Nucleosome assembly protein 1

DEVELOPMENTAL BIOLOGY

Embryonic

In the syncyial blastoderm prior to cell cycle 14 NAP1 is observed throughout the embryo, with strong nuclear staining. NAP1 is primary in the cytoplasm of cells in G2 phase of cell cycle 14. In cell cycle 15 NAP1 is present in both the nucleus and the cytoplasm during S phase and is primarily cytoplasmic in G2 (Ito, 1996a). By comparison, dNLP, the Drosophila Nucleoplasmin like protein, is located in the nucleus, as is Xenopus nucloeplasmin (Ito, 1996b). Both NAP1 and dNLP are present throughout development. They reach their highest levels in early embryos and remain at reduced, but significant levels throughout development (Ito, 1996b).

Effects of Mutation

Ends-in gene targeting was used to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1^KO knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes, conversion tracts in the recombination products were examined. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. It is concluded that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome (Lankenau, 2003).

Because the Nap1 gene is large, the donor did not possess additional genes whose altered expression pattern might affect a functional analysis of Nap1. Homozygous Nap1 flies did not express detectable amounts of NAP1 protein. First-generation homozygous mutant Nap1 flies (derived from heterozygous parents) developed until the adult stage, albeit at sub-Mendelian frequencies. These flies showed reduced viability, but they were weakly fertile and gave rise to a second generation of homozygous flies. In these flies, the phenotype became much stronger and more penetrant. The few escaper flies that developed to the adult stage showed impaired development and died a few days after eclosion. A functionally strong maternal component of Nap1 expression at low concentrations (undetectable by Western blot) is probably sufficient to sustain relatively normal development in a significant fraction of homozygous mutant flies derived from heterozygous parents. Only after depletion of the maternally supplied components does the lethal phenotype become fully penetrant. The lethal phenotypes therefore were similar to the phenotype of other gene products thought to be important in nucleosome remodeling. For example, imitation switch (ISWI) homozygotes, where ISWI is the catalytic subunit of three essential chromatin-remodeling complexes NURF, ACF, and CHRAC, die as late larvae or early pupae. The Nap1 knockout mutants may therefore point toward related functions of NAP1 (Lankenau, 2003).

acf1 genetically interacts with nap1 in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus

Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. This study investigated the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

Eukaryotic DNA is packaged into a periodic nucleoprotein complex termed chromatin. The nucleosome is the basic repeating unit of chromatin, and the nucleosomal core consists of 146 bp of DNA wrapped around an octamer of histones H2A, H2B, H3, and H4. In addition to the core histones, chromatin contains other components such as linker histones and high mobility group proteins. Chromatin is involved in the regulation of transcription and other DNA-directed processes via posttranslational modifications of core histones, the reorganization of nucleosomes by chromatin remodeling factors, and the alteration of higher-order structures (Fyodorov, 2004 and references therein).

The assembly of chromatin is a fundamental biological process that occurs in proliferating cells during DNA replication and in quiescent cells during maintenance and repair of chromosomes. During DNA replication, chromatin structure is transiently disrupted at the replication fork, and the preexisting nucleosomes are segregated randomly between the daughter DNA strands. Then, additional nucleosomes are formed with newly synthesized histones. In this process, it appears that histones H3 and H4 are deposited prior to the incorporation of histones H2A and H2B. Chromatin assembly also occurs in nonreplicating DNA, and several examples of replication-independent assembly of chromatin have been described. These latter processes may occur during histone replacement, DNA repair, and transcription (Fyodorov, 2004 and references therein).

The basic chromatin assembly process is mediated by core histone chaperones and an ATP-utilizing motor protein. The histone chaperones include CAF-1 (chromatin assembly factor-1), NAP-1 (nucleosome assembly protein-1), Asf1 (anti-silencing function-1), nucleoplasmin, N1/N2, and Hir (histone regulatory) proteins. These proteins appear to deliver the histones from the cytoplasm to the sites of chromatin assembly in the nucleus. The ATP-utilizing assembly factor ACF (ATP-utilizing chromatin assembly and remodeling factor) can catalyze the transfer of histones from the chaperones to the DNA to yield periodic nucleosome arrays. The assembly reaction can also be catalyzed by purified RSF (remodeling and spacing factor), which appears to possess both chaperone and motor activities (Fyodorov, 2004).

