BIOLOGICAL OVERVIEW

Chromatin is the DNA-protein complex that constitutes chromosomes. The major protein component of chromatin is the nucleosome octamer. One of the four proteins that comprise the nucleosome octamer is Histone H4. The special interest in Histone H4 derives from the fact that it is acetylated in several important processes, among them gene activation, chromatin assembly and histone displacement by protamines in spermatogenesis. Two of these processes are described below: gene activation and chromatin assembly. The evidence that Histone H4 acetylation is of fundamental biological importance is not confined to Drosophila, but has been gleaned from work with yeast, ciliates, flies, frogs and mammals. Histone acetylation is an evolutionarily conserved process, carrying out conserved biological functions.

Gene Activation: Histone acetylation plays a positive role in promoting access of transcription factors to nucleosomal DNA. The idea that acetylated histones are associated with transcriptionally active chromatin is more than three decades old. However, only in the last decade have experimental systems been sufficiently refined to provide convincing evidence. One such system is the 5s RNA gene of Xenopus. Whole histone octamers, consisting of (H2A/H2B/H3/H4)*2, prevent binding of transcription factor TFIIIA to the Xenopus 5s gene. Acetylation of the histones used to assemble the histone octamer onto the 5S RNA gene facilitates the association of TFIIIA with the gene. Removal of the N-terminal tails (the site of histone acetylation) from the core histones also facilitates the association of TFIIIA with nucleosomal templates. It is thought that histone tails have a major role in restricting transcriptional factor access to DNA and that their acetylation releases this restriction by directing dissociation of the tails from DNA or inducing a change in DNA configuration on the histone core to allow transcription factor binding (Lee, 1993).

Histone H4 isoforms can be found at four different lysine residues, acetylated in different combinations. When polytene chromsomes from Drosophila larva are examined with antisera specific for each of the four acetylated lysine residues, differently acetylated isoforms are found in distinct patterns of distribution. H4 molecules acetylated at lysines 5 and 8 are distributed in overlapping, but nonidentical islands throughout the euchromatic chromosome arms, suggesting that H4 acetylated at lysines 5 and 8 is associated with transcriptionally active genes. ß-Heterochromatin in the chromocenter is depleted in these isoforms, but relatively enriched in H4 acetylated at lysine 12. This suggests that H4 acetylated at lysine 12 is associated with transcriptionally silent ß-heterochromatin. H4 acetylated at lysine 16 is found at numerous sites along the transcriptionally hyperactive X chromosome in male larvae, but not in male autosomes or any chromosome in female cells.

The association of H4 acetylated at lysine 16 with male X chromosomes is intimately related to the process of dosage compensation. Males have only one X chromosome, compared with the two found in females. Were male X chromosomes to function at the same level of transcriptional activation as females, males would have only half the level of X chromosome coded gene products as females. The heightened activation of male X chromosomes is called dosage compensation (See Sex lethal). Dosage compensation is mediated by four loci, known as male-specific lethal genes (see also MSL-2). Histone H4 plays a role in dosage compensation. The specifically acetylated isoform of histone H4, H4Ac16, is detected predominantly on the male X chromosome. Two of the MSL proteins bind to the X chromosome in an identical pattern; the H4Ac16 pattern on the X is largely coincident with that of the MSL proteins. No H4HAc16 is found on X chromosomes in mutants of MSL genes. It has been suggested that acetylated Histone H4 plays a role in the heightened activation of the transcription of male X chromosomes (Bone, 1994).

Chromatin assembly: Control of gene accessability to transcription factors is not the only role of acetylation of H4 in the biology of the cell. Acetylation is involved in the process of histone assembly into nucleosomes. The cytoplasmic enzyme histone transacetylase B (HAT B) is involved in an evolutionarily conserved acetylation of newly synthesized Histone H4 on lysine 12 (Sobel, 1994 and 1995).

Hat B has been characterized from yeast, and it appears in the cytoplasm as a dimer consisting of two subunits, Hat1p and Hat2p. Hat1p is the histone transacetylase, while Hat2p is a member of an evolutionarily conserved family of p48 proteins. Members of the p48 family are histone escorts, accompanying newly synthesized histones from cytoplasm to nucleus. The p48 family members are conserved subfamily of WD-repeat proteins, possessing a motif involved in protein-protein interaction. p48 proteins are found in three contexts: associated with Hat B in the cytoplasm, associated with chromatin assembly factor (CAF-1) in the nucleus (Tyler, 1996 and Verreault, 1996), and associated with a histone deacetylase activity. It is likely that cytoplasmic H4 is acetylated by Hat B, carried to the nucleus by CAF-1 (See Nap1), where it is assembled into newly synthesized chromatin, and subsequently deacetylated in a process required for chromatin maturation. p48 family members act as histone escorts, accompanying the histones through the process of acetylation, assembly and deacetylation (Roth, 1996 and references).

A second histone transacetylase activity is found in the nucleus of yeast. GCN5p, a yeast protein involved in transcriptional activation, is homologous to tetrahymena HAT A, a nuclear histone acetyltransferase. Both the Tetrahymena protein and GCN5p possess histone acetyltransferase activity and a highly conserved bromodomain. p55 preferentially acetylates histone H3. The presence of a bromodomain in nuclear A-type histone acetyltransferases (but not in cytoplasmic B-type HATs), known to function in protein-protein interaction, suggests that HAT A is directed to chromatin through protein interaction to facilitate transcriptional activation (Brownell, 1996).

Thus histone acetylation plays biologically important roles in histone assembly, gene activation, and chromatin structure. The protein complexes responsible for orchestrating these funtions are only now being worked out. The payoff will be an understanding of the complex evolutionarily conserved machinery regulating chromatin dynamics and gene expression in living cells.

Sex-specific phenotypes of histone H4 point mutants establish dosage compensation as the critical function of H4K16 acetylation in Drosophila

Acetylation of histone H4 at lysine 16 (H4K16) modulates nucleosome-nucleosome interactions and directly affects nucleosome binding by certain proteins. In Drosophila, H4K16 acetylation by the dosage compensation complex subunit Mof is linked to increased transcription of genes on the single X chromosome in males. This study analyzed Drosophila containing different H4K16 mutations or lacking Mof protein. An H4K16A mutation causes embryonic lethality in both sexes, whereas an H4K16R mutation permits females to develop into adults but causes lethality in males. The acetyl-mimic mutation H4K16Q permits both females and males to develop into adults. Complementary analyses reveal that males lacking maternally deposited and zygotically expressed Mof protein arrest development during gastrulation, whereas females of the same genotype develop into adults. Together, this demonstrates the causative role of H4K16 acetylation by Mof for dosage compensation in Drosophila and uncovers a previously unrecognized requirement for this process already during the onset of zygotic gene transcription (Copur, 2018).

Mutational analyses of histone amino acid residues that are subject to posttranslational modifications provide a direct approach for probing the physiological role of these residues and their modification. This study investigated the function of H4K16 and its acetylation in Drosophila by generating animals in which all nucleosomes in their chromatin were altered to constitutively carry a positively charged H4R16, an acetyl-mimic H4Q16, or a short apolar H4A16 substitution. These three types of chromatin changes have different physiological consequences that lead to the following main conclusions. First, H4R16 and H4Q16 chromatin both support development of female zygotes into adults. This suggests that, in females, modulation of H4K16 by acetylation is a priori not essential for the regulation of gene expression and the chromatin folding that occurs during development of the zygote. Second, unlike in females, only H4Q16 but not H4R16 chromatin supports development of male embryos into adults. This difference between males and females directly supports the critical role of H4K16 acetylation for dosage compensation in males. Third, cells with H4A16 chromatin are viable, proliferate, and can differentiate to form normal tissues in both males and females, but animals that entirely consist of cells with H4A16 chromatin arrest development at the end of embryogenesis. This lethality contrasts with the viability of animals with H4K16, H4R16, or H4Q16 chromatin and suggests that presence of a long aliphatic side chain with a polar group (i.e., either K, R, or Q) at residue 16 is more important for H4 function than the ability to regulate the charge of this residue by acetylation. A fourth main conclusion of this work comes from the finding that males that completely lack Mof protein (i.e., mof ^m-z- males) arrest development during gastrulation, whereas females of the same genotype develop into morphologically normal adults. This uncovers a previously unknown critical requirement of Mof acetyltransferase activity in males, already during the onset of zygotic gene transcription. The following sections discuss the results reported in this study in the context of the current understanding of the role of H4K16 and its acetylation (Copur, 2018).

