Histone H3.3A
DEVELOPMENTAL BIOLOGY

To determine the histone composition of transcribed genes, the distribution of GFP-tagged H3 and H3.3 was examined in Drosophila polytene chromosome spreads. Use of the same epitope tag on these two histones eliminates possible differences in detection, and the fusion proteins were expressed throughout development to label all possible deposition sites. Quantitation on Western blots with an antibody to the C terminus of histone H3 showed that each of these fusion proteins constituted <0.5% of the total H3 in cells. These trace amounts are considered unlikely to interfere with chromatin functions (Schwartz, 2005).

The H3-GFP and H3.3-GFP histones show nearly opposite patterns of distribution in polytene chromosomes. H3-GFP localizes throughout the euchromatic arms and the heterochromatic chromocenter, and entirely overlaps with all DAPI-stained regions. In contrast, identically expressed H3.3-GFP localizes primarily to the interbands in euchromatin, as well as throughout the chromocenter. Tagged histone H3 in Drosophila Kc cells only deposits through the replication-coupled pathway (Ahmad, 2002), and the band pattern in polytene spreads is consistent with H3 incorporation during DNA replication in the developing salivary gland. H3.3 deposits through both replication-coupled and RI pathways, and its distribution in polytene chromosomes should result from the combination of these two processes (Schwartz, 2005).

To define sites that result solely from RI deposition, a truncated H3.3 protein (H3.3core-GFP) that can only participate in the RI pathway (Ahmad, 2002) was expressed. Constitutively expressed H3.3core-GFP localizes to interbands and is absent from the heterochromatic chromocenter. From comparison of these three patterns, it is concluded that the chromocenter and DAPI-banded regions are packaged with histones during DNA replication, while interbands are enriched for RI-deposited H3.3 (Schwartz, 2005).

Enrichment of H3.3 in interbands is consistent with the idea that this histone variant is enriched at active genes. There is occasional overlap between H3.3core-GFP sites and DAPI bands; however, these may be sites where closely juxtaposed sites and DAPI bands cannot be resolved. Alternatively, these sites may have been transcribed earlier in development and still retain tagged H3.3. Apart from these ambiguous sites, it is clear that the vast majority of RI histone deposition occurs in interbands (Schwartz, 2005).

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).

Effects of Mutation

Characteristic amino acid substitutions distinguish major histone H3 proteins from their replacement histone H3 paralogs, suggesting that some or all of these substitutions are responsible for differences in deposition. However, the replication-independent deposition of replacement histone H3 variants has been attributed entirely to its availability in the gap phases of the cell cycle. Instead, the current results producing H3-GFP and H3.3-GFP from the same inducible promoter argue that at least some of the characteristic substitutions specify which assembly pathway is used. It is clear that H3-GFP fails to undergo replication-independent deposition, because when the protein is produced after S phase, it does not deposit onto DNA. To identify which of the four differences between Drosophila H3 and H3.3 are responsible for differential deposition, site-directed mutagenesis was used to alter the histone-GFP fusion genes. Single mutations were introduced into the H3.3-GFP fusion gene (with amino acid residues S31 A87 I89 G90; abbreviated 'S/AIG') to match each of the H3 identities (A31 S87 V89 M90; 'A/SVM'). Each of these permutated templates was transfected and expressed in cells, and the ability of the resultant fusion proteins to participate in replication-coupled and replication-independent deposition was examined. All mutant proteins were efficiently deposited onto replicating DNA, demonstrating that these changes do not interfere with chromatin assembly. However, none of the mutations prevented deposition at the rDNA array, and thus it is concluded that no single identity is necessary for the replication-independent pathway. The converse mutations were introduced into the H3-GFP fusion gene to match each of the H3.3 identities. Strikingly, each of three mutations is sufficient by itself to confer partial replication-independent activity. All three of these positions lie in the core of the histone. To further confirm that these residues specify assembly pathways, all three positions in H3 were converted to the H3.3 identities (A/AIG). As expected, this mutant undergoes both replication-coupled and replication-independent deposition. Since any one change at these positions in H3 allows some replication-independent deposition, it appears that this is a default ability of H3 variants. It is concluded that the identities at these positions specify assembly pathways and that the combination of residues in the major H3 histone actively prevents replication-independent assembly (Ahmad, 2002).