This work investigates the biological function of ACF. ACF was purified from Drosophila embryos as an activity that mediates the ATP-dependent assembly of regularly spaced nucleosome arrays in vitro. During the assembly process, ACF commits to and translocates along the DNA template. ACF consists of two subunits, Acf1 and ISWI, which cooperatively catalyze nucleosome assembly in conjunction with histone chaperone proteins NAP-1 or CAF-1. Acf1 is the larger subunit of ACF, and it possesses WAC, DDT, WAKZ, PHD finger, and bromo-domain motifs. ISWI belongs to the SNF2-like family of DNA-dependent ATPases, and is a subunit of the ACF, CHRAC (chromatin accessibility complex), NURF, and TRF2 complexes. NURF and TRF2 complexes share only the ISWI subunit with ACF, whereas CHRAC is closely related to ACF. CHRAC was purified on the basis of its ability to increase the access of restriction enzymes to DNA in chromatin, and it consists of Acf1, ISWI, and two small subunits, CHRAC-14 and CHRAC-16, which are detected only during early embryonic development. The biochemical activities of ACF and CHRAC are indistinguishable. These Acf1-containing species will be referred to as 'ACF/CHRAC'. To study the function of ACF/CHRAC in vivo, a genetic analysis of the Drosophila acf1 gene was performed. The results indicate that Acf1 programs ACF/CHRAC to perform functions that are distinct from those of the NURF complex, which shares a common ISWI ATPase subunit with ACF/CHRAC. In addition, the phenotypes of flies lacking Acf1 suggest that ACF/CHRAC does not disrupt chromatin, as might be expected for a nucleosome remodeling factor, but rather promotes the formation of chromatin, as would be expected for a chromatin assembly factor (Fyodorov, 2004).

Polycomb regulation is caused by chromatin-dependent transcriptional silencing. The identity of body segments in Drosophila is specified by homeotic genes of the Antennapedia and bithorax complexes, which are in turn subject to regulation by Polycomb and trithorax group (PcG and trxG) genes. PcG genes encode protein complexes that can maintain chromatin-dependent transcriptional silencing via cis-acting DNA elements termed Polycomb response elements, or PREs (Fyodorov, 2004).

To determine the influence of Acf1 on Polycomb regulation, whether the loss of Acf1 affects transcriptional repression by the Ubx PRE in a PRE-miniwhite reporter gene was examined. In the wild-type control background(acf1³/acf1³), the expression of the PRE-miniwhite reporter gene was strongly repressed, with pigments limited to a small part of the adult fly eye. In the absence of Acf1 (acf1¹/acf1¹), partial activation was observed of the PRE-miniwhite reporter gene with pigments distributed over a larger area of the eye. This observed derepression in the homozygous acf1¹ background is comparable to derepression in a heterozygous Pc background (Fyodorov, 2004).

Whether acf1 interacts genetically with the segmentation function of Pc was investigatede. The appearance of extra sex combs on distal portions of the second and third legs in F1 males was scored in the progeny from a cross between males with a heterozygous deficiency for Pc (Df(3L)Asc) and females homozygous for acf1 alleles. The mutation of acf1 significantly enhanced this Pc phenotype in a manner similar to that seen with other enhancers of the Pc gene. Whereas only about 18% or 17% of the Df(3L)Asc/+; acf1³/+ or Df(3L)Asc/+; acf1⁴/+ males had extra sex combs on second and/or third pairs of legs (from the total number of male progeny scored, 61% or 58% of the Df(3L)Asc/+; acf1¹/+ or Df(3L)Asc/+; acf1²/+ male flies had the extra sex comb phenotype). In addition, >50% of males in the latter two crosses exhibited ectopic pigmentation of their A3 and A4 abdominal tergites, which was never observed in crosses with acf1³ or acf1⁴ mothers. These results, combined with the derepression of PRE-mediated miniwhite silencing, demonstrate that acf1 is a Polycomb enhancer and suggest that ACF/CHRAC is involved in the assembly and/or maintenance of repressive chromatin in Polycomb-responsive loci (Fyodorov, 2004).

