InteractiveFly: GeneBrief

Hira: Biological Overview | References

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm; Loppin, 2000), a unique Drosophila maternal effect mutant that prevents male pronucleus formation (Loppin, 2005).

ssm is a point mutation in the Hira gene (Kirov, 2008); the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. This study shows that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization (Loppin, 2005).

With the exception of its four centromeric regions, the needle-like, extremely condensed Drosophila sperm nucleus is devoid of the core histones (H2A, H2B, H3 and H4) that form the nucleosome particle. Indeed, in elongating spermatids, histones are replaced by sperm-specific chromosomal proteins including protamines. At fertilization, de novo nucleosome assembly in the male pronucleus is a crucial process that determines the ability of the paternal genome to participate in the formation of the diploid zygote. However, the molecular basis of this process in Drosophila is largely unknown. sésame (ssm), a unique maternal effect, embryonic-lethal mutation specifically affects the formation of the male pronucleus (Loppin 2000). In eggs laid by homozygous ssm females, maternal histones are not deposited in the male pronucleus, which remains abnormally condensed. Consequently, haploid embryos develop with only maternal chromosomes and invariably die before hatching (Loppin 2000). This dramatic phenotype suggests that ssm is involved in the replacement of protamines with histones in the male pronucleus (Loppin, 2001). To identify the ssm gene, the previously defined ssm genetic region (7B6-7C1, X chromosome) was aligned relative to the annotated genome. Of the ten candidate genes predicted in this region, the Hira gene was first tested in rescue experiments. A single copy of a Hira transgene completely rescued the fertility of homozygous ssm females, thereby demonstrating that Hira is the gene affected by the ssm mutation (Loppin, 2005).

Analysis of Hira transcripts by polymerase chain reaction with reverse transcription (RT-PCR) revealed no significant difference in expression between wild-type and ssm flies. HIRA proteins are characterized by seven conserved WD repeats in their amino-terminus, which are predicted to assemble into a ß-propeller structure known to mediate protein-protein interactions. Sequencing of the Hira gene amplified from ssm flies revealed a single point mutation that results in substitution of the highly conserved arginine 225 with a lysine residue (R225K). This residue is located between the fourth and fifth WD repeats of HIRA. The 100% penetrant phenotype of ssm arises from this subtle mutation, indicating that this residue sits in a crucial functional domain of the protein (Loppin, 2005).

HIRA has been shown to be critical for a replication-independent nucleosome assembly pathway, distinct from the major replication-coupled chromatin assembly that occurs during genome duplication (Ray-Gallet, 2002). In the decondensing sperm nucleus, chromatin assembly takes place before the onset of the first zygotic S phase. It was thus reasoned that this process could represent a peculiar case of replication-independent nucleosome assembly at the scale of a whole genome. To test this hypothesis, the distribution of maternal HIRA was studied in fertilized eggs. Transgenic flies were establised expressing HIRA fused to the 3 x Flag tag (HIRA-Flag). Anti-Flag Western blot analysis of early embryos from transgenic females revealed a single band corresponding to the expected size of HIRA-Flag. This fusion protein is functional; the Hira-Flag transgene fully rescues the fertility of ssm females (Loppin, 2005).

Immunofluorescence of eggs from transgenic females crossed with wild-type y w^67c males revealed bright HIRA-Flag staining in the male nucleus from the initiation of decondensation, when the sperm nucleus is still elongated, until the end of pronuclear formation. In contrast, HIRA-Flag was never detected in maternal chromosomes at any stage of meiosis or pronuclear formation, and was also absent from embryonic nuclei during early development. It was also verified that sperm from transgenic males did not contribute any detectable level of HIRA-Flag when crossed with wild-type females. Thus, the highly specific distribution of maternal HIRA in the male nucleus is consistent with the observed mutant phenotype (Loppin, 2005).

This result was confirmed using antibodies directed against two peptides from the Drosophila HIRA protein. In wild-type w¹¹¹⁸ flies, anti-HIRA antibodies stained the male nucleus exactly like HIRA-Flag did. In addition, anti-HIRA antibodies stained the sperm flagellum non-specifically, as is often the case for rabbit antibodies. In conclusion, the data show that maternal HIRA very efficiently targets the male nucleus immediately after sperm entry. This highly specific distribution of HIRA on paternal chromatin well before the onset of the first S phase makes a strong case for its potential role in replication-independent nucleosome assembly during male pronucleus formation (Loppin, 2005).

In eggs from mutant females stained with anti-HIRA antibodies, it was found that the mutant HIRA is still detected in the male nucleus at all stages of zygote formation. A version of the Hira-Flag transgene was constructed containing the R225K substitution (Hira^ssm-Flag). As expected, this construct was unable to rescue the ssm phenotype, despite the fact that HIRA^ssm-Flag brightly stained the male nucleus in mutant eggs. In conclusion, the ssm mutation does not affect the ability of HIRA to localize to the male nucleus (Loppin, 2005).

Increasing evidence supports an important role for histone H3 variants in specifying modes of nucleosome assembly. Whereas the major H3 histones (H3.1 and H3.2) are synthesized during S phase and are deposited on DNA strictly during DNA replication, the histone H3 replacement variant H3.3 is synthesized and deposited onto DNA throughout the cell cycle. Additionally, H3.3 deposition has been shown to mark transcriptionally active chromatin in vivo, suggesting that H3.3 nucleosomes may confer epigenetic inheritance of active chromatin states. Recently, the purification from human cells of complexes for replication-coupled deposition of H3.1 and replication-independent deposition of H3.3 has demonstrated that these alternative nucleosome assembly pathways depend on different chromatin assembly factors: the CAF-1 complex during DNA replication or DNA repair, and the HIRA complex, which is independent of DNA synthesis (Loppin, 2005).