Whether the N-terminal tail regions of histone H3 and H3.3 are required for either nucleosome assembly pathway was examined in Drosophila cells. A series of deletions was generated that removed portions of the histone tail from GFP fusion constructs. These constructs were transfected into cells and expression was induced. The distribution of histone-GFP was compared to the PCNA pattern in individual nuclei to determine whether replication-coupled deposition with the deleted protein would still occur. In this experimental system, it was found that the N-terminal tail of histone H3 is essential for in vivo replication-coupled nucleosome assembly. Histone H3 proteins deleted for this region localize poorly to replicating DNA or remain diffuse throughout the nucleus. Deletion of the N-terminal tail does not inhibit tetramer formation with histone H4 in vitro, and H3 continues to be imported into the nucleus, implying that these truncated proteins are defective for a later step in replication-coupled nucleosome assembly (Ahmad, 2002).

Uncovering a requirement for a region in the N-terminal tail of histone H3 for replication-coupled deposition prompted an examination of whether this region is also required for replication-independent deposition of histone H3.3. Most truncated histone H3.3-GFP proteins were efficiently used for replication-independent deposition and were resistant to salt extraction, although larger deletions produced aberrant protein aggregates in some nuclei. Only the most proximal deletion (deleting 40 of the 44 residues from the N-terminal tail) showed a reduced intensity of rDNA labeling. This deletion extends into a critical region of histone H3 that passes through the DNA gyres in the nucleosome. A similar deletion is lethal in yeast, suggesting that the region is required to form a proper nucleosomal particle. It is concluded that replication-independent nucleosome assembly machinery can deposit a truncated histone H3.3 protein (Ahmad, 2002).

Because the N-terminal tail is essential for replication-coupled but not for replication-independent deposition, deletion constructs of histone H3.3-GFP separate these two pathways of nucleosome assembly. Staining for PCNA in cells producing a truncated histone H3.3-GFP revealed that nucleosome assembly even in S phase cells is not limited to replicating DNA: some replication-independent deposition of histone H3.3 occurs in euchromatin and in the nucleolus. Thus, although the bulk of nucleosome assembly uses the vastly more abundant histone H3 and is coupled to DNA replication, at some sites nucleosomes are assembled continually throughout the cell cycle (Ahmad, 2002).

Transcriptional and developmental functions of the H3.3 histone variant in Drosophila

Changes in chromatin composition accompany cellular differentiation in eukaryotes. Although bulk chromatin is duplicated during DNA replication, replication-independent (RI) nucleosome replacement occurs in transcriptionally active chromatin and during specific developmental transitions where the genome is repackaged. In most animals, replacement uses the conserved H3.3 histone variant, but the functions of this variant have not been defined. Using mutations for the two H3.3 genes in Drosophila, widespread transcriptional defects were identified in H3.3-deficient animals. Mutant animals compensate for the lack of H3.3 in two ways: they upregulate the expression of the major histone H3 genes, and they maintain chromatin structure by using H3 protein for RI nucleosome replacement at active genes. Rescue experiments show that increased expression of H3 is sufficient to relieve transcriptional defects. In contrast, H3.3 is essential for male fertility, and germline cells specifically require the histone variant. Defects without H3.3 first occur around meiosis, resulting in a failure to condense, segregate, and reorganize chromatin. Rescue experiments with mutated transgenes demonstrate that H3.3-specific residues involved in RI nucleosome assembly-but not major histone modification sites-are required for male fertility. These results imply that the H3.3 variant plays an essential role in chromatin transitions in the male germline (Sakai, 2009).

Transcription in the absence of histone H3.2 and H3K4 methylation

Histone H3 proteins play fundamental roles in DNA packaging, gene transcription, and the transmission of epigenetic states. In addition to posttranslational modifications of their N termini, the use of H3 variants contributes to their regulatory repertoire. Canonical histone H3.2 is expressed during S phase and differs by four amino acid residues from the variant histone H3.3, which is synthesized in a cell-cycle-independent manner. Because H3.3 is enriched within actively transcribed loci, and because di- and trimethylation of H3 lysine 4 are hallmarks of chromatin at such sites in the genome, the H3.3K4 residue is considered to serve as the major regulatory determinant for the transcriptional state of a gene. This study used genetic approaches in Drosophila to replace all 46 gene copies of His3.2 with mutant derivatives and thereby demonstrate that canonical and variant H3 can functionally replace each other. Cells are able to divide and differentiate when H3.2 is entirely absent but replaced by S phase-expressed H3.3. Moreover, although slowed down in their proliferative capacity, cells that code for a nonmethylatable residue instead of K4 in all canonical and variant H3 genes are competent to respond to major developmental signaling pathways by activating target gene expression. Hence, the presence of different H3 protein species is not essential in Drosophila and transcriptional regulation can occur in the complete absence of H3K4 methylation (Hodl, 2012).