The identity of Drosophila abdominal segments A5-A8 is determined by homeotic selector genes of the bithorax complex. For instance, in Pc/acf1 males, the posteriorly directed homeotic transformation may be caused by an increase in the expression of the bithorax complex gene Abd-B on loss of Acf1. In contrast, the anterior transformation phenotype of ISWI/+; acf1/acf1 and nap1/nap1; acf1/acf1 animals is reminiscent of mutations in various trithorax group genes, which include the brm and kis genes that encode ATPase subunits of chromatin remodeling complexes. This anterior transformation is likely to result from a decrease in expression of Abd-B on loss of Acf1. These data suggest that Acf1 may be involved in repression or activation of Abd-B in different contexts. Transcriptional repression of Abd-B by Acf1 is consistent with its function in the assembly of repressive chromatin. In fact, genetic evidence in yeast as well as polytene chromosome localization studies in Drosophila primarily implicate ISWI-containing complexes in transcriptional repression in vivo. Transcriptional activation of Abd-B by Acf1 could be due to its chromatin remodeling function, which could potentially facilitate transcription, or to an indirect effect, such as the repression of a transcriptional repressor of Abd-B (Fyodorov, 2004).

Surprisingly, Acf1 is not absolutely required for viability. Chromatin from homozygous acf1 mutant embryos exhibits less nucleosomal periodicity as well as a shorter repeat length than chromatin from wild-type embryos. Extracts from Acf1-deficient embryos assemble nucleosomes in vitro much less efficiently than wild-type extracts, and also that the deficiency in chromatin assembly can be rescued on addition of purified recombinant ACF or Acf1. These findings indicate that ACF/CHRAC is a major chromatin assembly activity in Drosophila, but also that Acf1-deficient flies contain other ATP-utilizing chromatin assembly factor(s) that are able to sustain partial viability (Fyodorov, 2004).

The analysis of the Acf1 null flies revealed that ACF/CHRAC performs different biological functions than NURF, even though ACF/CHRAC and NURF both share a common ISWI ATPase. Hence, the unique subunits of ACF/CHRAC and NURF can program the basic motor function of ISWI to perform specific biological tasks in vivo (Fyodorov, 2004).

ATP-utilizing motor proteins could potentially assemble or disrupt chromatin structure. Through multiple lines of investigation, the function of ACF/CHRAC was studied in vivo. (1) Whether there are genetic interactions between acf1 and nap1 was investigated, because the ACF/CHRAC motor protein and the NAP-1 histone chaperone function together in chromatin assembly in vitro. Double mutant nap1/nap1; acf1/acf1 flies exhibit a homeotic transformation that is not seen in the corresponding single mutant flies. These results are consistent with the biochemical activities of ACF/CHRAC and NAP-1 in the chromatin assembly process (Fyodorov, 2004).

(2) The effect of Acf1 on heterochromatic transcriptional silencing was tested. In these experiments, suppression of pericentric position-effect variegation was detected on loss of Acf1. It was additionally found that Acf1-deficient flies exhibit reduced levels of Polycomb-mediated transcriptional silencing. These findings indicate that ACF/CHRAC is important for the establishment and/or maintenance of repressive chromatin states (Fyodorov, 2004).

(3) Whether Acf1 enhances or disrupts chromatin-mediated repression of DNA replication was investigated. Shortening of S phase was observed in Acf1-deficient embryos and larval neuroblasts, consistent with a role of ACF/CHRAC in the assembly rather than disruption of chromatin in vivo. The effect of chromatin structure on the duration of S phase in larvae was investigated with a deficiency that uncovers the histone gene cluster. These animals contain reduced levels of histones and exhibit an acceleration of late S phase progression in larval neuroblasts relative to that in wild-type flies. Thus, the mutation of acf1 as well as the reduction in the level of histones each correlate with an increase in the rate of S phase progression. These data collectively support a role of Acf1 in the assembly of histones into chromatin (Fyodorov, 2004).

In summary, several independent lines of experimentation implicate Acf1 in the formation of chromatin in vivo. These experiments provide evidence for the function of ACF/CHRAC (and other ATP-utilizing factors) in the assembly of chromatin in conjunction with the NAP-1 histone chaperone. They also include the unexpected finding of a role of ACF/CHRAC in Polycomb-mediated silencing as well as the discovery of mutations (acf1 and Df(2L)DS6) that result in an unusual increase in the rate of S phase. Lastly, the loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin, which support a role of Acf1 in the assembly of repressive chromatin. Hence, the collective biochemical and genetic data indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization

Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. This study identified four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. This study showed TAP/p32 to be required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, this study identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells (Emelyanov, 2014).