The evidence that vertebrate HIRA is specialized for the deposition of H3.3 nucleosomes on DNA prompted the possible implication of this histone variant in male pronucleus formation in Drosophila. H3, the unique Drosophila S phase variant, and H3.3 differ by only four amino acids, and no specific H3.3 antibody is currently available. Transgenic lines were thus established expressing 3 x Flag-tagged versions of both histone variants. Both constructs use the regulatory sequences of the Drosophila His3.3A gene, resulting in similar levels of both recombinant histones in eggs (Loppin, 2005).

The distribution of H3.3-Flag during fertilization and zygote formation was examined by crossing transgenic females with y w^67c males. In all cases, maternal H3.3-Flag specifically accumulated in the male nucleus at all stages of pronuclear formation. In contrast, the polar bodies and female pronucleus did not stain for H3.3-Flag, except for a weak staining that appeared by the time the pronuclei apposed. During the first zygotic mitosis, H3.3-Flag still strongly labelled paternal chromosomes, but the staining faded away after a few nuclear divisions. As expected, H3.3-Flag was absent or barely detected in the male nucleus in eggs from ssm mutant females (Loppin, 2005).

In clear contrast to H3.3-Flag, maternal H3-Flag was never detected in the decondensing male nucleus. However, shortly before pronuclear apposition, both pronuclei and the polar bodies were intensely labelled with H3-Flag. This stage corresponds to the onset of the first S phase in all nuclei, as confirmed by immunostaining of the replication factor PCNA. Thus, as expected, H3-Flag is strictly deposited during S phase. H3-Flag was not detected in maternal meiotic chromosomes, presumably because the transgene is not sufficiently expressed during oocyte differentiation to compete with the S-phase expression of the numerous endogenous H3 genes. After the first nuclear cycle, H3-Flag labelled all nuclei throughout embryonic development. Together, these results indicate that although both variants are present in the egg cytoplasm at fertilization, only H3.3 is deposited in the decondensing male nucleus, and this process requires HIRA (Loppin, 2005).

The absence of HIRA from maternal nuclei suggests that replication-coupled chromatin machinery, presumably involving the CAF-1 complex, is responsible for the limited deposition of H3.3 observed in the female pronucleus and polar bodies. In contrast, H3 is strictly deposited during DNA replication in all nuclei, independent of their origin. Considering that HIRA is expressed in all tissues, it is speculated that the ssm mutation only affects the function of HIRA in the male pronucleus. Indeed, H3.3-Flag deposition in other tissues is not affected by the mutation. A recent study has confirmed that in ssm eggs, the paternal chromatin remains associated with protamines (Raja, 2005). A model is whereby favored that the HIRA complex fulfils both protamine removal and replication-independent deposition of H3.3-H4 tetramers in the male nucleus. The R225K mutation would only affect the removal of protamines, thus preventing histone deposition. Stronger Hira alleles are expected to affect viability, as is the case in mouse (Roberts, 2002), for which the Hira-null mutation is embryonic-lethal (Loppin, 2005).

The HIRA-dependent deposition of H3.3 during sperm nucleus decondensation establishes an unsuspected level of epigenetic distinction between parental genomes within the diploid zygote. Considering the fact that most animal sperm nuclei must assemble nucleosomes independent of replication in the egg, it is expected that the HIRA-H3.3 chromatin assembly pathway is widely conserved for this process. This is supported by a recent study in mouse that pointed out the absence of H3.1 in the male pronucleus (van der Heijden, 2005). Moreover, it is well established that in mouse, histone H3 post-translational methylations are differentially distributed between the male and female pronuclei. In particular, di- or tri-methylation of lysine 4 and 9 of H3 are only found in the female pronucleus, at least during the earliest stages of pronuclear formation. Remarkably, these four H3 modifications (di- and tri-methyl K4, and di- and tri-methyl K9) are also restricted to maternal chromosomes in Drosophila eggs and are thus absent on paternal H3.3. Thus, the epigenetic distinction between parental genomes is remarkably conserved, both at the level of H3 variants and H3 lysine methylation. Future investigations should establish the functional significance of this H3.3 epigenetic mark in mammals with respect to the specific loss of paternal DNA methylation at fertilization or the inactivation of the paternal X chromosome in cleavage embryos. Finally, this work underlines the value in using the male pronucleus as a unique model for studying genome-wide replication-independent chromatin assembly in vivo (Loppin, 2005).

The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization

In many animal species, the sperm DNA is packaged with male germ line-specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, it was shown previously that HIRA, a conserved replication-independent chromatin assembly factor, is essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. This study reports the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, H3.3 deposition was shown not to be affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries can deposit this histone variant (Bonnefoy, 2007; full text of article).

Replacement of histones by protamines and Mst77F during chromatin condensation in late spermatids and role of Sesame in the removal of these proteins from the male pronucleus

Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little of this process is known for Drosophila. This study characterizes three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame (Raja, 2005).

For mammals, the somatic set of histones are modified, as these are in part replaced by specific variants during meiotic prophase. After meiosis, histones are replaced by major transition proteins TP1 and TP2 and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements leads to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing protamines as major chromatin condensing proteins and DNA. Some mammals have only one protamine gene, while mice and humans have two genes encoding two different protamines, both of which are essential for fertility and are haploinsufficient. HILS1 (spermatid-specific linker histone H1-like protein) has been proposed to participate in chromatin remodeling in mouse and human spermiogenesis. The transition between histone removal and its replacement by protamines in mice and humans is characterized by small 6- to 10-kDa transition proteins acting as a short-term chromosomal proteins. In mice, the transition proteins TP1 and TP2 are redundant in function. In fishes and birds, transition proteins are missing and protamines directly reorganize the chromatin. In annelids and echinoderms, the nucleosomal configuration is maintained in sperms, while protamine-like proteins have been described for mussels. These protamine-like proteins lack the typical high cysteine content necessary for disulfide bridges. Therefore, a doughnut-type chromatin structure as in mammals is unlikely to occur in mussels. It has been proposed that the protamine-like proteins in mussels belong to the histone H1 family. The sperm chromatin of mussels contain core histones and thus a nucleosomal configuration, but histone H1 is replaced by protamine-like molecules which organize the higher order structure of the chromatin (Raja, 2005).

