BIOLOGICAL OVERVIEW

Dosage compensation is a regulatory process that ensures that males and females have equal amounts of X-chromosome gene products. In Drosophila, this is achieved by a 2-fold enhancement of X-linked gene transcription in males, relative to females. The enhancement of transcription is mediated by the activity of a group of regulatory genes whose protein products form a complex that is preferentially associated with numerous sites on the X chromosome in somatic cells of males but not of females. These regulatory genes are referred to as male-specific lethals because of the male-specific lethality of their loss-of-function alleles. Binding of the dosage compensation complex is correlated with a significant increase in the presence of a specific histone isoform, histone 4 acetylated at Lys16, on this chromosome. The phenotype of male-specific lethality was used to screen the X chromosome of Drosophila melanogaster for ethyl methane sulfonate (EMS)-induced mutations, thereby identifying additional genes that might be involved in the regulatory process of dosage compensation. In one such mutation. males-absent on the first (mof), dying mutant males lack the X-associated isoform of H4Ac16. Mof exhibits the signature motif for the acetyl coenzyme A binding site found in numerous and diverse acetyl transferases. Mof is a histone acetyl transferase (HAT) responsible for the particular histone acetylation involved in the male-specific hypertranscription of X-linked genes (Hilfiker, 1997).

Mutant mof males can develop to the third larval instar or the prepupal stage but fail to metamorphose and to hatch; the viability of mutant females is unaffected. Two lines of evidence establish that this male-specific lethality is due to a defect in dosage compensation. The first involves the effect of the mof mutation on the binding of the other dosage compensation regulatory factors to the X chromosome, as well as the effect of this mutation on the normal consequences that this binding has on nucleosomal structure. The association of Msl-1 and Msl-2 with the X chromosome of mutant mof male larvae is somewhat reduced, while that of Mle is substantially reduced. The X-specific isoform of histone 4 (H4Ac16) appears to be absent. The apparent reduction in the level of Msl-1 and Msl-2 bound to the X chromosome may be the indirect result of the poor cytological condition of the salivary glands of moribund mutant male larvae. In contrast, the effect of the mutation on the level of Mle appears significant. Msl-1 and Msl-2 fail to associate with the X chromosome in male larvae homozygous for loss-of-function mle mutations. Yet, RNase treatment of the male X chromosome removes Mle while leaving Msl-1 and Msl-2 undisturbed. In light of these considerations it may appear that, while Mle is necessary for the initial binding of the dosage compensation complex to the X chromosome in normal males, its association with this chromosome may be stabilized through an interaction with nascent transcripts or with an unidentified RNA component of chromatin. By interfering with the presence of H4Ac16 on the X chromosome and with hypertranscription, the mof mutation may destabilize this interaction (Hilfiker, 1997).

The second line of evidence demonstrating that mof has a functional role in dosage compensation derives from the ability of the mof mutation to prevent the lethality caused by the ectopic expression of a particular dosage compensation regulatory factor in females. Females that carry a transduced msl-2 gene under the control of a heat shock promoter exhibit a very long developmental delay and a significant loss of viability. This is caused by the fact that the presence of Msl-2 is sufficient for the formation of the dosage compensation complex and its association with both X chromosomes, presumably leading to an abnormally high level of X-linked gene products. Normal development is restored in these females by the presence in their genome of mle or msl-3 null alleles in homozygous condition or by the presence of one Msl-1 loss-of-function allele, i.e. of a single dose of the wild-type Msl-1 gene. The same level of rescue is achieved by replacing one wild-type copy of mof with either a deficiency for the locus (L. Rastelli and M. Kuroda, personal communication to Hilfiker, 1997) or with the mof mutation (Hilfiker, 1997).

The presence of Mof homologs in organisms as divergent as yeast and humans suggests that these proteins play an important cellular role, presumably in the modulation of transcription. Such a role, documented with respect to Mof homologs Tip60, SAS2 and SAS3, is strongly indicated with respect to Mof by its involvement in dosage compensation. In Drosophila, because the mof mutation is not lethal in females, the general transcriptional function of the ancestral Mof protein appears to have been appropriated by the dosage compensation mechanism (which is male-specific) and to have been replaced in both males and females by the function of some other factor. The presence of the mof transcript in females, in which the Mof product is dispensable, is not an uncommon occurrence in Drosophila dosage compensation and, therefore, should not be interpreted as evidence of Mof function in this sex (Hilfiker, 1997).

