the two decisions.     

Expression of new KDN-gangliosides in rainbow trout testis during spermatogenesis and their structural identification

The developmental expression of 2-keto-3-deoxy-D-glycero-D-galacto-nononicacid-containing glycosphingolipids (KDN-gangliosides) in rainbowtrout testis during spermatogenesis was studied using a monoclonalantibody, mAb.kdn3G, which recognizes the KDN     

Immunochemical detection of changes in chromatin subunits induced by histone H4 acetylation.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non-acetylated H3, H2A, and H2B with either mono-, di-, or tri-acetylated H4 isolated from cuttle -fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C-terminal) peptide of H3 was considerably decreased when di- or tri-acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.     

Protamine messenger RNA: evidence for early synthesis and accumulation during spermatogenesis in rainbow trout

Trout testis cells were separated into various developmental classes by velocity sedimentation in bovine serum albumin gradients and were identified morphologically with particular stages of the process of spermatogenesis. The stage of testis cell differentiation at which protamine mRNA appears in the cell cytoplasm for the first time was determined by hybridization of RNA populations extracted from the separated cells to radioactively labeled protamine cDNA. Primary spermatocytes represent the earliest stage of differentiation at which protamine mRNA can be detected in large quantities in the cell cytoplasm, establishing that the synthesis of this class of mRNA occurs at a much earlier stage than the time of its translation at the spermatid stage. Protamine mRNA sequences were found in both the polysomes and postribosomal supernatant of the spermatid cells which are involved in the synthesis of protamine, while primary and secondary spermatocytes contained the mRNA sequences only in their postribosomal supernatant fractions. These findings strongly suggest that protamine mRNA is synthesized, accumulated, and stored in the cell sap of primary and secondary spermatocytes in the form of “inactive” messenger ribonucleoprotein particles, which are “activated” and translated at the spermatid stage.     

The condensation of chromatin and histone H1-depleted chromatin by spermine

At low ionic strength, spermine induces aggregation of native and H1-depleted chromatin at spermine/phosphate (Sp/P) ratios of 0.15 and 0.3, respectively. Physico-chemical methods (electric dichroism, circular dichroism and thermal denaturation) show that spermine, at Sp/P less than 0.15, does not appreciably alter the conformation of native chromatin and interacts unspecifically with all parts of chromatin DNA (linker as well as regions slightly or tightly bound to histones). In chromatin, the role of spermine could be more important in the stabilization of higher-order structure than in the condensation of the 30 nm solenoid. The addition of spermine to H1-depleted chromatin revealed two important features: (i) spermine can partially mimic the role of histone H1 in the condensation of chromatin; (ii) the core histone octamer does not appear to play any role in the aggregation process by spermine as DNA and H1-depleted chromatin aggregate at the same Sp/P ratio.     

Acetylation of histone H4 in complex structural transitions of spermiogenic chromatin

In spermiogenic nuclei of the cephalopod mollusc Sepia officinalis histones are replaced by a precursor-protamine molecule, which is later converted into protamine. Simultaneously, spermiogenic chromatin undergoes a complex structural change. Somatic-like chromatin belonging to the earliest spermatid is progressively reorganized into: (a) granules of 20 nm diameter, (b) fibres of 30-35 nm, and (c) fibres of 40-50 nm. In the final phases of spermiogenesis these fibres of 40-50 nm join to form larger structures of condensed chromatin, and lastly, the uniformly packed chromatin in the sperm nucleus. Using specific antibodies for mono- and hyperacetylated forms of histone H4, in this work we show that the first structural remodelling of chromatin (from somatic-like organization into 20 nm granules) is given concomitantly with a massive mono-acetylation of H4 (acetylation in lysine 12), whereas the structural remodelling from 30-35 to 40-50 nm fibres is produced simultaneously with hyperacetylation of H4 and the nuclear removal of histones.     

Expression of the mouse testicular histone gene H1t during spermatogenesis

 The testicular H1 histone variant, H1t, is synthesized during spermatogenesis in mammalian male germ cells. In situ hybridization and immunohistochemical techniques were used to assign the expression of either the H1t mRNA or the H1t protein to specific cell stages of spermatogenesis. Our results show the presence of the H1t mRNA only in the late and mid-pachytene stages, whereas the protein occurs first in pachytene spermatocytes, and persists until later stages from round up to elongated spermatids. Accepted: 1 March 1996     

Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4

The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A^Cnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A^Cnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A^Cnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A^Cnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A^Cnp1 for assembly into central domain chromatin, resulting in less CENP-A^Cnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A^Cnp1 influence the extent of DNA at centromeres that is packaged in CENP-A^Cnp1 chromatin and the composition of this chromatin. Thus, CENP-A^Cnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A^Cnp1 and other core histones.     

Changes in nuclear content of protein conjugate histone H2A-ubiquitin during rooster spermatogenesis

Electrophoretic analysis of acid-soluble chromosomal proteins isolated from rooster testis cell nuclei at different stages of spermatogenesis, revealed that the nuclear content of a protein identified by its solubility, electrophoretic mobility and amino acid analysis as the protein conjugate histone H2A-ubiquitin (uH2A, A24) changed markedly from meiotic cells to late spermatids. The protein was not detectable in tetraploid primary spermatocytes; it was present in 1.7% of the total amount of nucleosomal core histones in early spermatids and reached its maximum level (3.5% and 11%) at the end of spermiogenesis, when histones are replaced by the protamine galline.