Roles of transition nuclear proteins in spermiogenesis

The transition nuclear proteins (TPs) constitute 90% of the chromatin basic proteins during the steps of spermiogenesis between histone removal and the deposition of the protamines. We first summarize the properties of the two major transition nuclear proteins, TP1 and TP2, and present concepts, based on their time of appearance in vivo and in vitro properties, regarding their roles. Distinct roles for the two TPs in histone displacement, sperm nuclear shaping, chromatin condensation, and maintenance of DNA integrity have been proposed. More definitive information on their roles in spermiogenesis has recently been obtained using mice with null mutations in the Tnp1 or Tnp2 genes for TP1 and TP2, respectively. In these mice, histone displacement and sperm nuclear shaping appear to progress quite normally. Spermatid nuclear condensation occurs, albeit in an abnormal fashion, and the mature sperm of the Tnp -null mutants are not as condensed as wild-type sperm. There is also evidence that sperm from these mutant mice contain an elevated level of DNA strand breaks. The mutant sperm showed several unexpected phenotypes, including a high incidence of configurational defects, such as heads bent back on midpieces, midpieces in hairpin configurations, coils, and clumps, other midpiece defects, reduced levels of proteolytic processing of protamine 2 during maturation, and reduced motility. The two TPs appear partly to compensate for each other as both Tnp1 - and Tnp2 -null mice were able to produce offspring, and appear to have largely overlapping functions as the two mutants had similar phenotypes.     

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.     

Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice

Previous studies have demonstrated the importance of transition nuclear proteins, TP1 and TP2, in spermatogenesis and male fertility. However, importance of the overall level of transition proteins and their level of redundancy in the production of normal sperm is not clear. Epididymal sperm from the nine possible Tnp1 and Tnp2 null genotypes demonstrated a general decrease in normal morphology, motility, chromatin condensation, and degree of protamine 2 processing with decreasing levels of transition proteins in mutant sperm. Nuclei of some mutant epididymal sperm stained poorly with hematoxylin and DNA fluorochromes, suggesting that the DNA of these sperm underwent degradation during epididymal transport. When epididymal sperm were injected directly into oocytes, fertilization and embryonic development were reduced only in the two most severely affected genotypes. These phenotypes indicated some functional redundancy of transition proteins; however, redundancy of transition protein function was not complete, as, for example, sperm from double heterozygous males had fewer abnormalities than sperm from males homozygous for a single Tnp null mutation. Our study suggests that each TP fulfills some unique function during spermiogenesis even though sperm phenotypes strongly indicate defects are largely attributable to an overall gene dosage effect. Similarities between sperm defects found in Tnp mutants and infertile patients make the Tnp mutants a valuable tool with which to study outcomes following fertilization using sperm with compromised DNA integrity.     

Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12–15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4′,6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis. (J Histochem Cytochem 57:951–962, 2009)     

Protamine 3 shows evidence of weak, positive selection in mouse species (genus Mus)--but it is not a protamine

Protamines are short and highly basic sperm-specific nuclear proteins that replace somatic histones during spermiogenesis in a process that is crucial for sperm formation and function. Many mammals have two protamine genes (PRM1 and PRM2) located in a gene cluster, which appears to evolve fast. Another gene in this cluster (designated protamine 3 [PRM3]) encodes a protein that is conserved among mammals but that does not seem to be involved in chromatin condensation. We have compared protein sequences and amino acid compositions of protamines in this gene cluster, searched for evidence of positive selection of PRM3, and examined whether sexual selection (sperm competition) may drive the evolution of the PRM3 gene. Nucleotide and amino acid analyses of mouse sequences revealed that PRM3 was very different from PRM1 and from both the precursor and the mature sequences of PRM2. Among 10 mouse species, PRM3 showed weak evidence of positive selection in two species, but there was no clear association with levels of sperm competition. In analyses from among mammalian species, no evidence of positive selection was found in PRM3. We conclude that PRM3 exhibits several clear differences from other protamines and, furthermore, that it cannot be regarded as a true protamine.     