In yeast and flies, the comparison of the severities of the phenotypes caused by different amino acid substitutions at H4K16 highlights how the two organisms have evolved to use this conserved residue and its modification in different ways. In yeast, H4K16ac is present genome-wide and SIR silencing is the key physiological process that requires H4K16, in its deacetylated state. Yeast cells with H4K16R, H4K16Q, or H4K16A mutations are viable but they show defective SIR silencing. Silencing is much more strongly impaired in H4K16Q or H4K16A mutants than in H4K16R mutants. This is because SIR3 protein binding to deacetylated H4K16, a prerequisite for silencing, is probably less severely impaired by the arginine substitution than by the alanine or glutamine substitutions. In Drosophila, the phenotypic differences between H4K16R, H4K16Q, and H4K16A mutants suggest that H4K16 is associated with two other, distinct physiological functions that are critical for the organism. The male-specific lethality of H4K16R mutants and the restoration of male viability in H4K16Q mutants demonstrate that dosage compensation is one essential process that critically requires the acetylated form of H4K16. A reduction of internucleosomal contacts by H4K16ac to generate chromatin that is more conducive to gene transcription on the male X chromosome currently is the simplest mechanistic explanation for how H4K16 acetylation enables dosage compensation. The observation that an H4K16A mutation causes lethality in both sexes suggests that, unlike in yeast, a long aliphatic side chain at this residue is essential for H4 function in Drosophila. It is currently not known why Drosophila H4K16A mutants die. However, it is important to note that H4K16A mutant cells retain the capacity to proliferate and differentiate and the mutation therefore does not disrupt any fundamental process required for cell survival (Copur, 2018).

Previous studies that investigated the function of histone H3 modifications by histone replacement genetics showed that for modifications associated with transcriptionally active chromatin it is essential to remove not only the wild-type copies of the canonical histone genes but to also mutate the histone H3.3 variants. The analyses of H4K16R, H4K16Q, and H4K16A mutant phenotypes reported in this study were all performed in the genetic background of animals lacking His4r, the only histone H4 variant in Drosophila. Importantly, it was found that in a His4r⁺ background, where only the canonical H4 proteins are replaced with mutant H4, the modifiable His4r protein permitted H4K16R His4r⁺ mutant males and, surprisingly, also H4K16A His4r⁺ mutant females and males to develop into adults. These animals were therefore not analyzed further. Supporting these observations, a recent study that used a similar strategy for replacing canonical histone H4 with H4^K16R also found that H4K16R His4r⁺ mutant males develop into normal adults. This suggests that, like His3.3, the His4r protein might also preferentially be incorporated into transcriptionally active chromatin and become acetylated by Mof. Although the viable H4K16R His4r⁺ males have been reported to show a significant reduction of X-linked gene expression, a full assessment of transcriptional defects in animals containing only H4R16 nucleosomes would require that such molecular analyses be performed in H4K16R His4r^Δ mutant males (Copur, 2018).

A final point that should be noted here is that during the early stages of embryogenesis, H4K16R, H4K16Q, or H4K16A mutants also still contain maternally deposited wild-type H4 protein that becomes incorporated into chromatin during the preblastoderm mitoses and only eventually becomes fully replaced by mutant H4 proteins during postblastoderm cell divisions. During the earliest stages of embryogenesis it has therefore not been possible to assess the phenotype of animals with chromatin containing exclusively H4R16, H4Q16, or H4A16 nucleosomes. This needs to be kept in mind when considering comparisons between the phenotypes of H4K16 point mutants and mof ^m-z- mutants (Copur, 2018).

Males without Mof protein (i.e., mof ^m-z- males) arrest development during gastrulation while their female siblings develop into adults. Moreover, mof ^m-z+ males also fail to develop, demonstrating that zygotic expression of Mof protein is insufficient to rescue male embryos that lacked maternally deposited Mof protein. The most straightforward explanation for these observations is that H4K16 acetylation by Mof is critically required for hypertranscription of X-chromosomal genes that has been reported to occur already during the onset of zygotic gene transcription and that the early developmental arrest of males is a direct consequence of failed dosage compensation (Copur, 2018).

How does this early requirement for Mof activity at the blastoderm stage relate to current understanding of the temporal requirement for the DCC for dosage compensation? Previous studies showed that males lacking the DCC subunits Msl-1, Msl-2, Msl-3, or Mle complete embryogenesis and arrest development much later, around the stage of puparium formation. For example, Msl-1 protein null mutants (i.e., msl-1 ^m-z- mutants) die as late third instar larvae, yet Msl-1 directly interacts with Mof to incorporate it into the DCC and is critical for targeting the complex and H4K16ac accumulation on the X chromosome in larvae. One possible explanation for the conundrum that the lack of Mof but not that of Msl-1 or other DCC subunits results in lethality during gastrulation could be that during these early stages, H4K16 acetylation by Mof for dosage compensation is not as strictly dependent on the other DCC subunits as during later developmental stages, or that there is redundancy between Msl-1, Msl-2, or Msl-3 for targeting Mof to the X chromosome in the early embryo (Copur, 2018).

A final point worth noting is that Mof is also present in another protein assembly called the NSL complex. NSL was reported to act genome-wide for regulating housekeeping gene transcription in both sexes and several NSL subunits are essential for Drosophila viability. The finding that mof ^m-z- mutant females develop into morphologically normal adults shows that the NSL complex must exert regulatory functions that are essential for viability independently of Mof H4K16 acetyltransferase activity (Copur, 2018).

The acetylation of lysine residues in the N termini of histones is generally associated with chromatin that is conducive to gene transcription. Mutational studies in yeast showed that there is substantial functional redundancy between most of the different acetylated lysine residues in the N termini of histone H3 and H4 but that H4K16 has unique effects on transcriptional control, with well-defined phenotypic consequences. This study shows that in Drosophila the principal function of H4K16 acetylation is X-chromosome dosage compensation in males (Copur, 2018).

Probing the function of metazoan histones with a systematic library of H3 and H4 mutants

Replication-dependent histone genes often reside in tandemly arrayed gene clusters, hindering systematic loss-of-function analyses. This study used CRISPR/Cas9 and the attP/attB double-integration system to alter numbers and sequences of histone genes in their original genomic context in Drosophila melanogaster. As few as 8 copies of the histone gene unit supported embryo development and adult viability, whereas flies with 20 copies were indistinguishable from wild-types. By hierarchical assembly, 40 alanine-substitution mutations (covering all known modified residues in histones H3 and H4) were introduced and characterized. Mutations at multiple residues compromised viability, fertility, and DNA-damage responses. In particular, H4K16 was necessary for expression of male X-linked genes, male viability, and maintenance of ovarian germline stem cells, whereas H3K27 was essential for late embryogenesis. Simplified mosaic analysis showed that H3R26 is required for H3K27 trimethylation. This study has developed a powerful strategy and valuable reagents to systematically probe histone functions in D. melanogaster (Zhang, 2018).

Chromatin is essential for genome packaging and regulation. The basic unit of chromatin is the nucleosome, consisting of 147 base pairs of DNA wrapped around a histone octamer comprising two copies each of histone proteins H3, H4, H2A, and H2B. A fifth 'linker histone,' H1, dynamically binds DNA residing between histone octamers at a subset of nucleosomes. Histones do not merely provide a binding platform for DNA; they also actively participate in DNA-related processes, such as transcription. One mechanism for histones to carry out these functions is though post-translational modifications (PTMs) (Zhang, 2018).