The stunning evolutionary conservation of canonical versus variant H3 proteins, coupled to the finding that H3.3 distribution correlates with sites of active transcription, led to the notion that the two major H3 forms assume different roles in cells due to different protein sequences. This study shows that canonical and variant H3 can largely compensate for each other’s absence and that the major difference resides in their mode of transcriptional regulation. Specifically, animals or cell clones were generated that expressed exclusively either the H3.2 or the H3.3 type. Whereas the lack of H3.2 can only just be overcome by the H3.3 provided by the two wild-type His3.3 genes, significant rescue was observed if extra H3.3-encoding genes are introduced under the control of His3.2 regulatory sequences. Conversely, animals null mutant for both His3.3 genes are not only viable but even fertile if H3.2 is expressed under the His3.3B promoter. Hence, the four amino acids that distinguish H3.2 and H3.3 do not preclude the two proteins from assuming each other’s functions. This in turn indicates that neither the chaperones and machineries regulating H3 deposition, modification, and turnover nor the 'readers' that are recruited by a particular histone code discriminate between H3.2 and H3.3. Although biochemical measurements suggest that H3.3-containing nucleosomes are less stable and primarily incorporated around transcriptional start sites, no functional requirement is seen for either H3.2 or H3.3 for nucleosome stability or turnover in the course of standard gene activation (Hodl, 2012).

Furthermore, because correct transcriptional activation and repression were seen in wing disc cells exclusively expressing either H3.2 or H3.3, the notion that H3 identity determines the activity state of chromatin domains cannot be upheld. Thus, functional diversification of chromatin is largely established by posttranslational histone modifications and their readers. As the observations regarding H3K4me3 suggest, newly incorporated histone proteins are modified according to the local genomic context, irrespective of their identity (Hodl, 2012).

Finally, the recent increase in number of signal transduction components implicated in either reading or conferring di- and trimethylation of H3 lysine 4 prompted an extension of this analysis on the role of H3K4 in the transcriptional activation of genes targeted by these factors. Although the diminished proliferative behavior of cells mutant for all H3K4 residues clearly indicates a requirement for this residue, no obvious effect was observed on the transcriptional activity of typical targets of four major signaling pathways involved in imaginal disc development. Even Hox gene expression, which had been proposed to be controlled by H3K4 methylation, can occur in clones mutant for H3K4. It cannot be excluded that individual genes are reduced in their expression (like shown for Ubx), yet no ultimate requirement was observed for H3K4 methylation in the course of gene expression. These results do not support the classical model of a histone code, in which sequential or combinatorial histone marks specify unique biological outputs, nor do they support an ultimate role of H3K4 methylation in indexing nucleosomes for dynamic turnover which serves as a prerequisite for transcription. It is speculated that H3K4 methylation contributes to the robustness of transcriptional outputs, for example under stress conditions. A recent study reports that H3K4 and its methylation are involved in yeast DNA damage response, yet redundantly with the presence of the N terminus of H2A. It remains to be tested whether a similar redundancy exists in Drosophila. It can also be envisioned that effector proteins simultaneously recognize a combination of histone modifications, and that the diminished proliferative capacity observed for the H3K4A mutant cells could be a result of suboptimal binding of such factors and decreased downstream reaction kinetics. Further analysis will have to provide insights into the actual, subtle role of K4 methylation (Hodl, 2012).

Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling

Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. This study established a Drosophila animal model for the pathogenic histone H3K27M mutation and showed that its overexpression resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing derepression of PRC2 target genes and developmental perturbations. Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its misregulation in Drosophila results in changes of H3K9 methylation levels and heterochromatic silencing defects. This study has established histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin signaling pathways (Herz, 2014).