In Drosophila, the hypertranscription of the X chromosome in males appears to be directly correlated with the presence of a particular isoform of histone 4, H4Ac16, that is absent or significantly less abundant on the X chromosomes of females, and on the autosomes of both sexes. This feature of the male X-chromosome chromatin appears to be conserved throughout the genus (Bone, 1996; Steinemann, 1996). In D.melanogaster, the presence of H4Ac16 has been shown to result from the binding to the X chromosome of the complex responsible for the mechanism of dosage compensation (Hilfiker, 1994). The specific function of MOF in this regulatory mechanism is indicated by the presence of an apparent acetyl coenzyme A binding site. The functionality of this site is validated by the observation that replacement of Gly104 by an aspartate residue in the human spermidine/spermine acetyl transferase results in a protein with no measurable activity (Lu, 1996). Significantly, the glycine in question corresponds to Gly691 in MOF that, when replaced by a glutamic acid, leads to the absence of demonstrable H4Ac16 histone isoform on the X chromosome and to the male-lethal phenotype. These considerations have lead to the conclusion that MOF may be directly involved in the acetylation of histone 4 at Lys16 on the X chromosome of Drosophila males. MOF provides a functional link between this known nucleosomal modification and the transcriptional enhancement that is the basis of dosage compensation (Hilfiker, 1997).

Activation of transcription through histone H4 acetylation by Mof, an acetyltransferase essential for dosage compensation in Drosophila

While the link between acetylation of histone H4 at lysine 16 and the dosage compensated male X chromosome points to an involvement of the modification early on, a causal relationship between the two phenomena has been difficult to established. It has been considered possible that the same principle that promotes an increase in accessibility of genes on the male X chromosome to the transcription machinery may also increase the availability of the nucleosome substrate to a ubiquitous acetyltransferase. In this scenario, H4 acetylation would not be causal to the increased expression of X-linked genes in males, but both phenomena would profit from a common, yet unidentified cause. It has now been shown that acetylation of nucleosomes at H4 lysine 16 can lead to a remarkable relief of nucleosomal repression (Akhtar, 2000). Mof has been demonstrate to act is a histone acetyltransferase that acetylates chromatin specifically at histone H4 lysine 16. This acetylation relieves chromatin-mediated repression of transcription in vitro and in vivo if Mof is targeted to a promoter by fusion to a DNA-binding domain. Acetylation of chromatin by MOF, therefore, appears to be causally involved in transcriptional activation during dosage compensation. Dosage compensation in Drosophila therefore presents a strong case for a direct role of H4 acetylation on gene transcription in vivo (Akhtar, 2000).

Although required for dosage compensation in male flies, Mof is expressed in female cells to a similar extent as in males. While this observation raises the interesting issue of whether Mof has unknown functions in flies besides dosage compensation, it also highlights the importance of targeting of Mof specifically to the X chromosome in males. All histone acetyltransferases studied to date reside in large multiprotein complexes in cells. It is assumed that the histone acetyl transferase-associated subunits are involved in the targeting of HAT activity to specific sites of action, notably promoters. Targeting may involve direct interaction with promoter-bound transcription factors as has been shown for yeast ESA1. ESA1 resides in the large NuA4 complex for which an interaction with acidic activators, leading to targeted acetylation of histones, has been shown (Ikeda, 1999). Likewise, Mof is part of a multiprotein complex consisting of the known Msl proteins (Copps, 1998). In the case of dosage compensation that involves decondensation of an entire chromosome (a scenario whereby a key acetyltransferase is targeted to all promoters via factor interactions) appears less likely. Rather, the recent identification of high-affinity nucleation sites from which the dosage compensation complex may spread into the adjacent chromatin (Kelley, 1999) points to a targeting via X chromosome-specific sequence elements. Using indirect immunofluorescence on flies with varying mutant backgrounds, Lucchesi and colleagues (Gu, 1998) recently established a model for the assembly of the dosage compensation complex. According to their model, MSL1 and MSL2 first interact with chromatin in a site-specific manner. Recruitment of MOF to the complex requires prior interaction of the MLE protein (Gu, 1998). A possible scenario may be constructed in analogy to current models of long distance chromatin repression by the polycomb group (PcG) of proteins. It has been hypothesized that the PcG complex is initially targeted to dedicated polycomb response elements (PREs) and that a repressive chromatin structure spreads from there into the neighboring chromatin. The polycomb protein has recently been shown to directly contact nucleosomes via its C-terminal repression domain. It remains to be seen whether the observed interaction of Mof with nucleosomes solely reflects the substrate recognition, that is the interaction of the catalytic site with the histone H4 N-terminal tail, or whether second sites on the nucleosome or Mof are involved. Besides the HAT domain, a domain with similarity to the chromodomain, as well as a zinc finger of the Cys2-His-Cys type, has been noted in Mof (Akhtar, 2000 and references therein).