In the past two decades, over 20 types of PTMs have been identified on histones, including acetylation, methylation, phosphorylation, ubiquitination, and crotonylation. Among these PTMs, 12 are added to lysine residues. The N-terminal, flexible 'tail' domains are the most heavily modified portions of histones, presumably because they are more easily accessible to histone-modifying enzymes than other domains. However, PTMs have also been detected within the globular core domains of histones. Histone PTMs are thought to modulate chromatin structure and gene expression either directly or via recruitment of specific chromatin-associated proteins (Zhang, 2018).

Whether PTMs are always involved in chromatin structure remains controversial. Studies involving genetic or chemical interventions targeting histone-modifying enzymes have provided substantial evidence for biological functions of specific PTMs. For example, H3K27 methylation by the polycomb repressive complex 2 (PRC2) is involved in maintenance of cellular identity. Unfortunately, because these modifying enzymes generally have other protein substrates in addition to histones, and chromatin-regulating enzymes might also have functions unrelated to their enzymatic activities, these experimental data must be interpreted cautiously (Zhang, 2018).

The roles of PTMs can be directly queried by systematic mutation of histone residues. Such studies have been carried out in Saccharomyces cerevisiae, but experiments in higher organisms pose additional challenges. For example, there are 64 histone genes within the human genome, distributed at three major loci on different chromosomes, making it difficult to substantially alter levels of particular histone proteins inside human cells (Zhang, 2018).

Currently, the only multicellular organism in which histone mutagenesis has been performed is Drosophila melanogaster, in which all core-histone genes reside at a single locus on the left arm of chromosome 2, with ~100 copies of histone gene-repeat units (His-GUs) per chromosome. Each His-GU (~5 kb in length) contains the four core-histone genes in two pairs (His2A-His2B and His3-His4), each under the control of a divergent promoter, plus the linker-histone gene, His1, which is regulated independently (Zhang, 2018).

Histone residue function in D. melanogaster has been explored by removing the His-GU cluster (Df(2L)HisC, referred to as HisC hereafter) and complementing it with transgenes from plasmids or bacterial artificial chromosomes (BACs). These methods are labor intensive partly because four plasmids are needed for transgenic complementation and complex crossing procedures. Therefore, only limited sites within histone H3 and H4 have been analyzed. In addition, since the transgenes are randomly integrated, positional effects could confound data interpretation (Zhang, 2018).

This study generated an efficient histone-mutagenesis platform, enabling the functional study of each residue in all five histones with much higher throughput than with previous techniques. As a proof-of-concept study, H3 and H4 were targetted, revealing several interesting insights that would have been difficult to obtain by other means (Zhang, 2018).

This study has developed an efficient histone-mutagenesis system with several advantages over previous approaches. The histone-deletion line facilitates histone rescue in situ. A single plasmid is sufficient for complementation, and the plasmid is targeted to the original histone locus, which eliminates consideration of positional effects associated with random integration of plasmids and BACs. This high-throughput strategy to assemble multiple copies of His-GUs is fast and efficient and enables introduction of not only singular but also compound histone mutations (Zhang, 2018).

The results demonstrated that a low His-GU copy number causes developmental defects in both testes and ovaries, with more severe effects in ovary development. The ovarian defect was not the result of a loss of GSCs, and, instead, the budding processes were impaired), which leads to reduced fecundity or to sterility and which explains the severe fertility defects in females. The number of GSCs was only slightly reduced in testes from adult males with low histone copy numbers (compared with wild-type). Because histone copy numbers are altered globally in these flies, mosaic analysis could reveal whether reduced histone copies reflects an autonomous or non-autonomous effect on GSCs (Zhang, 2018).

The finding that H4K16 was critical for sex-dosage compensation and male development is consistent with the fact that MOF-MSL, which acetylates H4K16, contributes to male X-linked transcriptional activation. Notably, some H4K16A male adults were recovered and a weak homozygous mutant stock was generated under normal culture conditions, whereas the mof RNAi and mutant each lead to 100% male lethality. It is proposed that MOF has functions in male development beyond H4K16 acetylation (Zhang, 2018).

H4K16A mutation caused a severe sex bias (1:10 male:female) in homozygotes, reminiscent of that resulting from inactivation of the non-coding roX gene (another dosage compensation component) in D. melanogaster. Given that MOF-MSL-mediated H4K16 acetylation is roX-dependent, roX might act by stimulating H4K16 acetylation, directly or indirectly, which merits further exploration (Zhang, 2018).

H4K16A mutation severely depleted GSCs in the ovary, which presumably contributed to the infertility in the mutants. This finding is not surprising, given that MOF is involved in maintaining pluripotency and self-renewal of embryonic stem cells, and mof mutations lead to failure in the reprogramming of stem cells. The H4K16A mutation might additionally compromise follicle-cell development, as suggested by the fact that Chameau, another H4K16 acetyltransferase, regulates the developmental transition of follicle cells into the amplification stages of oogenesis (Zhang, 2018).

H3K27me3 is essential for gene repression involving polycomb-group (PcG) proteins, but it is not clear which other histone residues are also involved. Traditional mosaic cloning analysis has identified H3S28 as one such residue. This method requires the generation of fly mutants with a complex genotype, which is laborious as it involves multistep crosses. The current strategy for mosaic analysis is much faster and simpler, enabling readily screening of mutations of 19 essential histone residues. This study confirmed the previous finding about H3S28 and further demonstrated that H3R26 is also essential for PcG function, thereby validating this strategy (Zhang, 2018).

This study has shown that H3R26 is required for H3K27 trimethylation, which contributes to PcG-mediated gene repression. Additionally, H3R26 might stimulate PRC2 catalytic activity, as suggested by in vitro data showing that human PRC2 catalytic activity is partially dependent on H3R26. H3R26 may also facilitate PcG protein recognition, with the positive side chain of H3R26 contacting the SET domain of the E(z) methyltransferase. Whether H3R26 is modified remains unclear, although H3R26 methylation has been reported in mouse embryos. Further studies are needed to clarify these issues (Zhang, 2018).

Drosophila Prp40 localizes to the histone locus body and regulates gene transcription and development

In eukaryotes, a large amount of histones must be synthesized during the S phase of the cell cycle to package newly synthesized DNA into chromatin. The transcription and 3' end processing of histone pre-mRNA are controlled by the histone locus body (HLB), which is assembled in the H3/H4 promoter. This study identified the Drosophila Prp40 pre-mRNA processing factor (dPrp40) as a novel HLB component. dPrp40 is essential for Drosophila development, with functionally conserved activity in vertebrates and invertebrates. It was observed that dPrp40 is fundamental in endocycling cells, highlighting a role for this factor in mediating replication efficiency in vivo. The depletion of dPrp40 from fly cells inhibited the transcription but not the 3' end processing of histone mRNA. These results establish that dPrp40 is an essential gene for Drosophila development that can localize to the HLB and may participate in histone mRNA biosynthesis (Prieto-Sanchez, 2019).

In eukaryotic cells, the nucleus is compartmentalized and contains several dynamic nonmembrane-bound structures referred to as nuclear bodies or nuclear compartments, which are essential for the correct maintenance of nuclear architecture and the gene-regulatory processes that occur within the nucleus. The study of the constituents and the spatial and dynamic properties of these nuclear bodies is essential for understanding the regulation of gene expression programs, which are critical for cell stability and survival. Given the importance of nuclear bodies in controlling how gene expression is exerted, alterations in the regulation or biosynthesis of these structures can lead to pathological consequences (Prieto-Sanchez, 2019).

Because of the absence of a delineated membrane, the structural integrity of nuclear bodies is mediated by protein-protein and/or protein-RNA interactions. This property and the rapid dynamics of nuclear bodies are consistent with a self-organization model in which the structure of a body is determined by the global interactions among its constituents. Although significant progress has been made regarding the role of these nuclear bodies in gene expression, understanding how they are assembled in the cell is still far from being understood. Many studies have led to two main distinct but not exclusive assembly models. While one model posits that assembly occurs through an ordered, hierarchical process through which constituents are assembled around a primordial scaffolding factor or RNA, the other model considers that self-organization is accomplished randomly without any particular ordered or hierarchical nuclear body assembly. More recently, a third model for nuclear body formation involves intracellular phase separation to promote the assembly of droplets of nuclear protein/RNA has been proposed. This model posits the existence of different nucleoplasmic phases with distinct physical properties through which proteins may transition to gain favorable thermodynamic states so that nuclear body assembly is mediated by this phase transition (Prieto-Sanchez, 2019).