For Drosophila melanogaster, chromatin reorganization after meiosis has not been studied at the molecular level. At the light microscopic level, the Drosophila spermatid nucleus is initially round after meiosis and then is shaped to a thin needle-like structure with highly condensed chromatin, so that the volume of the nucleus is condensed over 200-fold. In mammals, the volume of the nucleus is reduced over 20-fold. In the mature sperms of Drosophila, core histones are not detectable by immunohistology. There is histochemical evidence for the presence of very basic proteins in sperm, but it still remains an open question whether histones are replaced by protamine-like basic proteins in Drosophila. The analysis of the Drosophila genome sequence revealed that the proteins encoded by two genes show similarity to mammalian protamines for which the male-specific transcripts Mst35Ba and Mst35Bb have been found and have been proposed to encode protamine-like proteins. Another male specifically transcribed gene, Mst77F, is a distant relative of the histone H1/H5 (linker histone) family and has been proposed to play a role either as a transition protein or as a replacement protein for compaction of the Drosophila sperm chromatin. With enhanced green fluorescent protein (eGFP) fusion for these abovementioned proteins, this study shows that Mst35Ba and Mst35Bb indeed encode protamines and Mst77F encodes a linker histone-like protein. The expression pattern of Mst77F overlaps the pattern of protamines as a chromatin component. Furthermore, during fertilization, the removal of protamines from the male pronucleus requires the function of the maternal component, Sesame, but not for the removal of Mst77F. It has been shown that sesame mutants cause impairment of the entry of histones into the male pronucleus (Raja, 2005).

Mst35Ba and Mst35Bb are present at cytological position 35B6 and 35B6-7, respectively, on the chromosome arm 2L. These two genes are arranged in tandem, and both consist of three exons. The 5'UTR, coding region, and the 3'UTR of these genes are highly identical; they probably arose from a recent gene duplication. The encoded protamines show over 94% identity to each other (Raja, 2005).

A remarkable feature of protamines is their ability to form intermolecular disulfide bridges, which is reflected by the conserved cysteine residues within mammalian protamines. The dProtA and dProtB are of 146 amino acids (aa) and 144 aa, respectively, and thus longer than even the human and mouse Protamine-2, which are 102 aa and 107 aa, respectively. Both Drosophila protamines contain 10 cysteines each and show significant similarity, particularly with respect to a high cysteine, lysine, and arginine content to mammalian protamines. Human and mouse Protamine-1 aligns to the N-terminal half of the Drosophila protamines (from aa positions 27 to 82), and four cysteine residues are conserved and regularly spaced. In contrast, Protamine-2 of human and mouse shows relatively high similarity to the C-terminal half of the Drosophila protamines, with four cysteines in this region that are conserved and regularly spaced, whereas one cysteine is shared with the mouse and human Protamine-1 (Raja, 2005).

Mst77F is present at the cytological position 77F on the chromosome arm 3L and lies within the large intron of PKA-R1. Mst77F is also male specifically transcribed, and the encoded protein has been proposed to be a linker histone H1/H5 type, which could also play the role of a transition protein or a protamine. The Mst77F protein shares a significant similarity to the HILS1 protein of mouse and human HILS1, where the percentages of cysteine, lysine, and arginine are similar to that of mHILS1 and hHILS1. HILS1 protein has been recently described as a component of the mammalian sperm nucleus. Drosophila Mst77F encodes a protein of 215 aa with a molecular mass of 24.5 kDa and with a pI of 9.86. mHILS1 is of 170 aa and shows 39% similarity to Mst77F. Mst77F contains 10 cystine residues as in Drosophila protamines, and mHILS1 contains eight cystine residues, of which four residues are conserved (Raja, 2005).

As there are considerable differences between the mammalian protamines as well as between the mammalian HILS1 proteins and the presumptive Drosophila homologue Mst77F, additional experiments are essential to clarify if these proteins are indeed involved in the condensation of sperm chromatin (Raja, 2005).

Drosophila protamine mRNAs are transcribed at the primary spermatocyte stage, whereas in mammals protamine mRNAs are synthesized at the round spermatid stage and translationally repressed until the elongated stage, which is mediated by 3'UTR. The Drosophila ProtamineA-eGFP and ProtamineB-eGFP constructs do not contain the 3'UTR of the respective protamine genes. Nevertheless, the transgenic flies carrying these constructs still show repression of translation. So, in Drosophila, the region responsible for the translational repression is most likely in the 5'UTR. Deletion constructs of Mst35Bb and Mst77F 5'UTRs fused to the reporter lacZ show that the translation repression element is indeed present in the 5'UTR. This holds true also for the mRNA of the Mst77F-eGFP fusion gene, as is the case for all mRNAs investigated concerning translational repression so far in male germ lines of Drosophila. In contrast to mammalian spermatogenesis, in Drosophila transcription ceases already with the entry into meiotic divisions. Since the protamines are made in the elongated spermatids, the transcriptional silencing in Drosophila spermatogenesis seems to be independent of protamines (Raja, 2005).