Hyperacetylation of histone H4 can lead to an increased access of transcription factors to nucleosomal DNA, to an unfolding of the nucleosomal fiber, and to activation of transcription on chromatin templates. However, hyperacetylation is an experimentally induced condition in cells that is not observed under physiological conditons. Histones are mainly monoacetylated in vivo, and the determinant of functional states appears to reside in the site specificity of acetylation rather than the extent of the modification. This has been nicely illustrated by the visualization of histone H4 on Drosophila polytene chromosomes: acetylation of H4 at lysine 16 paints the hyperactive X chromosome, while acetylation at lysine 12 is a hallmark of inactive heterochromatin. Site-specific acetylation may conceivably affect interactions of the histone H4 N terminus with either vicinal nucleosomes or nonhistone proteins. It is likely that these interactions may lead to alterations of the folding of the nucleosomal fiber and degree of chromatin compaction. Interactions of the H4 N-terminal amino acids K16-N25 with the H2A/H2B heterodimer occur in the nucleosome crystal, but it is unclear yet whether these would contribute to the folding of a physiological chromatin fiber. The quest for interacting factors with potential to discriminate between particular histone isoforms is ongoing, with no results reported to date (Akhtar, 2000 and references therein).

While it is clear that acetylation of H4 at lysine 16 suffices to increase transcription of model genes in vitro and in vivo, the mechanism is obscure by which it coordinately affects all genes on the male X chromosome. A change in higher order chromatin folding may affect the assembly of the basal transcription machinery in general or a step subsequent to initiation, such as the elongation rate. However, in vitro transcription reactions are not designed to monitor transcription elongation through nucleosomal arrays but rather to detect short transcripts. Furthermore, the ability of Mof to activate transcription in yeast when tethered to a promoter via a DNA-binding domain suggests that a rather local acetylation may lead to a significant stimulation of transcription. In vitro, recombinant MOF is able to boost transcription far more than just the 2-fold effect that it has in vivo. A precise doubling of X-derived RNA levels in males may hence either involve a feed-back mechanism or additional regulation at a posttranscriptional level, such as RNA stabilization (Akhtar, 2000).

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).

Transcriptional cofactors display specificity for distinct types of core promoters

Transcriptional cofactors (COFs) communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression. Although some COFs have been shown to prefer certain promoter types over others, the extent to which different COFs display intrinsic specificities for distinct promoters is unclear. This study used a high-throughput promoter-activity assay in Drosophila melanogaster S2 cells to screen 23 COFs for their ability to activate 72,000 candidate core promoters (CPs). Differential activation of CPs was observed, indicating distinct regulatory preferences or 'compatibilities' between COFs and specific types of CPs. These functionally distinct CP types are differentially enriched for known sequence elements, such as the TATA box, downstream promoter element (DPE) or TCT motif, and display distinct chromatin properties at endogenous loci. Notably, the CP types differ in their relative abundance of H3K4me3 and H3K4me1 marks, suggesting that these histone modifications might distinguish trans-regulatory factors rather than promoter- versus enhancer-type cis-regulatory elements. The existence was confirmed of distinct COF-CP compatibilities in two additional Drosophila cell lines and in human cells, for which COFs were found that prefer TATA-box or CpG-island promoters, respectively. Distinct compatibilities between COFs and promoters can explain how different enhancers specifically activate distinct sets of genes, alternative promoters within the same genes, and distinct transcription start sites within the same promoter. Thus, COF-promoter compatibilities may underlie distinct transcriptional programs in species as divergent as flies and humans (Haberle, 2019).