Some nuclear bodies are also associated with specific gene loci, and this association with a specific nuclear function or activity may be important for their formation and function. The histone locus body (HLB) is a chromatin-associated nuclear body that specifically associates with replication-dependent histone gene clusters to coordinate the transcription and 3' end processing of histone pre-mRNA. In Drosophila, the histone gene cluster is composed of ∼100 copies of tandemly arranged histone H1, H2a, H2b, H3 and H4 gene cassettes. Histones play a crucial role in the packaging of DNA into chromatin. Consistent with this role, histone expression is restricted to the early S phase of the cell cycle, which is tightly coupled to DNA synthesis. Defects in histone biosynthesis result in genomic instability, which may promote oncogenesis. Since the initial characterization of the HLB by the Gall laboratory, many factors have been identified as components of this nuclear body. Some of these factors are constitutively present in these nuclear bodies throughout the cell cycle, whereas others are recruited to the HLB only during the S phase when histone transcription is active. The first group of factors includes Multi sex combs (Mxc), FLASH, the U7 snRNP and Mute, whereas general and elongation transcription factors, such as RNA polymerase II (RNAPII), TBP, Spt6 and Myc, and factors regulating histone pre-mRNA processing, such as Symplekin and other proteins, associate with the HLB upon the activation of histone gene transcription. The emerging picture is that the Drosophila HLB assembles through the hierarchical recruitment of components; Mxc and FLASH form the foundational HLB that is detected in the early embryo at cycle 10, and U7 snRNP and Mute are recruited at cycle 11 in the absence of histone mRNA transcription. A sequence located between the histone H3 and H4 genes contains the shared H3 and H4 promoter (hereafter, denoted as the H3/H4 promoter), which is essential for histone gene expression, and is required for the recruitment of Mxc and FLASH. A significant number of proteins are subsequently joined to the HLB in a manner coupled to active histone gene transcription. How the initial interaction of Mxc and FLASH with the histone loci occurs and what the actual composition of a fully formed HLB is remain to be resolved (Prieto-Sanchez, 2019).

Prp40 was initially identified as an essential yeast factor that participates as a scaffold in the early steps of spliceosome complex formation. Prp40 has a characteristic domain organization, with two WW domains in the N-terminus and five FF domains in the C-terminus, which is a structure shared by a relatively small number of proteins. Strikingly, most of these structurally related proteins have been implicated in transcription and splicing regulation. There are two putative mammalian orthologs of Prp40, PRPF40A and PRPF40B. Based on phylogenomic data, PRPF40A appears to be more closely related to Prp40 than does PRPF40B, which emerged much later in evolutionary history probably due to a gene duplication event from an ancestral PRPF40A. PRPF40A and PRPF40B interact with the transcription and splicing machineries, and at least for PRPF40B, the modulation of alternative splice site selection in apoptosis-related genes has been shown. The Drosophila ortholog of Prp40, herein denoted dPrp40, encoded by the CG3542 gene, shares 23% and 41% sequence identity with the yeast Prp40 and the human PRPF40A proteins, which suggests that the function of these proteins in forming bridges between the 5' and 3' splice sites in the first spliceosomal complex might be conserved. In fact, the regulation of alternative pre-mRNA splicing of the glial-specific cell-adhesion molecule Neurexin IV by dPrp40 has been reported (Prieto-Sanchez, 2019 and references therein).

This study characterize dPrp40 and identifies a putative new role for this protein in histone mRNA transcription. dPrp40 localizes to the Drosophila HLB during prophase after the incorporation of the HLB primary protein components. dPrp40 is essential for Drosophila development. Moreover, dPrp40 and its human orthologs can rescue the phenotype of dPrp40 mutant flies, demonstrating a functional conservation of eukaryotic Prp40 activities in vivo. An essential requirement is shown for for dPrp40 in endocycling cells, highlighting a role for this factor in the replication efficiency in vivo. In a molecular context, this study shows that the depletion of dPrp40 from fly cells inhibits histone mRNA transcription without affecting the 3' maturation of histone mRNA. Furthermore, H3/H4-dependent transcription, which is essential for HLB assembly and high-level histone gene expression, is rescued by overexpressing dPrp40 in the depleted cells. Together, these results establish that dPrp40 is required for normal embryonic development and that dPrp40 can localize to the HLB and might regulate histone gene transcription, which could have important consequences for the cell cycle and maturation, development and viability (Prieto-Sanchez, 2019).

This study performed experiments to characterize the function of Prp40 (dPrp40) in Drosophila. dPrp40 was shown to be essential for Drosophila viability and development via siRNA-mediated depletion and single P element-mediated gene disruption approaches. Conditional knockdown of dPrp40 using different drivers resulted in abnormal phenotypes and increased apoptotic cells in certain regions of wing imaginal discs. These abnormal phenotypes were rescued by expression of the human orthologs of Prp40, indicating that the fly and human proteins have shared functions that affect cell viability. In agreement with this result, it was found previously that PRPF40B depletion increased both the number of Fas/CD95 receptors and cell apoptosis in mammalian cells, thus suggesting a role for this protein in programmed cell death. The pleiotropic effects caused by the lack of dPrp40 expression, however, may indicate that dPrp40 also regulates other genes. In fact, transgenic PRPF40A expression resulted in a faint Notch-like phenotype with wing margin 'notches', which may suggest either an effect of the overexpressed protein on Notch function or an involvement of dPrp40 in the Notch signaling pathway. Recently a transcriptome analysis of dPrp40 fly mutants was performed, and the preliminary results support the involvement of dPrp40 in the Notch signaling pathway. Further study is required to determine the molecular targets and signaling pathways regulated by dPrp40 (Prieto-Sanchez, 2019).

This study also identified a putative function for dPrp40 in the regulation of histone gene transcription. The localization of dPrp40 to the HLB pointed to a possible role of dPrp40 in the regulation of histone gene expression. This hypothesis is supported by the observation that dPrp40 loss-of-function mutants exhibited altered S phase progression and decreased histone gene mRNA expression. The 3' end processing of pre-mRNAs plays an important role in the regulation of histone mRNAs, and HLB components are required for the 3' end maturation of histone mRNAs. The results showed that dPrp40 depletion in Drosophila cells does not result in the polyadenylation of histone mRNAs, indicating that dPrp40 is not required for the 3' end processing of histone pre-mRNAs in vivo. These experiments, however, suggest that dPrp40 regulates histone mRNA expression by modulating transcription. An effect of dPrp40 on transcription synthesis is favored based on data using histone promoters and ChIP analysis. A possible effect of dPrp40 on RNA stability remains to be studied. The regulation of histone gene expression at the level of transcription by dPrp40 was an unexpected finding. The only function described for Prp40 in Drosophila is the regulation of alternative pre-mRNA splicing of the glial-specific cell adhesion molecule Neurexin IV This role of dPrp40 in the splicing process agrees with the proposed role for yeast Prp40 in the early steps of spliceosome formation and with previous data in Drosophila supporting a role for the mammalian Prp40 ortholog PRPF40B in pre-mRNA splicing. Other data challenge this view and suggest alternative mechanisms of action, including a role for this protein in the later steps of spliceosome assembly and in transcriptional regulation, which would be consistent with the unexpected data suggesting a role for dPrp40 in histone mRNA transcription. However, the possibility that dPrp40 is regulating other cellular processes and causing phenotypic defects by modulating the pre-mRNA alternative splicing of important Drosophila genes cannot be excluded. In fact, the FF domains that are critical for dPrp40 function are responsible for binding to Luc7 and Snu71, two proteins within the U1 snRNP complex. The effect of dPrp40 on splicing, however, seems not to be prominent in Drosophila tissues, according to the results of the current genome-wide analysis of transcript- and exon-level changes in siRNA flies. Further studies will be required to fully characterize the function of the dPrp40 protein in mRNA synthesis and processing (Prieto-Sanchez, 2019).