When primary amino acid sequences of Drosophila protamines are compared to mammalian protamines, it is quite evident that Drosophila protamines are relatively large. dProtA and dProtB are over 94% identical to each other. This could explain that both the protamines may be functionally redundant. Human and mouse Protamine-1 aligns with the N terminus of both Drosophila protamines, and Protamine-2 aligns more to the C terminus. It is possible that the Drosophila protamines undergo posttranslational cleavage at the N terminus, as is known for mammals. The cytoplasmic eGFP fused at the C terminus shows clear nuclear localization, indicating that the tagged protamine is functionally intact. Drosophila protamines each contain 10 cysteine residues at identical positions, while over 4 of 10 cysteines at the N terminus and the C terminus are conserved with human and mouse Protamine-1 and Protamine-2, respectively. With nine cysteines, the content is highest in Protamine-1 of mice. Inter- or intra-disulfide bridges can be formed between the cysteine-rich protamines to condense the DNA. For mice it is shown that mutation in protamine-1 or protamine-2 is haploinsufficient and causes male sterility. A haploid situation was analyzed for the Mst35Ba and Mst35Bb genes with the deficiency Df(2L)Exel8033/+; these flies are fertile males and show normal spermatogenesis. The large amount of identity that both dProtA and dProtB exhibit can contribute to the functional redundancy (Raja, 2005).

Chromatin reorganization is an essential feature during spermiogenesis. The functional significance of chromatin compaction during spermiogenesis is still unknown. The main explanation seems to be that compaction of the sperm nucleus is an essential factor for its mobility as well as for the penetration of sperm into the egg and genomic stability. In mammals, somatic histones are in part replaced by spermatid-specific variants during meiotic prophase, later by major transition proteins TP1 and TP2, and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements lead to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing major chromatin condensing proteins and DNA in the nucleus (Raja, 2005).

In Drosophila, so far no proteins have been identified that are involved in the packaging of the genome in the mature sperm nucleus. One observation, that Histone3.3 variant and the somatic H3 isoform in Drosophila are vanishing at the time of chromatin condensation, supports the view of histone displacement, but it was still a question of whether it is the real absence of histones at this stage in Drosophila or whether the antibodies are not accessible to the mature sperm due to the tight packaging of the chromatin. To circumvent this problem, the GFP fusion approach was chosen, use was made of the existing His2AvDGFP, and Protamine-eGFP and Mst77F-eGFP fusion transgenic flies were generated in order to analyze the situation in Drosophila. The results clearly show that histone His2AvD is lost from the spermatid nuclei at the time of appearance of protamines and Mst77F during later stages of spermatid differentiation. The exact molecular mechanisms underlying the histone displacement, degradation, and incorporation of protamines onto the chromatin are poorly understood. For mammals, evidence has been obtained that histone H2A is ubiquitinated in mouse spermatids around the developmental time period when histones are removed from the chromatin. The mammalian HR6B ubiquitin-conjugating enzyme is the homologue of yeast RAD6, and both can ubiquitinate histones in vitro. Thus far, the mechanism of histone displacement and protamine incorporation is unknown during spermiogenesis in Drosophila. In flies as well as in mammals, many questions remain unanswered that need to be addressed about these underlying mechanisms of chromatin remodeling during spermiogenesis (Raja, 2005).

In mammals, transition proteins act as intermediates in the histone-to-protamine transition. In mice, the onset of HILS1 and transition proteins TP1 and TP2 (major forms) overlaps with the pattern of Protamine-1 and later with Protamine-2 but HILS1 and the transition proteins are no longer present in the mature sperm. Mice lacking both TP1 and TP2 show normal transcriptional repression, histone displacement, nuclear shaping, and protamine deposition but show the loss of genomic integrity with large numbers of DNA breaks leading to male sterility. In Drosophila, histones are displaced with synchronous accumulation of protamines and Mst77F. Mst77F, a distant relative of the histone H1/H5 (linker histone) family, has been proposed to play a role either as a transition protein or as a protamine for compaction of the Drosophila sperm chromatin. Mst77F shows highest similarity to HILS1 with respect to the cysteines and basic amino acid content but not to mouse TP1, TP2, or H1t. Moreover, the results show that the pattern of expression of Mst77F in the nucleus is similar to that of mHILS1 in the nucleus, with the exception that Mst77F is also transiently detected in the flagella and persists in mature sperm nuclei, unlike mHILS1. In mammalian mature sperm nuclei, it is only the protamines that are the chromatin condensing proteins which persist. This again raises the question of whether Mst77F could also play the role of protamines. However, one additional copy of dProtB (dProtA and dProtB showing 94% identity may be functionally redundant) does not rescue the ms(3)nc3 phenotype, indicating that the role of Mst77F may be completely or partially different from that of protamines in the nucleus. However, a null mutation for Mst77F is required to answer this question with respect to chromatin condensation. In ms(3)nc3 mutants, the chromatin condensation with the native protamines continues to take place. When a closer look was taken at the deposition of ProtamineB-eGFP in ms(3)nc3/Df(3L)ri-79c trans-heterozygotes, it revealed that the condensed chromatin in the tid-shaped nuclei is concentrated at the two opposite ends, with a lightly stained chromatin spaced in the center. So the chromatin condensation takes place but may not be complete with the incorporation of the mutant Mst77F protein. The large amount of chromatin compaction or condensation seen in Drosophila mature sperm when compared to that of mouse and human sperm possibly could be the result of persistence of Mst77F in the mature sperm nuclei. It remains to be clarified whether the sperm nucleus contains further protamines that have not yet been properly annotated (Raja, 2005).

ms(3)nc3 is a second-site noncomplementation (nc) mutation that was isolated in an ethylmethanesulfonate screen to identify interacting proteins involved in microtubule function in Drosophila. This study shows that ms(3)nc3 is a single missense mutation from a T>A transition, causing the substitution of threonine instead of serine at aa position 149. Mst77F shows a pattern of expression similar to protamines in the nucleus and was also seen in the flagella until the individualization stage. Since ms(3)nc3 fails to complement class I alleles at the ß2 tubulin locus, it is possible that Mst77F has a dual role to play as a chromatin condensing protein in the nucleus and for the normal nuclear shaping. Nuclear shaping is a microtubule-based event. ms(3)nc3 leads to a tid-shaped nuclear phenotype, where the nucleus fails to shape into a needle-like nuclei. Similar defective nuclear shaping is seen with the few homozygous and heteroallelic combinations of class I alleles of ß2 tubulin. The incorporation of the defective subunit encoded by ms(3)nc3 may interfere with the function of the resulting complex. These data suggest the involvement of an Mst77F (a linker histone variant) in the microtubule dynamics during the nuclear shaping. This again complements the role of sea urchin histone H1 in the stabilization of flagellar microtubules (Raja, 2005).