To systematically test intrinsic COF-CP preferences for many CPs in a standardized setup, a plasmid-based high-throughput promoter-activity assay and self-transcribing active core promoter-sequencing (STAP-seq) were combined with the specific GAL4 DNA-binding-domain (GAL4-DBD)-mediated recruitment of individual COFs. Using this assay in S2 cells, is this study tested whether 13 different individually tethered D. melanogaster COFs, representing different functional classes and enzymatic activities (two acetyltransferases (P300/CBP and Mof), three H3K4-methyltransferase-complex components (Lpt, Trr and Trx), two chromo and chromo-shadow-domain COFs (Chro and Mof) and three bromodomain COFs (Brd4, Brd8 and Brd9), the mediator complex subunits MED15 and MED25, and two less well-characterized COFs (EMSY and Gfzf) could activate transcription from any of 72,000 CP candidates, 133 base pair (bp) long DNA fragments around a comprehensive genome-wide set of transcription start sites (TSSs) and negative controls. If a tethered COF activates a candidate CP, this generates reporter RNAs with a short 5' sequence tag, derived from the 3' end of the corresponding CP. These reporter transcripts were captured with a 5' RNA linker that includes a 10 nucleotide (nt) long unique molecular identifier (UMI), enabling counting of individual reporter RNA molecules and quantifying of productive transcription initiation events at single-base-pair resolution for all candidate CPs in the library (Haberle, 2019).

Three independent COF-STAP-seq screens for each of the 13 COFs and positive (P65) and negative (GFP) controls in S2 cells were highly similar (all pairwise Pearson's correlation coefficients (PCCs) ≥ 0.89) and showed more initiation events for P65 and the 13 COFs than for GFP, as expected. Initiation mainly occurred at CPs corresponding to annotated gene starts, whereas random negative controls showed the least initiation, corroborating previous findings that gene CPs are specialized sequences, able to strongly respond to activating enhancers (Haberle, 2019).

Each COF showed differential activation of CPs and activated a unique set of CPs. For example, within a representative genomic locus, MED25 and Lpt most strongly activated the CP of CG9782, Chro and Gfzf most strongly activated the CP of RpS19a, and Mof most strongly activated the CPs of mbt and SmG. Indeed, the activation profiles of the COFs across all CPs were characteristically different, as revealed by hierarchical clustering. The differential CP activation by luciferase reporter assays were validated with MED25, Lpt, Mof and Chro for 50 CPs. The two assays agreed well (PCCs ≥ 0.72 except Mof, with PCC = 0.58), and it was confirmed that COFs activate some CPs more strongly than others (MED25, for example, preferentially activates the CPs on the left, Mof preferentially activates those in the middle, and Chro preferentially activates those on the right), which are refered to as distinct preferences, specificities, or 'compatibilities' towards different CPs (Haberle, 2019).

To test whether the COF-CP compatibilities generalize beyond S2 cells, three independent COF-STAP-seq screens were performed for six COFs (MED25, P300, Lpt, Gfzf, Chro and Mof) in two additional D. melanogaster cell lines, one derived from embryos (Kc167) and one from adult ovaries (ovarian somatic cells (OSCs)). For each of the six COFs, the screens were highly similar across all three cell lines (all PCCs ≥ 0.69), validating the distinct CP preferences of the COFs and the observed COF-CP compatibilities. These results establish the observed COF-CP compatibilities as a cell-type-independent, COF- and CP-sequence-intrinsic regulatory principle (Haberle, 2019).

To test whether the COF-CP preferences reflect endogenous gene regulation, the binding was assessed of each COF to genomic CPs of genes expressed in S2 cells. Published chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for P300, Brd4, Trx18, Trr18, Lpt19 and Mof20 from S2 cells, and Chro from D. melanogaster embryos showed stronger COF binding at CPs that were strongly activated in STAP-seq by the respective COF (top 25%) and weaker binding at CPs that were more weakly activated (bottom 25%). Next, the COF-CP preferences were compared with the impact of COF inhibition or depletion on endogenous gene expression. Analyses of published gene expression data upon COF inhibition with small molecules (P300) or RNA interference (RNAi; Brd4 and Trx18) revealed that genes associated with the top 25% of CPs preferentially activated by P300, Brd4 or Trx displayed stronger downregulation upon inhibition of the respective COF, compared to genes associated with the bottom 25% of CPs. Conversely, the CPs of all genes that are downregulated upon inhibition of P300, Brd4 or Trx showed stronger activation by the respective COF in STAP-seq than the CPs of genes not affected by COF inhibition. Together, these results suggest that the distinct COF-CP preferences that were observed are employed during endogenous gene regulation in vivo (Haberle, 2019).

The observed COF-CP compatibilities suggest the existence of distinct CP classes that differentially respond to specific COFs. To address this, K-means clustering was used to define groups of CPs with similar responses. Around 75% of the variance can be explained by five CP groups, which were activated preferentially by: (1) MED25, P300, and strongly by P65; (2) MED25, P300, and weakly by P65; (3) Mof, and weakly by Lpt and Chro; (4) Chro and Gfzf; and (5) Gfzf. Although additional types of CPs are likely to exist in more specialized cell types such as germline cells, screening ten additional COFs—including subunits of prominent COF complexes with diverse enzymatic activities (for example, SAGA, ATAC, NuA4/Tip60 and Enok) and general transcription factors (GTFs; for example, TBP, Trf2 and Taf4)—did not reveal additional CP types in S2 cells, presumably because each of the additional COFs was highly similar to at least one of the original 13 COFs (Haberle, 2019).