The data demonstrate that dPrp40 depletion results in growth defects and that dPrp40 localizes to the HLB and regulates histone gene transcription. Although it is tempting to link the phenotypic defects resulting from dPrp40 loss of function with histone gene expression, it is believed that dPrp40 may regulate cell growth and proliferation by mechanism(s) other than the regulation of histone genes. The current data support this notion. First, this study showed that dPrp40 associates with the HLB during interphase and early mitosis. Despite the colocalization of dPrp40 with MPM2, which is associated with the HLB only during S phase when the bulk of histone protein synthesis occurs, dPrp40 staining was also detected during the starting phase of cell division, when DNA replication is over. These data are consistent with dPrp40 being present in the HLB throughout interphase and early mitosis and therefore disengaged from the activation of histone gene transcription. The small increase in dPrp40 binding at the promoter sequences in cells in late S phase compared to at the G1/S transition is not in disagreement with this hypothesis. Second, whereas the expression of the ΔWWdPrp40 construct resulted in the deficient accumulation of dPrp40 in the HLB, indicating a less-important role of the FF domains in the localization of dPrp40 to this nuclear compartment, the FF domains of dPrp40 were essential for rescuing the phenotype resulting from the loss of dPrp40 and were also important in the activation of the histone H3/H4 promoter. Therefore, and in the absence of convincing evidence of dPrp40 having a direct role in histone mRNA metabolism, these observations suggest that the growth defects resulting from dPrp40 loss of function were not linked to the localization of dPrp40 at the HLB and the regulation of histone gene expression (Prieto-Sanchez, 2019).

An interesting question that arises from this study regards the means by which dPrp40 might be targeted to the HLB. Seminal work by Duronio provided evidence for an ordered process in Drosophila HLB assembly. Mxc and FLASH are first recruited to the HLB, whereas the other components, including U7 snRNP, Mute and other transcription and mRNA factors, are subsequently recruited in a histone gene transcription-dependent fashion. Because of the reported association of the WW and FF domains of Prp40 with the phosphorylated C-terminal domain (phospho-CTD) of RNAPII, an exciting possibility is that dPrp40 might be recruited to the HLB via a mechanism involving the phospho-CTD. Importantly, phosphorylated RNAPII is highly associated with the HLB during the S phase, when histone mRNA transcription activation occurs. Several other interpretations are also possible. Interactions among HLB components are necessary for the ordered recruitment of additional HLB factors. For example, the C-terminal region of FLASH is necessary for the recruitment of U7 snRNP to the HLB. Similarly, dPRP40 might be recruited to the HLB complex through interactions of its WW domains with other components of the complex. Another mechanism potentially collaborating in the formation of the HLB complex involves phosphorylation by Cyclin E-Cdk2, which is essential for histone mRNA expression. Although Mxc is a target of this kinase, Mxc localization to the HLB does not require Cyclin E-Cdk2 activity. The Spt6 HLB component is specifically immunoprecipitated by the phosphoprotein epitope-specific MPM2 monoclonal antibody, and phosphate treatment of the extract disrupts the interaction of Spt6 with the HLB complex, thus suggesting a role of Cyclin E-Cdk2 activity in Spt6 localization to the HLB (White, 2011). Because of the cyclin-dependent kinase consensus motif at position 739 of dPrp40, assessing the localization of dPrp40 to the HLB with respect to Cyclin E-Cdk2 activity would be informative (Prieto-Sanchez, 2019).

In summary, this study has characterized the function of Prp40 in Drosophila and has identified dPrp40 as a new component of the HLB. dPrp40 was also shown to be required for normal embryonic development and might participate in histone mRNA biosynthesis. Further study of dPrp40 will clearly be useful to define the detailed mechanism of its function (Prieto-Sanchez, 2019).

Intergenerationally maintained histone H4 lysine 16 acetylation is instructive for future gene activation

Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. This study shows that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation (Samata, 2020).

The fusion of the maternal and paternal gametes triggers a remarkable transition from two fully differentiated cells to a totipotent zygote that gives rise to all tissues during embryogenesis. In flies, the development of the embryo during the first 13 synchronized nuclear divisions relies on maternally provided proteins and transcripts. These maternal elements are replaced by newly synthesized ones during the major wave of zygotic genome activation (ZGA) at the nuclear cycle (nc) 14 at embryonic stage (st) 5 when the zygotic genome has reformed to accommodate the transcriptional active status. Increase in nucleosome accessibility as well as gradual enrichment of RNA Polymerase II (RNA Pol II) are observed from nc11. The repressive mark H3K27me3 is inherited from the maternal germline restricting the activation of developmental genes, but most of the other acetyl and methyl marks only become prominent genome-wide at ZGA. The few transcripts that are activated before ZGA (during the minor zygotic wave) are under the control of the pioneer transcription factor Zelda, which mediates local chromatin accessibility. However, the mechanisms that guide the reprogramming of the entire genome are not fully understood (Samata, 2020).

Acetylation of histone tails is known to promote transcriptional activation. Among the histone modifications positively correlated with transcription activation, H4K16ac is unique because it prevents chromatin compaction in vitro. However, the developmental dynamics and the biological significance of this modification in the embryonic genome prior to ZGA remain unclear. H4K16ac is deposited by the histone acetyltransferase (HAT) males absent on the first (MOF). The MOF-containing male-specific lethal (MSL) complex is responsible for chromosome-wide upregulation of the male X chromosome to equalize its expression to the female X as well as to autosomal genes. The complex consists of five proteins (MSL1, MSL2, MSL3, MOF, and MLE) together with two long non-coding RNAs, RNA on the X 1 and 2 (roX1, roX2), and is capable of specifically recognizing the single male X chromosome. Interestingly, all MSL proteins, apart from MSL2, are maternally deposited as transcripts and proteins, which remain stable through the early embryonic stages. However, it has not been determined whether they form a functional complex (Samata, 2020).

By analyzing precisely staged Drosophila embryos before and after ZGA and by performing genetic and genomic experiments, this study shows that H4K16ac is intergenerationally transmitted from the female germline and has a fundamental role in controlling chromatin accessibility in the absence of ongoing transcription during early embryogenesis. Furthermore, it poises promoters for future gene activation (Samata, 2020).

MOF represents the major enzyme catalyzing H4K16ac. It is proposed that maternally provided MOF plays a dual role in 'depositing' and 'maintaining' H4K16ac. First, maternal MOF establishes H4K16ac in the maturing oocyte. Following fertilization, MOF exploits its unique ability to remain associated with mitotic chromosomes in order to actively propagate H4K16ac, hence acting as the maintenance factor for H4K16ac during the first and subsequent embryonic divisions. Continuous presence of MOF is essential because histone acetylation marks typically exhibit fast turnover. Thus, the proposed intergenerational H4K16ac transmission model is distinct from the mechanisms of inheritance of methylation marks, many of which rely on different catalytic modes for de novo deposition and propagation (Samata, 2020).

The combination of maternal deposition and early embryonic maintenance of the H4K16ac information is critical for marking genes prior to ZGA for future activation. Absence of this information leads to misregulation of H4K16ac targets and subsequently to increased embryonic lethality. Other acetyl histone marks were not able to restore proper gene expression in the absence of MOF, demonstrating a specific requirement for H4K16ac in oocytes. Furthermore, expression of the 'maintaining' (zygotic) MOF upon ZGA cannot compensate for loss of the 'depositing' (maternal) MOF function. It will be interesting to characterize the specificities of maternal and zygotic MOF and further explore whether genome structure, developmental timing, or other determinants affect the H4K16ac deposition pattern (Samata, 2020).

Maternally deposited H4K16ac primes a subset of genes for subsequent transcriptional activation upon the onset of ZGA and later in development. The transcription factor Zelda is responsible for activation of the first zygotic transcripts. However, neither Zelda-mediated transcription nor the chromatin marks around these Zelda-dependent regions explain the global emergence of chromatin accessibility in early embryos. The current data indicate that the establishment of H4K16ac-mediated nucleosome accessibility on numerous Zelda-independent promoters before ZGA creates a permissive chromatin state that enhances RNA Pol II recruitment and facilitates gene expression activation (Samata, 2020).