The DNA of metazoan somatic cells is packaged into a compact nucleoprotein complex termed chromatin. Chromatin fiber is comprised of highly conserved repetitive units (nucleosomes) that contain an octamer of four core histones and 145-147 base pairs (bp) of DNA wrapped around the octamer in 1.65 turns of a left-handed superhelix. Nucleosomes are assembled in vivo in an ATP-dependent fashion through a concerted and sequential action of core histone chaperones and motor proteins that belong to the Snf2 family of DNA-dependent ATPases. For instance, Drosophila ACF/CHRAC can mediate chromatin assembly in conjunction with histone chaperone NAP-1. Other known ATP-dependent chromatin assembly factors include RSF, CHD1, ATRX, and ToRC/NoRC (Emelyanov, 2014).

The most abundant chromatin component in male germline cells is protamines-small positively charged arginine- and cysteine-rich protein. During spermiogenesis, protamines replace 85%-95% of DNA-bound histones in the nucleus to achieve a higher density of sperm nuclear DNA. Crystalline-like sperm chromatin structure is sixfold more compact than metaphase chromosomes and renders sperm DNA enzymatically inert. At fertilization, the oocyte remodels the condensed sperm chromatin into a transcriptionally competent chromatin of the male pronucleus. During this process, protamines are expelled and replaced with oocyte-supplied histones, which are then organized into nucleosomes. Sperm chromatin remodeling (SCR) is controlled by biochemical activities in the early oocyte, but components of these activities remain largely unknown. However various protein factors have been implicated in SCR, including core histone chaperones from Xenopus and Drosophila (NAP-1, p22, DF31, HIRA, and Yemanuclein). In mammals, members of nucleoplasmin/nucleophosmin family proteins (NPM1-3) function in sperm chromatin decondensation in vitro. In addition, Npm2 knockout female mice exhibit fertility defects consistent with a role of NPM2 in nuclear and nucleolar chromatin organization. It was also suggested that sperm chromatin decondensation is ATP-dependent (Emelyanov, 2014).

Drosophila sperm cells contain two major protamines (A and B) encoded by male-specific transcripts Mst35Ba and Mst35Bb, respectively. The Drosophila maternal effect mutant sésame (ssm) prevents male pronucleus formation. ssm encodes the histone variant H3.3-specific chaperone HIRA, postulated to be required for replication-independent deposition of histones in the male pronucleus during sperm decondensation . In eggs from homozygous ssm females, maternal histones are not deposited in the chromatin of male pronuclei, preventing normal mitosis and resulting in the development of gynogenetic haploid embryos and embryonic stage lethality. A similar phenotype is observed in null mutants of the gene encoding ATP-dependent chromatin assembly factor CHD1. Thus, CHD1 and HIRA act cooperatively and are required for nucleosome assembly during SCR. Intriguingly, protamines are efficiently expelled from the DNA of nascent male pronuclei in Chd1 and ssm eggs, suggesting that protamine removal and histone deposition are functionally distinct steps (Emelyanov, 2014).

This study used a biochemical approach to identify specific protein components of the Drosophila egg machinery that promote the dissociation of protamine-DNA complexes of sperm chromatin. These factors turn out to be two known core histone chaperones (NAP-1 and NLP), a homolog of mammalian nucleophosmin, and a novel Drosophila histone chaperone (TAP/p32). These putative 'protamine chaperones' facilitate SCR independently of CHD1 and HIRA, which mediate nucleosome assembly in nascent male pronuclei. Of note, TAP/p32 is specifically required to expel Drosophila protamine B from sperm chromatin in vitro, whereas NAP-1, NLP, and nucleophosmin share roles in removal of protamine A. In vivo evidence is provided that TAP/p32 functions in Drosophila egg SCR. In conclusion, this study has characterized protein factors that mediate the first obligatory step of SCR (protamine dissociation) and reconstituted the complete SCR reaction (reorganization of protamine-containing sperm chromatin into core histone-containing nucleosome arrays) in a purified defined system in vitro (Emelyanov, 2014).

Although recent studies provide details of sperm chromatin composition (Hammoud 2009; Miller 2010) and assembly during spermiogenesis, relatively little is known about the protein machinery that mediates SCR during fertilization. In vivo analyses in Drosophila suggest that removal of protamines from sperm chromatin is biochemically uncoupled from subsequent nucleosome assembly because male pronucleus-specific nucleosome assembly factors CHD1 and HIRA are not required for protamine removal. This study demonstrates that, indeed, a separate set of protein factors (protamine chaperones) is required for protamine eviction. Using assay-based biochemical approaches, four Drosophila proteins were identified that are sufficient for unraveling of DNA-protamine complexes in vitro. Their biochemical activities and mechanisms of SCR were analyzed. Significantly, it was possible to recapitulate the entire process of SCR (protamine eviction and nucleosome assembly) in a defined purified system. Biological functions of TAP/p32 were further analyzed in Drosophila, and evidence was obtained of its proposed roles in SCR in vivo. Finally, it was discovered that all four proteins additionally share a function as core histone chaperones (Emelyanov, 2014).