Given that COF-STAP-seq measures COF-CP compatibility in an otherwise constant reporter setup, distinct compatibilities are likely to arise from differences in CP sequences. Indeed, the five groups of CPs displayed marked differences in the occurrence of known CP motifs. Group 1 is strongly enriched for the TATA box and a variant of the DPE, whereas group 2 is enriched for a different DPE variant. By contrast, groups 1 and 2 are depleted in motifs Ohler 1, 6 and 7, and in the DNA replication-related element (DRE), all of which are enriched in group 3 and to a lesser extent in group 4. Group 4 is the only group with a strong enrichment for the TCT motif that is known to occur in the promoters of genes encoding ribosomal proteins and other proteins involved in translation, which are indeed among the top 10% of CPs preferentially activated by Chro. In accordance with the differential occurrence of CP motifs, published datasets reveal differential binding of GTFs to these CPs in their endogenous genomic contexts. For instance: the TATA-binding protein (TBP) bound more strongly to group 1 CPs, which are enriched for the TATA box; TAF1 bound more strongly to group 1 and 2 CPs, which are enriched for the Inr motif; and motif 1-binding protein (M1BP) and DRE factor (DREF) bound more strongly to group 3 CPs, which are enriched for motif 1 and DRE. Last, the TBP paralogue TRF2 bound more strongly to group 3, 4 and 5 CPs, consistent with reports that TRF2 regulates ribosomal protein genes. The differences in motif occurrence and GTF binding between the CP groups suggest that COF compatibility might relate to GTF composition at the CP, which is determined by the CP sequence (Haberle, 2019).

The CP groups defined by their COF responsiveness are reminiscent of groups previously defined on the basis of motif content and transcription initiation patterns that differ in chromatin properties, gene function and expression, and enhancer responsiveness. The dataset used in this study might provide a functional link between these observations and the activation of distinct CP types by specific COFs. Indeed, group 1 and 2 CPs are associated with genes that are expressed highly variably across cells in Drosophila embryos and have cell-type-specific or developmental functions, whereas group 3 and 4 CPs are associated with genes that are expressed more uniformly and have housekeeping functions. Furthermore, both the upstream sequences and the nearest enhancers of these CPs were enriched for transcription-factor binding motifs known to occur preferentially in developmental versus housekeeping enhancers, and developmental and housekeeping enhancers indeed preferentially activated group 1 and 2 versus group 3 and 4 CPs, respectively, when tested by STAP-seq. Together, these results directly link enhancer-CP specificity to COF-CP compatibility (Haberle, 2019).

Because COFs can modify nucleosomes and alter the chromatin structure, the endogenous genomic contexts of the five CP groups were tested in S2 cells (only considering CPs of active genes). Nucleosome positioning, DNA accessibility and histone modifications all differed between the CP groups: group 1 and 2 CPs have broader DNA accessible regions around the TSSs and lower nucleosome occupancy and nucleosome phasing downstream of the TSS, compared with group 3 and 4 CPs, which have more narrow nucleosome-depleted regions around the TSS and strongly phased downstream nucleosomes (Haberle, 2019).

Unexpectedly, the CP groups also differed in the methylation status of histone 3 lysine 4 (H3K4). H3K4me3 is thought to be universally associated with active promoters, and indeed strongly marks CPs of groups 3, 4 and 5. By contrast, group 1 and 2 CPs have lower levels of H3K4me3 but higher levels of H3K4me1 compared with group 3, 4 and 5 CPs, a modification that is typically considered an enhancer mark. This difference is consistent with the differential binding of Trr and Set1, which deposit H3K4me1 and H3K4me3, respectively, and does not seem to stem from higher levels of Pol II binding or transcription at group 3, 4 or 5 CPs. Consistent with reports that developmental promoters lack H3K4me3, these results suggest that high levels of H3K4me3 versus H3K4me1 might not be a universal feature of promoters that distinguishes them from enhancers as previously suggested, and instead might depend on the COFs that regulate the respective promoters. Indeed, ranking all active CPs in S2 cells by their H3K4me1:H3K4me3 ratio revealed that those with the highest ratio are preferentially activated by P300 and MED25, and those with the lowest ratio are preferentially activated by Mof or Chro (Haberle, 2019).