Histone proteins constitute the core of eukaryotic chromatin. SET domain-containing histone methyltransferase complexes such as complex of proteins associated with Set1 (COMPASS) and polycomb repressive complex 2 (PRC2) methylate lysine residues within the histone H3 amino-terminal tail and are essential for normal development. Establishing direct functions for modified lysine residues in histones is difficult because there are multiple histone gene copies in metazoans. Moreover, histone methyltransferase enzymes occur in multimember families with potential redundant activities and histone methylation-independent functions. In Drosophila, replacing all copies of histone H3 with H3 Lys27-to-Arg27 (H3K27R) in a clonal substitution recapitulates the phenotype of mutating E(z), the PRC2 H3 Lys27 methyltransferase gene, suggesting this mark is indeed required for PRC2-mediated repression. Single-allele mutations of histone H3.3 Lys27-to-Met27 (H3.3K27M) occur in a subtype of aggressive pediatric brain cancers and act in a dominant manner to deplete H3K27 methylation by inhibiting PRC2 methyltransferase activity. Other histone H3 lysine-to-methionine mutants also possess dominant gain-of-function activities, making them attractive tools for in vivo functional studies of histone lysine modifications. Trimethylation of histone H3 Lys27 (H3K27me3) and Lys9 (H3K9me3) are associated with distinct forms of transcriptionally silenced chromatin. Histone H3K27me3 catalyzed by PRC2 is enriched at so-called facultative heterochromatin and is implicated in the silencing of key developmental genes, in particular the homoeotic gene clusters. By contrast, H3K9me3 is associated with 'constitutive' heterochromatin at telomeres and centromeres (Herz, 2014).

This study established wild-type histone H3.3, H3.3K27M, and H3.3K9M constructs with a C-terminal FLAG-hemagglutinin (HA) tag for tissue-specific overexpression in Drosophila. Overexpression of H3.3K27M in the posterior compartment of wing imaginal discs driven by engrailed-GAL4 caused a strong reduction in all three H3K27 methylation states and derepression of the PRC2 target gene Ultrabithorax (Ubx), thus phenocopying knockdown of the catalytic PRC2 subunit E(z). Also, increased H3K27 acetylation was observed for H3.3K27M overexpression in Drosophila and mammalian cells and E(z)-RNAi in Drosophila. Genome-wide RNA sequencing (RNA-seq) analysis of H3.3K27M-overexpressing wing imaginal discs revealed up-regulated RNA transcripts for known polycomb target genes, including Ubx, wingless (wg), and the PRC1 subunits Posterior sex combs (Psc) and Suppressor of zeste 2 ([Su(z)2]). Other Homeobox (Hox)-containing genes such as engrailed (en) and invected (in), and signaling pathway components such as cubitus interruptus (ci), were down-regulated upon H3.3K27M overexpression. Moreover, flies expressing H3.3K27M under a tissue-specific Distal-less-GAL4 driver exhibit gross morphological defects-such as severe leg malformations and fusion phenotypes and malformed, reduced, or missing proboscis-and die around eclosion, phenocopying E(z)-RNAi under the same conditions (Herz, 2014).

Trimethylation of histone H3K9me3 by supressor of variegation 3-9 [Su(var)3-9] proteins is a hallmark of constitutive heterochromatin. Histone H3K9me3 serves as a binding substrate for heterochromatin protein 1 α (HP1α, also known as CBX5) and establishes a transcriptionally repressed state. Euchromatic genes that become abnormally juxtaposed to heterochromatic regions are subject to transcriptional silencing through position-effect variegation (PEV). Less is known about the direct role of H3K9 methylation in the regulation of gene expression. Indeed, studies in fission yeast point to H3K9 methylation-independent functions for the Su(var)3-9 homolog Clr4 in chromatin silencing. To test a direct role for H3K9 methylation in the regulation of gene expression in metazoans, H3.3K9M was overexpressed in Drosophila wing imaginal discs and mammalian cells, and a global depletion of H3K9 methylation levels was observed but no effect was seen on H3K27 methylation. In contrast, H3K9 mono- and dimethylation were slightly reduced when H3.3K27M was overexpressed. Mononucleosomes were purified from wild-type H3.3-, H3.3K9M-, and H3.3K27M-overexpressing human embryonic kidney (HEK) 293 cells, and these samples were subjected to multidimensional protein identification technology (MudPIT) mass spectrometry). The bindings of HP1α (CBX5), HP1β (CBX1), and HP1γ (CBX3) were substantially reduced for H3.3K9M-containing mononucleosomes, as were the interactions of the HP1-associated proteins chromatin assembly factor 1a (CHAF1A/p150) and CHAF1B/p60. Substantially increased association of the H3K9 demethylase KDM3B and the H3K9/K56 deacetylase SIRT6 were found with H3K9M-containing mononucleosomes (Herz, 2014).