After the first steps in the fertilization process, the male gamete is still in the highly compact protamine-based chromatin structure. In a wild-type egg, the paternal pronucleus changes the shape from the needle-like to a spherical structure. Furthermore, the male pronucleus acquires a nucleosome-based structure before zygote formation and thus is transformed into a replication-competent male pronucleus. sesame is a maternal effect mutation in HIRA and had been mapped to 7C1. HIRA family of genes (named after yeast HIR genes; HIR is an acronym for 'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. In Drosophila, HIRA is expressed in the female germ line and a high level of HIRA mRNA is deposited in the egg. Human HIRA is shown to bind to histone H2B and H4. The WD repeats present at the N-terminal part of HIRA could probably function as a part of a multiprotein complex. Xenopus HIRA proteins are also known in promoting chromatin assembly that is independent of DNA synthesis in vitro. The corresponding maternal effect mutant sesame, in which the sperm fertilizes the egg but no zygote is formed, has been analyzed. Although the shape change of the nucleus to the spherical structure occurs in these mutants, maternal histones are not incorporated into the male pronucleus, which strengthens the function of HIRA in binding to the core histones. This study shows that neither Drosophila protamine is removed from the male pronucleus in sesame mutants. This leads to the proposal that the transport and incorporation of histones onto the chromatin in some manner is coupled to the removal of protamines in which HIRA could play an important role in the multiprotein complex required in this chromatin reconstitution process. Mst77F removal from the male pronucleus in contrast to protamines is independent of HIRA (Raja, 2005). During spermiogenesis, chromatin reorganization of the complete genome is an essential feature for male fertility. This process leads to an extremely condensed state of the haploid genome in the sperm and requires a reorganization of the paternal genome in the male pronucleus during fertilization and before zygote formation. With the characterization of the chromatin condensing proteins in Drosophila, it would be possible to gain more insight into the mechanisms of sperm chromatin reorganization during spermiogenesis and fertilization (Raja, 2005).

Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading

Epigenetic maintenance of the expression state of the genome is critical for development. Drosophila GAGA factor interacts with FACT and modulates chromatin structure for the maintenance of gene expression. This study shows that the GAGA factor-FACT complex (Fact is a heterodimer of dSPT16 and dSSRP1; Shimojima, 2003) and its binding site just downstream from the white gene are crucial for position effect variegation. Interestingly there is a dip of histone H3 Lys 9 methylation and a peak of H3 Lys 4 methylation at this site. The GAGA factor and FACT direct replacement of histone H3 by H3.3 through association of HIRA at this site, and maintain white expression under the heterochromatin environment. Based on these findings it is proposed that the GAGA factor and FACT-dependent replacement of Lys 9-methylated histone H3 by H3.3 counteracts the spreading of silent chromatin (Nakayama, 2007).

This study shows that the GAGA factor-FACT complex is present on the GAGA factor-binding DNase-hypersensitive site d1, a site just downstream from w, and participates in PEV. d1 appears to be a peculiar site where histone H3 K4 methylation peaks and H3 K9 methylation dips, and necessary and sufficient to counteract the heterochromatin spreading. The GAGA factor and FACT contribute to replacement of histone H3 by H3.3 through association of histone H3.3 chaperone HIRA to d1, and maintains w expression under the heterochromatin environment. Based on these data, the following model is proposed for the maintenance of the active state against the spreading of silent chromatin. Heterochromatin is marked by K9-methylated histone H3 and its binding protein HP1, and has a tendency to spread into neighboring regions. Histone H3.3 replacement is thought to be achieved through either eviction of a nucleosome and deposition of a H3.3-containing nucleosome or stepwise disassembly-reassembly without eviction of a nucleosome. Since the GAGA factor-FACT complex facilitates chromatin remodeling and the GAGA factor is known to generate a nucleosome-free region around its binding site, it is most likely that eviction or disassembly of a nucleosome occurs at the DNase-hypersensitive site of d1. The GAGA factor and FACT participate association of HIRA to d1 and the histone replacement would be accomplished by subsequent deposition or reassembly of a H3.3-containing nucleosome. This process would be repeated constantly to eliminate K9-methylated histone H3 at d1 and counteract the spreading of silent chromatin (Nakayama, 2007).

It has been reported that histone H3.3 replacement is triggered by transcription elongation. However, genome-wide profiling has shown histone H3.3 replacement from upstream of to downstream from transcription units. Although some of the replacement may be explained by elongation during intergenic transcription, the histone H3.3 replacement at d1 appears to occur independent of transcription elongation. Thus, the present study indicates a distinct pathway for histone H3.3 replacement (Nakayama, 2007).