Sequential Expression of Nucleoproteins during Rat Spermiogenesis

Transition proteins and protamines are highly basic sperm-specific nuclear proteins that serve to compact the DNA during late spermiogenesis. To understand their sequential role in this function, transition protein 1 (TP1), transition protein 2 (TP2), and protamine 1 (P1) were assayed by polyacrylamide gel electrophoresis in pools of microdissected, staged seminiferous tubule segments in the rat. The results were compared with immunocytochemical analyses of squash preparations from accurately identified stages of the epithelial cycle. TP2 was the first to appear as a faint band at stages IX–XI, followed by high levels at stages XII–XIV of the cycle. TP1 showed a low expression at stage XII of the cycle and peaked at stages XIII–I, whereas protamine 1 first appeared at stage I of the cycle and remained high throughout the rest of spermiogenesis. Immunocytochemical analyses and Western blots largely confirmed these results: TP2 in steps 9–14, TP1 in steps 12–15, and P1 from late step 11 to step 19 of spermiogenesis. We propose that TP2 is the first nucleoprotein that replaces histones from the spermatid nucleus, and its appearance is associated with the onset of nuclear elongation. TP1 shows up along with the compaction of the chromatin. The two transition proteins seem to have distinct roles during transformation of the nuclei and compaction of spermatid DNA.     

Nuclear and nucleoid localization are independently conserved functions in bacteriophage terminal proteins

Bacteriophage terminal proteins (TPs) prime DNA replication and become covalently linked to the DNA 5′‐ends. In addition, they are DNA‐binding proteins that direct early organization of phage DNA replication at the bacterial nucleoid and, unexpectedly, contain nuclear localization signals (NLSs), which localize them to the nucleus when expressed in mammalian cells. In spite of the lack of sequence homology among the phage TPs, these three properties share some common features, suggesting a possible evolutionary common origin of TPs. We show here that NLSs of three different phage TPs, Φ29, PRD1 and Cp‐1, are mapped within the protein region required for nucleoid targeting in bacteria, in agreement with a previously proposed common origin of DNA‐binding domains and NLSs. Furthermore, previously reported point mutants of Φ29 TP with no nuclear localization still can target the bacterial nucleoid, and Cp‐1 TP contains two independent NLSs, only one of them required for nucleoid localization. Altogether, our results show that nucleoid and nucleus localization sequence requirements partially overlap, but they can be uncoupled, suggesting that conservation of both features could have a common origin but, at the same time, they have been independently conserved during evolution.     

Involvement of importin-4 in the transport of transition protein 2 into the spermatid nucleus

Mammalian spermiogenesis is characterized by a unique chromatin-remodeling process in which histones are replaced by transition protein 1 (TP1), TP2, and TP4, which are further replaced by protamines. We showed previously that the import of TP2 into the haploid spermatid nucleus requires the components of cytosol and ATP. We have now carried out a detailed analysis to characterize the molecular components underlying the nuclear translocation of TP2. Real-time PCR analysis of the expression of different importins in testicular germ cells revealed that importin-4 and importin-beta3 are significantly up-regulated in tetraploid and haploid germ cells. We carried out physical interaction studies as well as an in vitro nuclear transport assay using recombinant TP2 and the nuclear localization signal of TP2 (TP2(NLS)) fused to glutathione S-transferase in digitonin-permeabilized, haploid, round spermatids and identified importin-4 to be involved in the import of TP2. A three-dimensional model of the importin-4 protein was generated using the crystal structure of importin-beta1 as the template. Molecular docking simulations of TP2(NLS) with the importin-4 structure led to the identification of a TP2(NLS) binding pocket spanning the three helices (helices 21 to 23) of importin-4, which was experimentally confirmed by in vitro interaction and import studies with different deletion mutants of importin-4. In contrast to TP2, TP1 import was accomplished through a passive diffusion process.     

