Núria Saperas

Histone H1 and the origin of protamines

We present evidence that chordate protamines have evolved from histone H1. During the final stages of spermatogenesis, the compaction of DNA in many organisms is accomplished by the replacement of histones with a class of arginine-rich proteins called protamines. In other organisms, however, condensation of sperm DNA can occur with comparable efficiency in the presence of somatic-type histones or, alternatively, an intermediate class of proteins called protamine-like proteins. The idea that the highly specialized sperm chromosomal proteins (protamines) and somatic chromosomal proteins (histones) could be related dates back almost to the discovery of these proteins. Although this notion has frequently been revisited since that time, there has been a complete lack of supporting experimental evidence. Here we show that the emergence of protamines in chordates occurred very quickly, as a result of the conversion of a lysine-rich histone H1 to an arginine-rich protamine. We have characterized the sperm nuclear basic proteins of the tunicate Styela montereyensis, which we show consists of both a protamine and a sperm-specific histone H1 with a protamine tail. Comparison of the genes encoding these proteins to that of a sister protochordate, Ciona intestinalis, has indicated this rapid and dramatic change is most likely the result of frameshift mutations in the tail of the sperm-specific histone H1. By establishing an evolutionary link between the chromatin-condensing histone H1s of somatic tissues and the chromatin-condensing proteins of the sperm, these results provide unequivocal support to the notion that vertebrate protamines evolved from histones.

On the evolution of protamines in bony Fish: Alternatives to the “Retroviral horizontal transmission” hypothesis

Fish protamines are highly specialized molecules which are responsible for chromatin condensation during the last stages of spermatogenesis (spermiogenesis). However, not all fish contain protamines in their sperm nuclei; rather, there seems to be a random distribution of protamines within this group. The origin of this sporadic presence of protamines in the sperm and its significance have not yet been precisely determined. In this paper we have conducted an exhaustive survey of the literature available on the different types of nuclear protein composition of the sperm of teleost fish in order to try to correlate these data with what is presently known about the taxonomy of this group. The results of this analysis have allowed us to make the following observations. The divergence between protamines and histones has occurred several times during the evolution of the bony fish. However, the relative frequency of this divergence is almost negligible during the differentiation of genera and species (intrafamily variation) and is very small during the differentiation of families (interfamily variation). Nevertheless, the divergence is very noticeable among the different orders. It is therefore possible to conclude from all this that the sporadic distribution of protamines in bony fish is not a random event as initially believed. Furthermore, such a heterogeneous distribution of protamines cannot be easily accounted for by a mechanism of horizontal retroviral transmission through repeated and independent acquisition of a prot amine gene as has been recently proposed (Jankowski, Stater, Dixon (1986) J Mol Evol 23:1–10). Rather, it could possibly be explained by a repeated and independent loss of the expression of the protamine gene (or loss of the gene itself) which mainly occurred during the diversification of the orders of this group.

Crystal structure of a complex of DNA with one AT-hook of HMGA1.

We present here for the first time the crystal structure of an AT-hook domain. We show the structure of an AT-hook of the ubiquitous nuclear protein HMGA1, combined with the oligonucleotide d(CGAATTAATTCG)2, which has two potential AATT interacting groups. Interaction with only one of them is found. The structure presents analogies and significant differences with previous NMR studies: the AT-hook forms hydrogen bonds between main-chain NH groups and thymines in the minor groove, DNA is bent and the minor groove is widened.

Spermiogenic nuclear protein transitions and chromatin condensation. Proposal for an ancestral model of nuclear spermiogenesis

We have chosen three species (Sparus aurata, Dicentrarchus labrax, and Monodonta turbinata) that represent different transition patterns in the composition and structure of spermiogenic nuclei. The transition patterns of these species are representative of spermiogenesis in a large number of animal species. We analyze: (a) nuclear protein exchange; (b) chromatin condensation pattern; and (c) histone acetylation during spermiogenic development. In the simplest spermiogenesis histones and nucleosomes remain in mature sperm. Chromatin of spermatids is organized into 20 nm granules, simultaneous with a nuclear volume reduction. The granules coalesce in the final stage of spermiogenesis. Granular chromatin is correlated with acetylation of histones H3 and H4, whereas final coalescence is associated with histone deacetylation. We also studied two other spermiogenesis where a basic protein substitutes histones. Each species has a very different substituting protein. One has a typical protamine of 34 amino acids; the other has a sperm nuclear basic proteins (SNBP) of 106 amino acids. In both, the structural transitions and histone acetylation pattern are similar: in early spermiogenesis chromatin is organized into 20 nm granules, and histones are significantly acetylated, while the nuclear volume decreases. Subsequently, acetylated histones are displaced by the protamine or SNBP. Histone substitution causes chromatin remodelling and additional reduction in nuclear volume. We analyze these three cases together with earlier works and propose that the formation of 20 nm granules containing acetylated H3 and H4 accomplishes the minimum functional requirement to be considered the most evolutionarily ancestral chromatin conformation preceding condensation in animal spermiogenesis.