Transcription of the w adjacent gene CG32795 has been reported to start immediately after the GAGA factor-binding sequence of d1, suggesting that d1 is a part of the promoter region of CG32795. Therefore, the effect was examined of Trl and spt16 mutations on expression of CG32795. The reduction of a single dose of Trl or spt16 affect the CG32795 expression in the w^m4 context but not in the w⁺ context. These data are consistent with the idea that d1 is a functional promoter element of CG32795 in the w⁺ context, although Trl and spt16 become haplo-insufficient only when the accessibility of the GAGA factor-FACT complex to d1 decreased under the heterochromatin environment. This raises the possibility that the protection from heterochromatin spreading by the GAGA factor and FACT at d1 is a consequence of their function within the CG32795 promoter. However, conventional promoters do not have a barrier function against heterochromatin silencing. For example, the presence of GAL4 (or E2F) on a promoter carrying GAL4 (or E2F)-binding site did not modify PEV of the attached reporter gene. Genome-wide profiling of H3.3 replacement in Drosophila has revealed the clear dip of H3.3-containing nucleosomes at immediately upstream of the transcription start sites of active genes. This is in sharp contrast with the case of d1, where peaks were observed of both the H3.3/H3 ratio and the actual H3.3 level, and illuminates the difference between d1 and ordinary promoters. Furthermore, the GAGA factor-dependent histone H3.3 replacement was detected also at the DNase HS1 in the Fab-7 boundary of Abd-B, where no promoter activity has been demonstrated. These findings indicate that the GAGA factor and FACT-dependent histone H3.3 replacement can occur without promoter functions. Nevertheless, the barrier function could be assisted by the putative promoter activity of d1 such as formation of a transcription initiation complex (Nakayama, 2007).

The GAGA factor-binding sequence at d1 consists of (GA)₈. Since GAGA factor forms an oligomer through its BTB domain, the factor can make a cooperative and stable binding to closely spaced GAGAG elements. This is presumably the reason why d1 gave a prominent signal among the GAGAG sequences around w in the ChIP assay. Because the GAGA factor occupies many closely spaced GAGAG sequences within the Drosophila genome including the Polycomb/trithorax response elements of Hox genes, the proposed mechanism may operate not only in loci juxtaposed with heterochromatin but also in other loci such as the regulatory regions of Hox genes. Indeed GAGA factor and FACT-dependent histone H3.3 replacement were observed in the Fab-7 boundary of Abd-B. High levels of histone H3.3 have been also reported at the locus control region of the chicken folate receptor gene, suggesting that the barrier function against the chromatin silencing via histone H3.3 replacement may be evolutionarily conserved up to vertebrates (Nakayama, 2007).

CHD1 is required for deposition of histone variant H3.3 into chromatin in vivo

The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of chromatin remodeling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses, and leads to the development of haploid embryos. Furthermore, CHD1, but not ISWI, interacts with HIRA in cytoplasmic extracts. These findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, these results point to the general requirement of adenosine triphosphate (ATP)-utilizing motor proteins for histone deposition in vivo (Konev, 2007).

Histone-DNA interactions constantly change during various processes of DNA metabolism. Recent studies have highlighted the importance of histone variants, such as H3.3, CENP-A (centromere protein A), or H2A.Z, in chromatin dynamics. Incorporation of replacement histones into chromatin occurs throughout the cell cycle, whereas nucleosomes containing canonical histones are assembled exclusively during DNA replication. A thorough understanding of the replication-independent mechanisms of chromatin assembly, however, is lacking (Konev, 2007).

In vitro, chromatin assembly requires the action of histone chaperones and adenosine triphosphate (ATP)-utilizing factors. Histone chaperones may specialize for certain histone variants. For example, H3.3 associates with a complex containing HIRA, whereas canonical H3 is in a complex with CAF-1 (chromatin assembly factor 1) (Tagami, 2004). The molecular motors known to assemble nucleosomes are ACF (ATP-utilizing chromatin assembly and remodeling factor), CHRAC (chromatin accessibility complex), and RSF (nucleosome-remodeling and spacing factor), which contain the Snf2 family member ISWI as the catalytic subunit, and CHD1, which belongs to the CHD subfamily of Snf2-like adenosine triphosphatases (ATPases). These factors have not been shown to mediate deposition of histones in vivo. It has been demonstrated that CHD1, together with the chaperone NAP-1, assembles nucleosome arrays from DNA and histones in vitro (Lusser, 2005). This study investigated the role of CHD1 in chromatin assembly in vivo in Drosophila (Konev, 2007).

Chd1 alleles were generated by P element-mediated mutagenesis. Two excisions, Df(2L)Chd1[1] and Df(2L)Chd1[2], deleted fragments of the Chd1 gene and fragments of unrelated adjacent genes. Heterozygous combinations, however, of Chd1[1] or Chd1[2] with Df(2L)Exel7014 affect both copies of the Chd1 gene only. Also a single point mutation was identified that results in premature translation termination of Chd1 (Q1394*) in a previously described lethal allele, l(2)23Cd[A7-4]. Hence, l(2)23Cd[A7-4] was renamed Chd1[3] (Konev, 2007).

Analysis of Western blots of embryos from heterozygous Chd1[3] fruit flies revealed the presence of a truncated polypeptide besides full-length CHD1. No truncated polypeptides were detected in heterozygous Chd1[1] or Chd1[2] embryos. Therefore, the corresponding deficiencies result in null mutations of Chd1. Crosses of heterozygous Chd1 mutant alleles with Df(2L)JS17/CyO or Df(2L)Exel7014/CyO produced subviable adult homozygous mutant progeny. Both males and females were sterile. Homozygous null females mated to wildtype males laid fertilized eggs that died before hatching. Therefore, maternal CHD1 is essential for embryonic development (Konev, 2007).

When the chromosome structure of 0- to 4-hour-old embryos laid by Chd1-null females were examined, it was observed that, during syncytial mitoses (cycles 3 to 13), the nuclei appeared to be abnormally small. The observed numbers of anaphase chromosomes suggested that they were haploid. To confirm this observation, wild-type or Chd1-null females were mated with males that carried a green fluorescent protein (GFP) transgene. Embryonic DNA was amplified with primers detecting male-specific GFP and a reference gene, Asf1. In wild-type embryos, both primer pairs produced polymerase chain reaction (PCR)-amplified products, whereas only the Asf1 fragment was amplified in the mutants. Thus, Chd1 embryos develop with haploid, maternally derived chromosome content (Konev, 2007).

