Lindsay J. Frehlick

Protamines, in the Footsteps of Linker Histone Evolution

It was perhaps a lucky coincidence that the early attempts to establish the chemical composition of the cell nucleus were initially carried out on such diverse biological systems as salmon sperm heads (1) and geese and chicken erythrocytes (2). Examination of sperm and erythrocyte systems, respectively, lead to the protamine and histone concepts (3). We know with certainty that, with the exception of the male gametes, all somatic cells exclusively contain histones. Hence, in metazoans, protamines (4, 5) are confined to the sperm nuclear chromatin, and even among sperm, protamines are not always present. Indeed, a large number of metazoans contain somatic-like histones in their sperm, and some crustaceans (order Decapoda) lack any chromosomal proteins in their sperm (6, 7). Therefore, in contrast to the somatic nucleus, sperm chromatin may have a much more diverse protein composition. It was not until the first attempt of classification of the sperm nuclear basic proteins (SNBPs)2 by David Bloch (6, 8), an effort later on extended by Harold Kasinsky (7), that a clearer picture started to emerge in this regard. More recently, an enormous effort has been carried out in several laboratories, including our own (9–15), to extend this analysis to a large number of representative organisms from the different phylogenetic groups. With a broader perspective now available, SNBP heterogeneity can be restricted to three major groups or types: histone (H), protamine (P), and protamine-like (PL) (16). Histones consist of core histones (histones H2A, H2B, H3, and H4) and linker histones (histone H1 family). The names refer to the structural role of these proteins. Core histones are responsible for constraining DNA wrapped about a histone core to produce a nucleoprotein complex (chromatin subunit) known as a nucleosome core particle. Linker histones bind to the linker DNA regions connecting adjacent nucleosome core particles and assist in the folding of the chromatin fiber (17). Protamines are a highly compositionally and structurally heterogeneous group of proteins (9). They exhibit a high charge density and a prevalence of arginine in their composition (13), a fact that it is most likely related to the higher affinity with which this basic amino acid binds to DNA (18). They lack any secondary structure in solution butmay adopt a folded conformation upon interactionwithDNA. Protamine-like proteins share compositional and structural similarities between histones and protamines (9, 16). Hence they represent a structurally intermediate group that will be discussed more extensively in the following sections of this review. To a certain extent, all three types of SNBP can be considered structurally analogous as all of them produce folded chromatin fibers of 30–50 nm (19) regardless of the particular structure of the individual nucleoprotamine complexes. At the functional level, somatic histones bind to DNA in a highly dynamic way that not only helps in the folding of the genome but also has an important role in the epigenetic regulation of gene expression (20, 21). In contrast, protamine and protamine-like SNBPs of the spermatozoa bind very tightly to the genome to produce a maximal genome compaction, which completely abolishes the epigenetic information of the paternal histones (9) in this terminally differentiated system. This epigenetic silencing can be reverted only with the assistance of highly specialized protamine-removing proteins such as nucleoplasmin during the molecular events involved in nuclear metabolism during early fertilization (22). The analogous structural DNA condensation potential of the three types of SNBPs raises a question as to the extent of structural homology between them. In the next sections we are going to discuss a series of recent papers that suggest that protamines and protamine-like proteins are evolutionarily related to linker histones.

Long-term evolution and functional diversification in the members of the nucleophosmin/nucleoplasmin family of nuclear chaperones

The proper assembly of basic proteins with nucleic acids is a reaction that must be facilitated to prevent protein aggregation and formation of nonspecific nucleoprotein complexes. The proteins that mediate this orderly protein assembly are generally termed molecular (or nuclear) chaperones. The nucleophosmin/nucleoplasmin (NPM) family of molecular chaperones encompasses members ubiquitously expressed in many somatic tissues (NPM1 and -3) or specific to oocytes and eggs (NPM2). The study of this family of molecular chaperones has experienced a renewed interest in the past few years. However, there is a lack of information regarding the molecular evolution of these proteins. This work represents the first attempt to characterize the long-term evolution followed by the members of this family. Our analysis shows that there is extensive silent divergence at the nucleotide level suggesting that this family has been subject to strong purifying selection at the protein level. In contrast to NPM1 and NPM-like proteins in invertebrates, NPM2 and NPM3 have a polyphyletic origin. Furthermore, the presence of selection for high frequencies of acidic residues as well as the existence of higher levels of codon bias was detected at the C-terminal ends, which can be ascribed to the critical role played by these residues in constituting the acidic tracts and to the preferred codon usage for phosphorylatable amino acids at these regions.

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.