Reduced dosage of Drosophila HP1 α [also known as Su(var)205] and Su(var)3-9 results in suppression of PEV. By using a heat shock-inducible lacZ construct inserted within Y-chromosomal heterochromatin, this study found that overexpression of H3.3K9M results in suppression of PEV in both Drosophila salivary glands and eye-antenna imaginal discs. Bulk histone H3K9 methylation levels were decreased in H3.3K9M-overexpressing salivary glands. The effects of H3.3K9M overexpression on heterochromatic silencing were assessed in Drosophila ovaries. The gypsy-lacZ construct is normally silenced in almost all follicle cells but is up-regulated upon loss of heterochromatin function. Overexpression of H3.3K9M results in derepression of lacZ. Thus, the H3.3K9M mutation disrupts heterochromatic silencing of retroelements (Herz, 2014).

KDM3B is a JumonjiC domain-containing histone demethylase that shows specificity toward H3K9 and is involved in gene activation in leukemia cells. Because KDM3B specifically interacts with H3.3K9M-containing nucleosomes, it was of interest to test whether changes in KDM3B levels would alter H3K9 methylation by knocking down or overexpressing its Drosophila homolog, JHDM2, in wing imaginal discs. Depletion of JHDM2 results in increased H3K9 mono- and dimethylation. Conversely, the overexpression of JHDM2 in wing imaginal discs results in depletion in H3K9 dimethylation levels and, to a lesser extent, H3K9 trimethylation and suppresses PEV in both Drosophila salivary glands and eye-antenna imaginal discs. JHDM2 and SIRT6 also globally affect H3K9 acetylation to a similar degree as H3.3K9M overexpression. Sirt6 is not a major regulator of PEV in eye-antenna imaginal discs and salivary glands, but Sirt6-RNAi results in a somewhat modest derepression of the gypsy-lacZ reporter (Herz, 2014).

Histone lysine-to-methionine mutants were used to globally modulate histone methylation in vivo. A Drosophila animal model of the H3K27M mutation was established, that may help elucidate the molecular pathogenesis of pediatric gliomas. To gain mechanistic insight into the molecular function of these mutants, an unbiased proteomic strategy was used to identify histone lysine-to-methionine-interacting partners. Biochemical studies do not identify PRC2 components, such as EZH2, SUZ12, and EED, as significantly enriched on H3.3K27M-containing nucleosomes as previously suggested. However, an increase in H3K27 acetylation levels was detected, along with association of bromodomain-containing protein 1 (BRD1) and BRD4 to H3.3K27M-containing nucleosomes. These findings suggest that inhibitors of H3K27 acetylation or BRD4 inhibitors, such as JQ1 and iBET, could be useful for the treatment of the H3.3K27M-mutated subtype of aggressive pediatric glioblastomas (Herz, 2014).

It was also demonstrated that H3K9M globally depletes H3K9 methylation levels in vivo, disrupts interaction of HP1 proteins, and thus suppresses PEV. Via an unbiased proteomic strategy, KDM3B/JHDM2 and Sirt6 were identified as regulators of H3K9 methylation-dependent heterochromatic silencing. Indeed, JHDM2 acts as a suppressor of variegation in multiple tissues in these assays, whereas Sirt6 function seems to be restricted to retroelement silencing. Mutations of histone H3.3K36M were recently discovered in a subtype of bone cancer. Thus, histone lysine-to-methionine mutations are associated with highly tissue-specific cancer types. Given the importance of heterochromatin in maintaining genomic stability, it is plausible that as-yet-uncharacterized H3K9M mutations might occur in some cancers. The system that was established will provide a powerful tool to inhibit histone lysine modifications at specific residues in vivo and allow to biochemically capture the molecular players involved in chromatin signaling pathways (Herz, 2014).


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Histone H3.3A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 23 August 2017

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