Separation of spermatogenic cells from adult transgenic mouse testes using unit-gravity sedimentation

During the final stage of spermatogenesis (i.e., spermiogenesis), round spermatids differentiate into mature spermatozoa. This transformation is mediated by a suite of nuclear packaging proteins. These include the transition proteins and the protamines. The two human protamines PRM1 and PRM2, and transition protein TNP2, are encoded by a single chromatin domain bounded by two regions of matrix attachment. Previous transgenic studies in our laboratory have shown that mice harboring a 40-kb segment of human chromosome 16p13.13 containing the PRM1 → PRM2 → TNP2 domain express the transgene in a haploid-specific, copy number-dependent, and position-independent manner. While these results indicate that this segment of the genome is a complete structural and functional regulatory unit, the elements governing the haploid expression of this suite of genes remain to be clearly defined. The preparation of spermatogenic cells is required to begin to address this mechanism. The CELSEP (Wescor/Dupont Inc. Wilmington, DE) unit-gravity sedimentation apparatus provides a simple, efficient, and reproducible means to separate testicular germ cells at all stages along this differentiative pathway. The high quality and integrity of germ cells obtained by this means provides a valuable resource for characterizing the molecular mechanisms governing the regulation of the PRM1 → PRM2 → TNP2 domain during spermatogenesis. A discussion of the CELSEP apparatus and the application of this methodology in our laboratory are presented.     

Transition nuclear proteins during spermiogenesis: unrepaired DNA breaks not allowed

The completion of spermiogenesis requires condensation of the haploid spermatid genome. This task is accomplished in a gradual and relentless manner by first erasing the nucleosomal organization of chromatin while the DNA is protected by transient nuclear proteins TP1 and TP2. Then, the more permanent protamines come into play to stabilize the spermatid genome until fertilization occurs. Mice lacking TPI manage to produce relatively structurally normal sperm, although fertility is reduced and chromatin condensation is abnormal despite the compensatory expression of TP2. TP1 and TP2 appear to have the house-keeping function of reestablishing continuity when chromatin breaks take place during the remodeling process. DNA single-strand breaks are frequently observed when spermiogenesis is half completed. There is a temporal relationship between TP1 and DNA breaks: TP1 nuclear levels increase and the frequency of DNA breaks become less prominent as spermiogenesis is reaching completion. TP1 seems to hold the broken ends together until an as-yet-unidentified ligase bridges the gap.     

Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene

Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.     

Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis

During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.     

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.     

Chromatin organization during spermiogenesis in Octopus vulgaris. II: DNA-interacting proteins

In this article we study the proteins responsible for chromatin condensation during spermiogenesis in the cephalopod Octopus vulgaris. The DNA of ripe sperm nuclei in this species is condensed by a set of five different proteins. Four of these proteins are protamines. The main protamine (Po2), a protein of 44 amino acid residues, is extraordinarily simple (composed of only three different amino acid types: arginine (R), serine (S), and glycine (G). It is a basic molecule consisting of 79.5 mol% arginine residues. The rest of the protamines (Po3, Po4, Po5) are smaller molecules (33, 28, and 30 amino acid residues, respectively) that are homologous among themselves and probably with the main Po2 protamine. The ripe sperm nucleus of O. vulgaris also contains a small quantity of a molecule (Po1) that is similar to Po2 protamine. This protein could represent a Po2 protamine-precursor in a very advanced step of its processing. We discuss the characteristics of these proteins, as well as the relation between the complexity of chromatin condensation and the transitions of nuclear proteins during spermiogenesis in O. vulgaris.     

Mapping of Post-translational Modifications of Transition Proteins,TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2

In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg⁷¹, Arg⁷⁵, and Arg⁹² residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys⁸⁸ and Lys⁹¹ residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome.     

Characterization of the specific DNA-binding properties of Tnp26, the transposase of insertion sequence IS26

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1–P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix–turn–helix domain (amino acids I13–R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1–D12) did not bind. The N-terminal M1–P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1–M234) and Tnp26 M1–P56 fragment. These findings indicate that the helix–turn–helix and disordered domains of Tnp26 play a role in Tnp26–TIR complex formation. Both domains are conserved in all members of the IS26 family.