Maternal depletion of MOF also led to profound chromatin architecture changes. Even though the structure of TAD boundaries remained unaffected, substantial defects were observed in compartmentalization during ZGA. Analysis of Hi-C datasets from Drosophila st5 embryos whose transcription was abrogated by drug treatment (Hug, 2017) revealed similar phenotypes to those observed in embryos after maternal H4K16ac loss. However, the compartmentalization defects in maternal mof RNAi offspring were apparent only in early and not late embryos, despite persistent transcription misregulation at both stages. Thus, an aberrant transcriptional program is not the primary driving force for the genome compartmentalization defects observed upon H4K16ac loss early on. It is concluded that although maternal H4K16ac contributes toward establishing global genome organization early on, other factors can compensate for this loss at later embryonic stages (Samata, 2020).

The 'future' dosage compensated genes on the X chromosome are among the numerous targets that show H4K16ac signal prior to the onset of their transcription. By characterizing the developmental dynamics of H4K16ac, this study describes the sequence of events that leads to establishment of dosage compensation on the male X chromosome. H4K16ac decorates all chromosomes prior to ZGA but becomes strongly enriched on the male X chromosome during later stages. It is proposed that initiation of dosage compensation at both X-linked genes and high-affinity sites (HASs) relies on the instructive H4K16ac signal from the mother. Without maternal H4K16ac, MSL complex targeting is compromised in males and the mature dosage-compensated phase cannot be reached. Thus, the X chromosome serves as a readout of H4K16ac memory on a chromosomal scale (Samata, 2020).

H4K16ac is deposited in oocytes by a maternal MSL sub-complex composed of MSL1, MOF, and MSL3. This first step prepares the chromatin landscape for establishment of nucleosome accessibility and dosage compensation initiation in a sex-independent manner. Assembly of the canonical MSL complex requires the expression of the male-specific protein MSL2, whose expression starts at stage 5. MSL2 targets the X chromosome at ZGA and together with MOF mediates transcriptional activation of genes close to HASs in a male-specific manner. Interestingly though, no MSL complex 'spreading' in the vicinity of HASs is observed at this stage. Because X-chromosome territory formation coincides with the expression of the roX2 long non-coding RNA, it is possible that efficient MSL complex spreading is mediated by the contribution of roX2/MSL interactions. Thus, the MSL complex targeting and spreading on the X chromosome represent two temporally discrete steps with distinct requirements. Moreover, the three-dimensional organization of HASs may function as an additional stabilizing factor for X-territory maturation. Indeed, interactions that were observed between HASs were more abundant in stage 15 compared to stage 5 embryos, possibly because of the stronger chromatin compartmentalization in late embryos . It is therefore possible that maturation of the active compartment is required for the stronger clustering of HASs (Samata, 2020).

Although the dosage compensation defects represent a clear readout of the importance of maternal H4K16ac, the influence of this early mark is not restricted to male progeny. Furthermore, this study found the H4K16ac marking of the oocyte to be evolutionarily conserved in three Drosophila species (Drosophila melanogaster, D. virilis, and D. busckii) as well as in mammals. Given that mammals have a different dosage compensation mechanism, retention of H4K16ac in the early mammalian zygote likely indicates the importance of this histone modification in embryogenesis (Samata, 2020).

Maternal inheritance of H3K27me3 mediates gene silencing in both Drosophila and mammals. DNA methylation, H3K4me3, and H3K36me3 mediate zygotic genome activation in other organisms. However, these modifications are absent from the young Drosophila zygotes. A variety of mechanisms have thus evolved to propagate instructions to the next generation via histone modifications in the germline. Future work will elucidate the function of H4K16ac early presence in mice and human (Samata, 2020).

Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions

This study reports that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, a mutant H3T3A was expressed to compromise asymmetric histone inheritance. Under this condition, an increase was observed in Delta-symmetric cell pairs and overpopulated ISC-like, Delta-positive cells. Single-cell RNA-seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, these results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells (Zion, 2023).

In multicellular organisms, asymmetric cell division (ACD) of adult stem cells serves as an important mechanism for tissue homeostasis and regeneration. Disruption of this precisely regulated cell division mode can result in the dysregulation of stem cells, leading to cancer or tissue degeneration. Epigenetic mechanisms enable different cell types within a multicellular organism to establish distinct cellular identities while carrying the identical genetic information. Canonical histone proteins H3, H4, H2A, and H2B are synthesized and incorporated into DNA during replication as an octamer structure, forming the fundamental unit of chromatin, the nucleosome. It is well known that nucleosomes and chromatin structure can affect cell fate decisions; however, it remains largely unclear how epigenetic information is retained or altered during cell divisions to produce cells with different identities in multicellular organisms. Previous studies have shown that H3 and H4 histones are asymmetrically inherited during ACD of the Drosophila male germline stem cells (GSCs), where preexisting (old) histones are retained in the self‐renewed stem cell, while newly synthesized (new) histones are enriched in the differentiating daughter cell. In contrast, old and new H2A and H2B are inherited more symmetrically during ACD of male GSCs. It is hypothesized that the old H3 and H4 histones retain an epigenetic memory that is inherited by the self‐renewed stem cell, while the newly synthesized histones lacking this information can be used to establish a new gene expression program in the differentiating cell. Complementary to this hypothesis, previous studies have reported differences in post‐translational modifications between preexisting and newly synthesized histones. Additionally, it has been shown that nucleosomal density displays differences between old and new histone‐enriched sister chromatids, with the old histone side having higher overall nucleosomes than the new histone side. Nucleosome density and position have profound impacts on many cellular processes by modulating DNA accessibility to different regulators, including pioneer factors, transcription factors, and cell cycle regulators. One functional readout of the inheritance of asymmetric chromatin statuses in GSC divisions is the asymmetric recruitment of the DNA replication component Cdc6, which allows asynchronous cell cycle progression in the resulting daughter cells. Furthermore, when asymmetric H3 segregation is disrupted, progenitor germline tumors and germ cell loss phenotypes are both detected, suggesting that this process is required for both stem cell maintenance and proper germline differentiation. Recently, a new study revealed that proper interactions between homologous chromosomes at a critical 'stemness' gene, stat92E locus, depend on asymmetric H3 inheritance, without which stat92E gene expression becomes misregulated. The findings of asymmetric histone inheritance in Drosophila male GSCs set a precedent in studying epigenetic inheritance modes in multicellular organisms. The question remains, however, of whether this phenomenon is germ cell‐specific or if it serves as a more general mechanism. It also remains unclear whether asymmetrically inherited histones aid in defining distinct cell fates at a single‐cell level. Addressing these questions will not only greatly enhance our current understanding of how epigenetic inheritance modes dictate cell fates, but it will also help establish new methods to identify bona fide stem cells and asymmetrically dividing cells in vivo (Zion, 2023).

To investigate the generality of asymmetric histone inheritance, this study use the Drosophila intestinal stem cells (ISCs) in the midgut as a model system. One feature of ISCs is that they can alternate between ACD, which produces a self‐renewed ISC and a differentiating daughter of either an enteroblast (EB) or a pre‐enteroendocrine (pre‐ee) cell, and symmetric cell division (SCD), which results in two self‐renewed ISCs. As the Notch (N) signaling pathway is critical for cellular differentiation in the ISC lineage, the expression of Delta can be used as an ISC‐enriched cellular marker. It is proposed that the ISC lineage is a great system to study histone inheritance due to its well‐characterized lineage, clearly distinguishable ISC‐specific mitosis, and abundant ISCs in vivo. Indeed, previous studies have shown asymmetric inheritance of old versus new centromere‐specific histone H3 variant CENP‐A (i.e., CID in Drosophila) in the ISCs. Using this system this study reports that asymmetric canonical histone inheritance applies to this somatic stem cell lineage during ACD of ISCs and that misregulation of this process leads to midgut hyperplasia with ISC‐like cells. Collectively, these results demonstrate that asymmetric histone inheritance could be a more general feature for asymmetric stem cell divisions. This study also offers insight into how histone inheritance could influence the establishment or maintenance of cell identities and how misinheritance could lead to diseases such as cancer (Zion, 2023).