The characterization of amphibian nucleoplasmins yields new insight into their role in sperm chromatin remodeling

BackgroundNucleoplasmin is a nuclear chaperone protein that has been shown to participate in the remodeling of sperm chromatin immediately after fertilization by displacing highly specialized sperm nuclear basic proteins (SNBPs), such as protamine (P type) and protamine-like (PL type) proteins, from the sperm chromatin and by the transfer of histone H2A-H2B. The presence of SNBPs of the histone type (H type) in some organisms (very similar to the histones found in somatic tissues) raises uncertainty about the need for a nucleoplasmin-mediated removal process in such cases and poses a very interesting question regarding the appearance and further differentiation of the sperm chromatin remodeling function of nucleoplasmin and the implicit relationship with SNBP diversity The amphibians represent an unique opportunity to address this issue as they contain genera with SNBPs representative of each of the three main types: Rana (H type); Xenopus (PL type) and Bufo (P type).ResultsIn this work, the presence of nucleoplasmin in oocyte extracts from these three organisms has been assessed using Western Blotting. We have used mass spectrometry and cloning techniques to characterize the full-length cDNA sequences of Rana catesbeiana and Bufo marinus nucleoplasmin. Northern dot blot analysis shows that nucleoplasmin is mainly transcribed in the egg of the former species. Phylogenetic analysis of nucleoplasmin family members from various metazoans suggests that amphibian nucleoplasmins group closely with mammalian NPM2 proteins.ConclusionWe have shown that these organisms, in striking contrast to their SNBPs, all contain nucleoplasmins with very similar primary structures. This result has important implications as it suggests that nucleoplasmins role in chromatin assembly during early zygote development could have been complemented by the acquisition of a new function of non-specifically removing SNBPs in sperm chromatin remodeling. This acquired function would have been strongly determined by the constraints imposed by the appearance and differentiation of SNBPs in the sperm.

Histone H2A (H2A.X and H2A.Z) variants in molluscs: molecular characterization and potential implications for chromatin dynamics.

Histone variants are used by the cell to build specialized nucleosomes, replacing canonical histones and generating functionally specialized chromatin domains. Among many other processes, the specialization imparted by histone H2A (H2A.X and H2A.Z) variants to the nucleosome core particle constitutes the earliest response to DNA damage in the cell. Consequently, chromatin-based genotoxicity tests have been developed in those cases where enough information pertaining chromatin structure and dynamics is available (i.e., human and mouse). However, detailed chromatin knowledge is almost absent in most organisms, specially protostome animals. Molluscs (which represent sentinel organisms for the study of pollution) are not an exception to this lack of knowledge. In the present work we first identified the existence of functionally differentiated histone H2A.X and H2A.Z variants in the mussel Mytilus galloprovincialis (MgH2A.X and MgH2A.Z), a marine organism widely used in biomonitoring programs. Our results support the functional specialization of these variants based on: a) their active expression in different tissues, as revealed by the isolation of native MgH2A.X and MgH2A.Z proteins in gonad and hepatopancreas; b) the evolutionary conservation of different residues encompassing functional relevance; and c) their ability to confer specialization to nucleosomes, as revealed by nucleosome reconstitution experiments using recombinant MgH2A.X and MgH2A.Z histones. Given the seminal role of these variants in maintaining genomic integrity and regulating gene expression, their preliminary characterization opens up new potential applications for the future development of chromatin-based genotoxicity tests in pollution biomonitoring programs.

Genome organization by vertebrate sperm nuclear basic proteins (SNBPs)

DNA is tightly packed in the sperm nucleus through its association with specific chromosomal proteins that are heterogeneous in size and known as sperm nuclear basic proteins (SNBPs). Despite their structural diversity, SNBPs are evolutionarily related and can be classified into three major groups or types: histone (H-type); protamine (P-type), and protamine-like (PL-type). The three types are widespread amongst vertebrates. During spermiogenesis these proteins replace the somatic histones that are present at the onset of spermatogenesis. Mammals exhibit an increased level of complexity as transition proteins (TPs) temporarily bind to DNA before being replaced by protamines in the mature sperm. The proper sequential chromatin remodeling is dependent on global post-translational modifications (PTMs) of the chromosomal proteins involved, including acetylation and phosphorylation. A transient 20nm chromatin fiber that is independent of the SNBP type is often formed. The temporally and spatially organized compaction of chromatin during spermiogenesis may be important for the understanding of post-meiotic events such as gene expression, Huntington’s disease CAG expansion, and the remnant histones that are present in the mature sperm of certain mammals, including humans. Alterations in SNBP composition result in DNA damage and infertility, underscoring the importance of these proteins for male germline genome integrity.