Recent microarray analysis of mating-responsive genes in Drosophila revealed that CG6459/P32 expression is strongly activated in the female lower reproductive tract within 6 h of mating. In fact, CG6459/P32 exhibits the strongest response of all genes identified in the study. The up-regulation is transient and is reversed 24 h after mating. This temporal expression pattern of TAP/p32 further supports its proposed role during fertilization (Emelyanov, 2014).

Intriguingly, orthologs of protamine chaperones are expressed in unicellular organisms, such as S. cerevisiae, which do not express protamines and whose gametes do not undergo the chromatin reorganization characteristic of metazoan sperm cells. Evidence is provided that Mam33p, an S. cerevisiae ortholog of TAP/p32, is involved in chromatin remodeling and DNA compaction and/or repair. It is likely that protamine chaperone homologs in unicellular eukaryotes perform conserved functions of core histone chaperones and are involved in nucleosome assembly and remodeling. During metazoan evolution, however, their biochemical activities may have been harnessed for SCR owing to biochemical similarities (net charge and amino acid composition) and functional roles (DNA compaction) of histones and protamines (Emelyanov, 2014).

Although yeast TAP/p32 ortholog Mam33p is clearly involved in regulation of DNA integrity/repair in response to treatment with mutagens and genetically interacts with factors of chromatin remodeling and DNA compaction and repair, it is also required for metabolism of alternative carbon sources. Similarly, mammalian TAP/p32 has also been implicated in mitochondrial function. Furthermore, Drosophila TAP/p32 and NAP-1, although subject to nuclear translocation, are also efficiently recruited to mitochondria. Hence, it is interesting to consider the apparent dual role of TAP/p32 in mitochondrial function and nuclear DNA compaction. Mature sperm cells in Drosophila and vertebrates contain a stack of mitochondrial structures in the midpiece at the junction of the head and tail. (In Drosophila, the sperm mitochondria are depleted of DNA, and, in most metazoan species during fertilization, sperm mitochondria undergo rapid ubiquitination and degradation by autophagocytosis) Thus, the elevated affinity of TAP/p32 and NAP-1 to protein components of mitochondria may be used and adapted for rapid and specific recruitment of the TAP/p32 and NAP-1 to the sperm head, which would facilitate their loading onto sperm chromatin for its processing (Emelyanov, 2014).

It has been suggested that Xenopus nucleoplasmin is sufficient for the initial stage of SCR (decondensation of demembranated sperm and removal of sperm basic proteins SP1-6 in vitro). However, the removal of sperm proteins (and their replacement by histones) in the presence of nucleoplasmin does not appear complete/quantitative. Furthermore, the Xenopus sperm decondensation assay is prone to artifacts: It is frequently performed (and works) with heterologous proteins and extracts, including those from yeast. In contrast, the current analyses suggest that a family of several factors may share partially redundant roles in protamine removal, and their cooperative action is necessary and sufficient for complete protamine eviction from sperm chromatin substrates. On the other hand, considering poor evolutionary conservation of protamine number and identities, it is possible that species other than Drosophila use smaller or larger sets of factors for SCR (Emelyanov, 2014).

Despite being ~94% identical, protamines A and B require different chaperones for their removal. For instance, in the absence of TAP/p32, a mixture of NAP-1, NLP, and Nph is incapable of protamine B eviction. Protamine polypeptides are extremely evolutionarily divergent. In fact, it is rarely possible to assign a protamine function based on a sequence conservation search of related proteins in distinct metazoan species. For example, a closely related organism, Drosophila simulans, expresses one protein homologous to D. melanogaster protamines. It is more closely related to protamine B and shares with it only 77% identity. D. simulans also express orthologs of protamine chaperones. A high degree of functional/sequence specificity makes it unlikely that D. melanogaster protamine chaperones will be able to remodel MSC assembled from more divergent, evolutionarily distant protamines. This specificity may contribute to gametic isolation of distinct species. In the future, it will be interesting to analyze cross-reactivity of protamines and protamine chaperones from these species in MSC remodeling in vitro and in vivo (Emelyanov, 2014).

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date revised: 25 October 2014 

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