To study histone distribution and inheritance patterns during ISC divisions, a dual‐color histone labeling and tracking system was optimized in the ISCs, similar to what has been previously used. In this study, the expression of labeled histones was driven by a cell type‐specific escargot‐Gal4 (esg‐Gal4) to turn on the UAS‐histone transgene in the ISC lineage. After a heat‐shock‐induced switch from eGFP to mCherry‐labeled histone expression, ISCs were allowed to undergo at least a complete round of DNA replication after an approximately 18‐h recovery time, shown by robust incorporation of new replication‐dependent canonical histones genome‐wide. This time frame was determined through a time course experiment, where the expression and incorporation of new H3‐mCherry began around 12 h after heat shock. By 24 h, robust signals of both H3‐eGFP and H3‐mCherry could be detected. However, by 36 h old H3‐eGFP signals became almost undetectable. This time lapse required for new H3 incorporation is consistent with the replication‐dependent incorporation mode for canonical histones. Contrastingly, the incorporation of new histone variant H3.3‐mCherry which is independent of DNA replication showed robust signals 12 h after heat shock. Quantification showed minimal flipped‐out without heat shock but efficient flipped‐out at 18 h after heat shock, which is the time point at which most imaging‐based analysis was performed. These results are consistent with previous data using GFP‐tagged H3 versus GFP‐tagged H3.3 that show distinct incorporation modes between DNA replication‐dependent incorporation of the canonical H3 and DNA replication‐independent incorporation of the H3.3 histone variant in Drosophila. The ISCs co‐labeled with both eGFP (old histones) and mCherry (new histones) signals entered the subsequent mitosis, when sister chromatids are condensed and segregated equally into the daughter cells. The mitotic ISCs can be labeled using a mitotically enriched H3S10ph mark (phosphorylation at Serine 10 of H3). Using this mitotic mark, esg driver‐labeled ISCs can be distinguished in this histone tracer assay. Old versus new histone distribution patterns were studied at different mitotic stages of ISCs (Zion, 2023).

A long‐standing biological question is how distinct cell fates are established, maintained, and changed by epigenetic mechanisms at the single‐cell level in multicellular organisms, where cells have identical genomes. In this study, using the Drosophila ISC lineage as a model system, the inheritance of different canonical histones H3, H4, and H2A was studied during the ISC divisions. In postmitotic pairs of cells with an asymmetric Delta expression pattern, asymmetric histone inheritance patterns are detected, where the Delta‐high cell inherits more old histones and the Delta‐low cell inherits more new histones. In contrast, histones are distributed more symmetrically between the postmitotic pair of cells with similar Delta expression. It was hypothesized that the observed differences in Delta expression patterns are indicative of distinct cell identities resulting from different cell division modes. An asymmetric Delta expression pattern, with one Delta‐high cell and one Delta‐low cell, may indicate that these two cells result from an asymmetric ISC division, where the Delta‐high cell will maintain an ISC identity while the Delta‐low cell will take on differentiation. In contrast, a symmetric Delta expression pattern could result from a symmetric ISC division, where both Delta‐expressing cells will maintain the ISC identity. Interestingly, the percentages of asymmetric versus symmetric Delta expression patterns in the postmitotic pairs (79.6% versus 20.4%) are largely consistent with the ratios of asymmetric versus symmetric ISC divisions as reported in other studies, as well as the H3 inheritance modes in the mitotic ISCs observed in this study (75.0% versus 25.0%). These findings link the asymmetric histone inheritance mode with the establishment of distinct cell identities after one cell division. Furthermore, this asymmetric inheritance mode is specific to H3 and to the less extent to H4, as old and new H2A histones are almost always inherited symmetrically. This molecular specificity could be explained by the incorporation of old H3 and H4 into chromatin as a (H3‐H4)2 tetramer, while old H2A and H2B are incorporated as (H2A‐H2B) dimers. Since H3 and H4 carry most of the post‐translational modifications that influence gene expression, the asymmetric inheritance of old versus new (H3‐H4)2 serves as an elegant mechanism for establishing distinct epigenomes in the daughter cells that arise from stem cell ACD, possibly leading to differential gene expression programs and potentially other distinct cellular features (see Model for old (green) and new (red) histone inheritance patterns and their potential roles in the ISC system (Zion, 2023).

The biological significance of the asymmetric histone inheritance is further exemplified when this pattern is disrupted by the H3^T3A histone mutant, where an increase was detected in Delta‐symmetric postmitotic pairs of cells as well as the overpopulated ISC‐like cells. Interestingly, based on intestinal morphology, hyperplasia was observed in the H3T3A mutant more often in the posterior midgut. Previous work in the field has identified the posterior midgut as a region that is more sensitive to tumor formation, acting as a 'tumor hotspot'. In the current experiments, the posterior midgut appears to be more sensitized to the H3T3A mutant, consistent with these findings. These data connect misregulated histone inheritance with potential changes in cell fate determination. Recently, large‐scale analysis identified histone mutations in 3.8% of human tumor samples, a ratio similar to the mutations of known cancer‐associated genes such as BRCA2 and NOTCH1. In particular, mutations at the Thr3 residue of H3 have been found in a variety of human tumor samples, including lung, breast, skin, bladder, and liver cancers. However, the molecular mechanisms underlying these 'oncohistones' are not fully understood. The current findings on the oncohistone H3T3A illuminate how this mutation could lead to the loss of proper epigenetic inheritance and the onset of tumor formation (Zion, 2023).

The asymmetric inheritance mode of histones was first reported during the ACD of male Drosophila GSCs, which opened a new avenue of research; however, many essential questions remained, such as whether this asymmetric histone inheritance mode is specific to germ cells, stem cells, and/or asymmetrically dividing cells. The results reported in this paper provide a solid basis to addressing these questions. Despite significant differences in the niche structure, signaling cascades for regulating stem cell activity, and cellular differentiation pathways between the ISC and male GSC lineages, several key features of asymmetric histone inheritance are common between these two stem cell systems. First, the cellular specificity in the GSC lineage has been demonstrated by asymmetric histone inheritance mode in asymmetrically dividing GSCs but not in symmetrically dividing spermatogonial cells. In the ISC lineage, this cellular specificity is manifested by ISCs displaying an asymmetric histone inheritance mode in Delta‐asymmetric postmitotic pairs but not for Delta‐symmetric postmitotic pairs. Second, this asymmetry has the molecular specificity for H3 and H4 histones in both systems, emphasizing the importance of these two canonical histones in carrying and passing on or resetting an 'epigenetic memory.' Finally, expression of the mutant histone H3T3A abolishes asymmetric histone inheritances in both systems, resulting in stem cell or progenitor cell hyperplasia. Therefore, these results demonstrate that the asymmetric histone inheritance mode is not specific to either germ cells or stem cells, but likely contingent on the asymmetric mode of mitosis with the mission to generate two distinct daughter cells (Zion, 2023).

Zdditionally, asymmetric histone inheritance could occur at a local level, likely at critical genes that regulate stem cell fate or proper cellular differentiation. Similar local asymmetry of histone inheritance was shown in induced asymmetrically dividing mouse embryonic stem cells, with the H3K27me3 as a key histone modification displaying distinct distribution at the differentially expressed genes between the two daughter cells\. It will be intriguing to explore in the future what histone modifications are associated with old versus new histones (Zion, 2023).