A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin organization

Protamine‐like proteins constitute a group of sperm nuclear basic proteins that have been shown to be related to somatic linker histones (histone H1 family). Like protamines, they usually replace the chromatin somatic histone complement during spermiogenesis; hence their name. Several of these proteins have been characterized to date in invertebrate organisms, but information about their occurrence and characterization in vertebrates is still lacking. In this sense, the genus Mullus is unique, as it is the only known vertebrate that has its sperm chromatin organized by virtually only protamine‐like proteins. We show that the sperm chromatin of this organism is organized by two type I protamine‐like proteins (PL‐I), and we characterize the major protamine‐like component of the fish Mullus surmuletus (striped red mullet). The native chromatin structure resulting from the association of these proteins with DNA was studied by micrococcal nuclease digestion as well as electron microscopy and X‐ray diffraction. It is shown that the PL‐I proteins organize chromatin in parallel DNA bundles of different thickness in a quite distinct arrangement that is reminiscent of the chromatin organization of those organisms that contain protamines (but not histones) in their sperm.

Mutation of the small acidic tract A1 drastically reduces nucleoplasmin activity

Xenopus laevis nucleoplasmin is a molecular chaperone that mediates sperm decondensation and nucleosome assembly. Nucleoplasmin has three acidic tracts (A1, A2 and A3) and until recent years the long polyglutamic tract A2 was thought to be the binding site for basic proteins. However, the latest publications in this field show that neither A2 nor A3 is indispensable for histone and sperm‐specific protein binding. In this work, we show that the mutation of only four acidic amino acid residues of the small A1 tract drastically reduces nucleoplasmin decondensing activity, pointing out this region as the potential binding site for sperm proteins.

Sperm-Specific Basic Proteins in the Holocephalan Fish Hydrolagus colliei (Chondrichthyes, Chimaeriformes) and Comparison with Protamines from an Elasmobranch

Seven basic proteins can be isolated from sperm nuclei of the holocephalan ratfish Hydrolagus colliei. Two of these proteins (R3 and m0) are devoid of cysteine, whereas five of them (R1, R2, m1, m2, and m3) contain low levels of this amino acid residue. The proteins R1, R2, and R3 are major ones in the sperm nuclei of H. colliei, and they are analogous to basic proteins Z1, Z2, and Z3 (scylliorhinines) from the sperm of the elasmobranch Scyliorhinus canicula. However, taking into account the partial sequence of R3 protein and the number of cysteines in R1 and R2, these proteins do not seem to be homologous to the scylliorhinines. A comparison of sperm basic proteins between H. colliei (a holocephalan) and S. canicula (an elasmobranch) suggests a remarkable divergence of these proteins from a common ancestral pattern during the evolution of Chondrichthyes.

Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: Examples from mollusks☆

In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and conclude that the appearance of precursor (intermediate) molecules facilitated the development of complex patterns of condensation and, as a consequence, a great diversity of forms in the sperm cell nuclei

The primary structure of a chondrichthyan protamine: A new apparent contradiction in protamine evolution

We have determined the primary structure of protamine R3 from ratfish (Hydrolagus colliei), a species belonging to the order Chimaeriformes (an old phylogenetic line among the chondrichthyes). Protamine R3 contains 48 residues organized as follows: ARRRH SMKKK RKSVR RRKTR KNQRK RKNSL GRSFK (Q/A)HGFL KQPPR FRP. Comparison of this sequence with both protamine Z3 fromScyliorhinus canicula (a chondrichthyan) and typical protamines from bony fish generates an apparent contradiction: Two relatively close species (H. colhei andS. canicula, both chondichthyes) display different protamines, whereas species more distant in evolution (S. canicula and bony fish) contain very similar protamine molecules. We note that this is not an isolated case in the evolution of sperm nuclear basic proteins (SNBPs) and discuss the possible significance of this fact.

Chromosomal Proteins of the Sperm of a Cephalochordate (Branchiostoma floridae) and an Agnathan (Petromyzon marinus): Compositional Variability of the Nuclear Sperm Proteins of Deuterostomes

We have isolated and characterized for the first time the chromosomal proteins from the nucleus of the sperm of a lancelet (amphioxus) Branchiostoma floridae (Hubbs, 1922) (Phylum Chordata: Subphylum Cephalochordata) and of a lamprey Petromyzon marinus (Linnaeus, 1758) (Phylum Chordata: Subphylum Vertebrata: Class Agnatha). In the first case, the major protein component of the sperm-chromatin of a lancelet is a highly specialized protamine-like (PL) protein that has structural and compositional features similar to those of PL-III from bivalve mollusks. In contrast, the chromatin of the sperm of the lamprey has a structural arrangement and protein composition (histones) very similar to that found in the somatic cells of all eukaryotic organisms. Among the deuterostomes, chromosomal protein variability is considerably greater in representatives of the Phylum Chordata than in echinoderms. The possible evolutionary significance of these findings is discussed.