To investigate the causes of haploidy in mutant animals, distributions of various developmental stages were compared in samples of wildtype and Chd1-null embryos. The lack of maternal CHD1 dramatically changed this distribution. Most notably, at 0 to 4 hours after egg deposition, the majority of Chd1 embryos (56%) remained at a very early stage of development in contrast to the wild type (24%) (Konev, 2007).

In Drosophila eggs, meiosis gives rise to four haploid nuclei. When the egg is fertilized, one of them is selected as a female pronucleus; the remaining three form the polar body. After breakdown of the sperm nuclear envelope, the compacted sperm chromatin is decondensed, and sperm-specific protamines are replaced with maternal histones. The male and female pronuclei juxtapose in the middle of the embryo and undergo one round of separate haploid mitoses. The resulting products fuse with their counterparts to give two diploid nuclei. In the majority of Chd1 embryos, partial decondensation of the sperm chromatin and normal apposition of parental pronuclei were observed. Then, however, one pronucleus underwent mitosis; the other one did not. Considering the subsequent loss of paternal DNA, it is concluded that mitotic progression of the male pronucleus is hindered in Chd1 embryos (Konev, 2007).

Because CHD1 can assemble nucleosomes in vitro, it was asked whether the absence of CHD1 affects histone incorporation into the male pronucleus. Embryos from wild-type or Chd1-null females were stained with an antibody against histone H3. In wild-type embryos, uniform staining was observed in both parental pronuclei. In contrast, in Chd1-null embryos only the female chromatin was brightly stained. The male pronucleus contained considerably less histone H3. These observations indicate that CHD1 is necessary for nucleosome assembly during sperm decondensation (Konev, 2007).

Sperm DNA does not replicate during decondensation, and histones are deposited by replication-independent assembly mechanisms, which involve the variant histone H3.3 but not canonical H3. It has been shown in Drosophila and mice that H3.3 is specifically present in the male pronucleus (Loppin, 2005; Torres-Padilla, 2006). The distribution of H3.3 was analyzed in embryos derived from Chd1-null females that carry a FLAG-tagged H3.3 transgene. In wild-type embryos, colocalization of the H3.3-FLAG signal was observed with male pronuclear DNA during migration and apposition. No H3.3-FLAG was detectable in the maternal pronucleus. In Chd1-null embryos, the male pronucleus showed altered H3.3-FLAG staining. The signal did not co-localize with the DNA but remained constrained to the nuclear periphery in a sacshaped pattern (Konev, 2007).

These findings suggest that in the earliest phases of Drosophila development CHD1 is essential for the incorporation of H3.3 and normal assembly of paternal chromatin. In contrast, CHD1 does not appear to affect the organization of maternal chromatin. It is concluded that CHD1 is required for replication-independent nucleosome assembly in the decondensing male pronucleus, but is dispensable for replication-coupled incorporation of H3 (Konev, 2007).

It has recently been shown that the sèsame (ssm) mutation of Drosophila histone chaperone HIRA causes the development of haploid embryos and abolished H3.3 deposition into the male pronucleus. Chd1 and ssm mutants, however, differ profoundly in the manifestation of this phenotype. In ssm embryos, H3.3 is absent from the male pronucleus. In contrast, in Chd1-null embryos, H3.3 delivery to the male pronucleus appears to be unaffected. Thus, these observations allow the roles of CHD1 and HIRA to be mechanistically discerned. Whereas HIRA is essential for histone delivery to the sites of nucleosome assembly, CHD1 directly facilitates histone deposition. These findings are consistent with observations in vitro that histone chaperones either do not assemble nucleosomes or assemble them at a greatly reduced rate in the absence of ATP-utilizing factors. These data provide evidence that histone deposition in vivo also transpires through an ATP-dependent mechanism (Konev, 2007).

CHD1 has been implicated in transcription elongation-related chromatin remodeling (Sims, 2004). This study demonstrates that CHD1 functions in nucleosome assembly in the early Drosophila embryo, which is transcriptionally silent. The biological role of CHD1, therefore, is not confined to transcription-related processes. The Schizosaccharomyces pombe homolog of CHD1, Hrp1, has been shown to function in loading of the centromere-specific H3 variant CENP-A (Walfridsson, 2005). Similarly to H3.3, incorporation of CENP-A into chromatin is not restricted to S phase. Therefore, CHD1 may have a general role in replication-independent nucleosome assembly (Konev, 2007).

Sperm decondensation involves not only histone incorporation, but also eviction of protamines. To discern whether CHD1 has a role in this process, the fate of protamine B (Mst35Bb) was analyzed in Chd1-null embryos. Although GFP-tagged Mst35Bb was detected in the sperm head immediately upon fertilization, no Mst35Bb-GFP signal was detected in the male pronucleus. Thus, like HIRA, CHD1 is dispensable for protamine removal. This study has shown that the male pronucleus in Chd1-null embryos contains very low amounts of histones, yet the DNA is not packaged with protamines. It remains an open question whether other DNA-protein complexes exist in the male pronucleus (Konev, 2007).

Drosophila eggs contain stores of both known chromatin assembly factors CHD1 and ISWI. Nevertheless, ISWI is unable to substitute for CHD1 in the deposition of H3.3. To examine whether CHD1 and ISWI differ in their ability to interact with the H3.3 chaperone HIRA, coimmunoprecipitation experiments were performed with extracts from embryos expressing FLAG-HIRA. CHD1 signal was readily detected in FLAG-specific immunoprecipitates, whereas ISWI did not coimmunoprecipitate with HIRA. Thus, a fraction of CHD1, but not ISWI, physically associates with HIRA. This property of CHD1 may account for its unique function in the H3.3 deposition process (Konev, 2007).