Furthermore, asymmetric sister centromere in recognizing sister chromatids during ACD has also been reported in Drosophila male GSCs and female GSCs. Asymmetric inheritance of old versus new centromere‐specific histone variant CENP‐A/CID has also been reported in the Drosophila ISCs. More studies in the future are needed to understand how global (i.e., in fly male GSCs and ISCs) and local (i.e., in fly female GSCs and mESCs) canonical histone asymmetries are established, which is likely by developmentally programmed DNA replication. Furthermore, how canonical histone asymmetries on sister chromatids are recognized and differentially inherited is likely due to the differential attachment of the mitotic spindle to asymmetric sister centromeres. Finally, the distinct distribution of epigenetic information between the two daughter cells derived from ACD probably prepares them for distinct cellular behaviors, which is crucial to development, tissue homeostasis, and regeneration of multicellular organisms (Zion, 2023).

It has been debated whether the two cells resulting from ISC division are intrinsically asymmetric, or only become asymmetric through the extrinsic signaling cues after ISC division. Previous studies demonstrate that intrinsic polarity mechanisms result in the asymmetric distribution and inheritance of Par proteins to the apical daughter cell during ACD of ISCs, in order to promote differentiation. Furthermore, differential Notch activities due to the polarized Par complex induce distinct cellular differentiation pathways. Recent work has also shown that the spindle orientation in ISCs is tightly linked with cell fate, where planar orientation gives rise to two ISCs and angular orientation generates the ISC/EB pair of daughter cells\. Through analyzing different mitotic stages of ISCs, separable old versus new H3 distribution is detectable in prophase and prometaphase ISCs regardless of the ISC division modes, indicating that this asymmetry is likely intrinsically established prior to mitosis. Interestingly, old versus new H3T3A signals are still separable in prophase and prometaphase ISCs, similar to that of wild‐type H3, suggesting that this mutant histone does not interfere with differential histone incorporation before mitosis. However, increased symmetric segregation patterns in anaphase and telophase H3T3A‐expressing ISCs indicate that sister chromatids differentially enriched with old versus new H3T3A signals cannot be properly recognized and segregated. Because flies have two major autosomes (2nd and 3rd chromosomes) in addition to the sex chromosomes, even the randomized segregation of sister chromatids could lead to an asymmetric pattern at a low percentage, as shown previously. However, even though rare asymmetric histone inheritance occurs with randomized segregation pattern, the daughter cell enriched with old histone is still the cell expressing a higher level of Delta, indicative of ISC identity. Together, these findings indicate that ISC cell fate is likely specified by the intrinsic epigenetic information and the polarized extrinsic cues possibly act to ensure their differential segregation pattern. In summary, given the unique features of the ISC system, such as the ability to precisely label each derivative cell in the entire lineage, the large number of ISCs in their endogenous niche, and the sensitivity of ISC activity to environmental changes such as nutrition as well as aging, it will become a new in vivo model system to study the fundamental principles of different histone inheritance modes and relevant biological consequences under physiological and pathological conditions (Zion, 2023).

GENE STRUCTURE

The plasmid cDm500 consists of a 4.8-kb sequence of genes coding for five histone genes, H1, H3, H4, H2a, and H2b repeated in tandem 1.8 times. The five genes are consecutively oriented on alternate strands, and thus each successive gene is transcribed in alternate directions. Three genes (H3, H2A and H1) are transcribed from one DNA strand, and two (H4 and H2B) from the other strand. The reassociation kinetics of this repeat unit indicates that its sequence is repeated approximately 100 times per haploid genome. Virtually all copies of the DNA sequence are located in the region 39DE of salivary gland polytene chromosomes, a region that appears to span most of the 12 chromomeres associated with 39DE. In several species of sea urchin these five genes are likewise tandemly repeated, but all the genes are transcribed in the same direction. The finding that both sea urchin and the fly contain all five genes arranged in such a way, leads to the belief that the five histone genes are linked in species whose descendents subsequently diverged to give rise to Protosomia and Deuterostomia (Lifton, 1977).

PROTEIN STRUCTURE

Structural Domains

By searching the current protein sequence databases using sequences from human and chicken histones H1/H5, H2A, H2B, H3 and H4, a database was constructed of aligned histone protein sequences with statistically significant sequence similarity to the search sequence. In addition, a nucleotide sequence database of the corresponding coding regions for these proteins has been assembled. The region of each of the core histones containing the histone fold motif has been identified in the protein alignments. The database contains >1300 protein and nucleotide sequences. All sequences and alignments in this database are available through the World Wide Web: see Histone fold motif (Baxevanis, 1996).

The histone octamer is a tripartite assembly in which two dimers (H2A-H2B) flank a centrally located tetramer (H3-H4)(H3-H4). The histone octamer appears either as a wedge or as a flat disc. The folded histone chains are elongated rather than globular and are assembled in a characteristic "handshake" motif; that is, rather than assembling like the globular domains of the alpha and beta chains of the hemoglobin dimer, which have small local contacts, the histone chains, by clasping each other, develop an extensive molecular contact interface. The four types of core histone chains have very low sequence homology but share the histone fold, a common motif of tertiary structure. This common motif consists of a long central helix flanked on either side by a loop segment and a shorter helix. This structure suggests a common evolutinary origin for the four core histones. Each histone fold appears to be the result of a tandem duplication that divides it into two similar and contiguous helix-strand helix (HSH) motifs.

The histone fold residues can be classified in one of four ways: surface, self, pair or interface. "Surface" residues are located on the sides of the dimer subunits facing the exterior of the fully assembled octamer and either are exposed to the solvent or interact with DNA. "Self" residues are involved in contacts within one chain. "Pair" residues contribute to the contacts used to establish histone dimers - i.e., between H3 and H4 or between H2A and H2B. "Interface" residues are involved in contacts between the histone dimers - i.e., at the H3-H3 interface or at the H2A-H2B dimer-(H3-H4)2 tetramer interfaces. The histone fold is engaged directly in the formation of the histone dimers and specifies the paired-element motifs that guide the docking of the DNA to the octamer. Since the HSH motif is seen twice per histone and is present in all four core histone classes, it emerges as the basis from which eight classes of successful variations on the original motif have evolved over time. It appears that evolution allows consideable variation in primary structure, but only to the extent that the pattern of the histone fold is preserved. The overall configuation of the fold within the octamer is strictly maintained through evolution by the requirement that three well-separated regions of the fold (docking pads) be spaced so as to interact with the three consecutive turns of the phosphate backbones of a tightly curved double helix (Arents, 1995).

The transcription factor TFIID is a multimeric protein complex containing the TATA box-binding polypeptide (TBP) and TBP-associated factors. The N-terminal regions of dTAFII62 and dTAFII42 have sequence similarities with histones H4 and H3. The histone-homologous regions of dTAFII62 and dTAFII42 form a heteromeric complex both in vitro and in a yeast two-hybrid system. Neither dTAFII62 nor dTAFII42 forms a homomeric complex, in agreement with a nucleosomal histone character. Moreover, circular dichroism measurements show that the heteromeric complex is dominated by alpha-helical secondary structure. These results strongly suggest the existence of a histone-like surface on TFIID (Nakatani, 1996).

A complex of two TFIID TATA box-binding protein-associated factors (TAFIIs) has been observed by X-ray crystallography. The amino-terminal portions of dTAFII42 and dTAFII62 from Drosophila adopt the canonical histone fold, consisting of two short alpha-helices flanking a long central alpha-helix. Like histones H3 and H4, dTAFII42 and dTAFII62 form an intimate heterodimer by extensive hydrophobic contacts between the paired molecules. In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure (Xie, 1996).

Using the yeast two-hybrid system, a human cDNA was isolated that encodes a protein (hp22) interacting with TATA box-binding factor TFIID subunit p80 containing similarity with histone H4. Sequence analysis showed that the open reading frame (ORF) specifies a 161-amino-acid (aa) polypeptide homologous to Drosophila TFIID subunit p22 (dp22). Comparison of the aa sequence of human TFIID subunit p22 (hp22) with that of dp22 reveals that p22 is composed of two distinct regions; the less conserved N-terminal (20% identity) and the highly conserved C-terminal (65% identity). Additionally, the C-terminal region was found to contain similarities with histones H2B and H3. Northern blot analysis shows mRNA corresponding to hp22 is expressed in all tissues examined (Choi, 1996).

date revised: 1 March 2024 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.