To test whether regulatory compatibilities between COFs and CPs exist in other species, proof-of-principle screens were performed in human HCT116 cells for five human COFs (BRD4, MED15, EP300, MLL3 and EMSY) and P65, using a focused library containing 12,000 human CP candidates selected to cover the diversity of human CPs. These screens reveal that CPs also respond differently to different COFs in human cells: whereas the TATA-box-containing CP of REN is, for example, only activated by MED15 and P65, the CpG-island CP of IRAK1 responds most strongly to MLL3; and the tested COFs consistently displayed distinct CP-preferences across the entire CP library. Overall, the CPs most strongly activated by MED15 are enriched for TATA boxes, whereas CPs preferentially activated by MLL3 exhibit a higher GC and CpG content, suggesting that MLL3--but not MED15--preferentially activates CpG-island promoters. Together, this establishes that sequence-encoded COF-CP compatibilities exist in species as divergent as fly and human, suggesting that they constitute a general principle with important implications for transcriptional regulation (Haberle, 2019).

The regulatory compatibilities between COFs and CPs that were observed enable separate transcriptional programs to independently regulate not only different genes, but also alternative promoters and thus different isoforms of the same gene. Notably, composite promoters with differentially activated closely spaced TSSs exist and enable regulation by different COFs and programs, potentially in different developmental contexts. As the CP types differ in sequence elements, these might instruct the assembly of functionally distinct pre-initiation complexes (PICs) that differ in GTF composition or create distinct rate-limiting steps that require activation by different COFs, enabling specific and synergistic regulation. The existence of regulatory COF-CP compatibilities impacts promoter activation and gene expression in endogenous contexts and biotechnological applications and, together with other mechanisms that determine enhancer-promoter targeting in the context of the three-dimensional chromatinized genome, helps to explain how different genes or alternative promoters can be distinctly regulated in species as divergent as flies and humans (Haberle, 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).

GENE STRUCTURE

Exons - 1

PROTEIN STRUCTURE

Amino Acids - 827

Structural Domains

A striking similarity exists between the MOF protein and Tip60, a recently identified human protein that appears to interact with the HIV-1 Tat transactivator. Discovered by means of the yeast two-hybrid selection system, Tip60 has been demonstrated to greatly enhance Tat transactivation of the HIV-1 promoter in transient expression assays (Kamine, 1996). MOF also displays extended amino acid homology to MOZ, the human monocytic leukemia zinc finger protein. The MOZ gene was recently identified as one of the two breakpoint-associated genes in the translocation found in the M4/M5 subtype of acute myeloid leukemia. The chromosome translocation fuses MOZ in-frame to CBP, the CREB transcriptional factor-binding protein (Borrow, 1996). Finally, a significant level of sequence similarity is found between MOF, the SAS2 and SAS3 gene products of Saccharomyces cerevisiae, and other proteins of as yet unidentified function. In yeast, SAS2 is involved in silencing the telomeres; SAS2 and, to a lesser extent SAS3, are also involved in HMR locus silencing (Reifsnyder, 1996). Present within this region of homology, which extends for ~250 amino acids, is a domain common to many acetyl transferases that has been shown to be required for the binding of acetyl coenzyme A. Deduced from its homology to mammalian spermidine/spermine acetyl transferases and microbial antibiotic acetyl transferases, this domain is also found in enzymes known to acetylate histones, such as histone acetyl transferase 1 of yeast, histone acetyl transferase A of Tetrahymena and its yeast homolog Gcn5p, and p300/CBP-associated factor P/CAF. A second domain, identified as a C₂HC/H zinc finger found in a variety of transcription factors and in oncogenes, is present in all of the MOF-related proteins, with the exception of ScYOR244w. An additional region of homology is shared by MOF, Tip60 and the S.cerevisiae YOR244w. This region is very similar to the chromo domain contained within a large number of proteins. With respect to this domain, the highest level of similarity with MOF is exhibited by human retinoblastoma-binding proteins RBP-1 and RBP-2 and the frog XNF7 and newt PwA33 nuclear factors. The latter is a maternal protein associated with nascent transcripts on the lampbrush chromosome loops of the oocyte. The chromo-like domain and the single zinc finger may represent sites of protein-protein interaction, although binding to DNA should not necessarily be ruled out (Hilfiker, 1997).

date revised: 15 May 2000

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