A subpopulation of Chd1 mutant haploid embryos survives beyond apposition stage. Therefore, it was asked whether H3.3 deposition is altered in Chd1 mutant embryos during later developmental stages. In wild-type nuclei, the H3.3-FLAG signal originating from the male pronucleus becomes undetectable after 2 to 3 divisions. Most maternal H3.3 remains distributed diffusely throughout the syncytium. After cycle 11 (roughly correlating with the onset of zygotic transcription) H3.3-FLAG is redistributed into the nuclei, where it colocalizes with the DNA. In contrast, incorporation of H3.3 into Chd1 mutant nuclei was impaired. H3.3-FLAG produced a speckled staining with numerous bright dots that poorly overlapped with the maxima of DNA staining. It is important to note that, in the ssm (HIRA) mutant, H3.3 incorporation defects in tissues or developmental stages other than the apposition stage were not observed. This result is consistent with the idea that misincorporation of H3.3 in Chd1 embryos is a direct effect of CHD1 deletion rather than a consequence of haploid development. It is also concludes that CHD1 functions in H3.3 deposition during later stages of embryonic development, possibly in a HIRA-independent fashion (Konev, 2007).

This study provides evidence that ATP-dependent mechanisms are used for histone deposition during chromatin assembly in vivo. Thus, molecular motor proteins, such as CHD1, function not only in remodeling of existing nucleosomes but also in de novo nucleosome assembly from DNA and histones. Finally, this work identifies CHD1 as a specific factor in the assembly of nucleosomes that contain variant histone H3.3 (Konev, 2007).

Cloning, chromosome mapping and expression analysis of the HIRA gene from Drosophila melanogaster

The HIRA family of genes (named after yeast HIR genes; HIR is an acronym for 'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. This study describes a new member of the HIRA family, Dhh, for the Drosophila homolog of HIRA. Northern analysis with poly (A)+ mRNA isolated from different developmental stages of Drosophila shows hybridization with a single Dhh transcript of 4.1kb. Hybridization is strong in female adults, unfertilized eggs and 0-3-h-old embryos, then diminishes, but is still detectable, during later stages of development and in adult males. More specifically, in-situ hybridization shows that Dhh transcripts, which are initially detected in nurse cells during mid-oogenesis, become localized to the developing oocyte at high levels. Transcripts persist strongly during early blastoderm stages then fade dramatically by 3h of development. The Dhh cDNA encodes an open reading frame of 1061 amino acids with high similarity scores to the HIRA polypeptides, as well as two hypothetical polypeptides from C. elegans and S. pombe, in a protein database search. They all share three highly homologous regions: a WD-repeat cluster, a small domain with clustered positively charged amino acids, and a domain comprising two repeats with close resemblance to WD repeats plus a region with no homology outside of the family. The conservation of these homologous regions in HIRA-encoded proteins from evolutionary distant organisms suggests that they are important for the activity of the members of the family (Kirov, 1998).

The HIRA gene from Drosophila melanogaster corresponds to a 3374 nucleotide cDNA that encodes a protein of 1047 aa, that exhibits a 42% identity with the human protein. Alignment with the predicted HIRA proteins from human, mouse, chick and pufferfish reveals strong conservation within the N-terminal region which contains seven WD domains, with less conservation of C-terminal sequences. In situ hybridisation to salivary gland chromosomes indicates that the gene resides in region 7B2-3 of the X chromosome. Drosophila hira is expressed through embryonic development and at lower levels during larval and pupal development. The expression of hira is dramatically increased in early embryos and in females, suggesting that the hira mRNA could be maternally deposited in the embryos (Llevadot, 1998)

Search PubMed for articles about Drosophila HIRA

Bonnefoy, E., Orsi, G. A., Couble, P. and Loppin, B. (2007). The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization. PLoS Genet. 3(10): 1991-2006. Medline abstract: 17967064

Kirov, N., Shtilbans, A. and Rushlow, C. (1998). Isolation and characterization of a new gene encoding a member of the HIRA family of proteins from Drosophila melanogaster. Gene 212(2): 323-32. Medline abstract: 9611274

Konev, A. Y., et al. (2007). CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo. Science 317(5841): 1087-90. Medline abstract: 17717186

Llevadot, R., et al. (1998). Cloning, chromosome mapping and expression analysis of the HIRA gene from Drosophila melanogaster. Biochem. Biophys. Res. Commun. 249(2): 486-91. Medline abstract: 9712723

Loppin, B., Docquier, M., Bonneton, F. and Couble, P. (2000). The maternal effect mutation sÈsame affects the formation of the male pronucleus in Drosophila melanogaster. Dev. Biol. 222: 392-404. 10837127

Loppin, B., Berger, F. and Couble, P. (2001). The Drosophila maternal gene sésame is required for sperm chromatin remodeling at fertilization. Chromosoma 110: 430-440. 11735001

Loppin, B., Bonnefoy, E., Anselme, C., Laurencon, A., Karr, T. L. and Couble, P. (2005). The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. Nature 437(7063): 1386-90. Medline abstract: 16251970

Nakayama, T., et al. (2007). Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading. Genes Dev. 21: 552-561. Medline abstract: 17344416

Raja, S. J. and Renkawitz-Pohl, R. (2005). Replacement by Drosophila melanogaster protamines and Mst77F of histones during chromatin condensation in late spermatids and role of Sesame in the removal of these proteins from the male pronucleus. Molec. Cell. Biol. 25: 6165-6177. 15988027

Ray-Gallet, D. et al. (2002). HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis. Mol. Cell 9: 1091-1100. 12049744

Shimojima, T., et al. (2003). Drosophila FACT contributes to Hox gene expression through physical and functional interactions with GAGA factor. Genes Dev. 17: 1605-1616. 12815073

van der Heijden, G. W., et al. (2005). Asymmetry in histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote. Mech. Dev. 122(9): 1008-22. Medline abstract: 15922569

date revised: 13 December 2007

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