InteractiveFly: GeneBrief

enoki mushroom: Biological Overview | References

The piRNA pathway is a highly conserved mechanism to repress transposon activation in the germline in Drosophila and mammals. This pathway starts from transcribing piRNA clusters to generate long piRNA precursors. The majority of piRNA clusters lack conventional promoters, and utilize heterochromatin- and HP1D/Rhino-dependent noncanonical mechanisms for transcription. However, information regarding the transcriptional regulation of piRNA clusters is limited. This study reports that the Drosophila acetyltransferase Enok, which can activate transcription by acetylating H3K23, is critical for piRNA production from 54% of piRNA clusters including 42AB, the major piRNA source. Surprisingly, it was found that Enok not only promotes rhino expression by acetylating H3K23, but also directly enhances transcription of piRNA clusters by facilitating Rhino recruitment. Taken together, this study provides novel insights into the regulation of noncanonical transcription at piRNA clusters and transposon silencing (Tsai, 2021).

In a wide range of organisms, repressing the activation of transposon insertions is essential for maintenance of genome stability. Small RNA-mediated heterochromatin formation plays important roles in silencing transposons in eukaryotic genomes. Mammals and Drosophila utilize the PIWI-interacting RNA (piRNA) pathway to achieve transcriptional and post-transcriptional silencing of transposons in the germline. In Drosophila, the piRNA pathway starts from transcription of the 142 piRNA clusters, usually ranging from 50 to a few hundred kilobases and containing multiple copies of truncated or full-length transposons, which produce long piRNA precursors. The long RNA precursors would then be processed through slicer- and Zucchini (Zuc)-dependent mechanisms into mature 23-29 nt piRNAs that get loaded to the Piwi protein. In addition, another two PIWI-clade Argonaute proteins, Ago3 and Aubergine (Aub), function in the ping-pong cycle to specifically amplify piRNAs against active transposons. Guided by complementary piRNAs, Ago3 and Aub can mediate degradation of transposon transcripts, and the Piwi-piRNA complex can also direct heterochromatin formation at the loci of transposons and piRNA clusters by recruiting epigenetic factors, resulting in effective repression of transposons both transcriptionally and post-transcriptionally (Tsai, 2021).

The Drosophila piRNA clusters can be divided into two classes: uni-strand, which produces piRNAs mainly from one genomic strand, and dual-strand, which produces piRNAs from both genomic strands. Transcription of the uni-strand clusters is proposed to be similar to the canonical transcription of protein-coding genes, as they contain clear promoter structures with enriched H3K4me2 and peaks of RNA polymerase II (Pol II). In contrast, dual-strand clusters lack clear Pol II promoter regions and are enriched for the heterochromatic H3K9me3 mark. Therefore, these clusters undergo noncanonical transcription that utilizes multiple internal initiation sites via heterochromatin- and Rhino (Rhi)-dependent mechanisms (Tsai, 2021).

Rhi is the germline-specific heterochromatin protein 1D (HP1D), and it associates with Deadlock (Del) and Cutoff (Cuff) to form the RDC complex. The RDC complex is recruited to dual-strand clusters by the interaction between H3K9me3 and the chromodomain of Rhi. At dual-strand clusters, the RDC complex licenses and promotes their transcription through four mechanisms. First, Del interacts with the germline-specific paralog of transcription factor IIA (TFIIA)-L, Moonshiner (Moon), and in turn recruits TFIIA and the TATA-box binding protein (TBP)-related factor TRF2 for transcription initiation. Second, the RDC complex has been shown to suppress the splicing of piRNA cluster transcripts, which is proposed to facilitate piRNA production. Third, Cuff recruits the transcription-export (TREX) complex to nascent transcripts to promote efficient transcription at piRNA clusters. Fourth, Cuff interferes with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex, and therefore prevents premature termination during transcription of piRNA precursors. While the positive roles of the RDC complex in noncanonical transcription of piRNA clusters were studied extensively, further transcriptional regulation upstream to the recruitment of this complex to piRNA clusters is still unclear (Tsai, 2021).

The KAT6 acetyltransferases are highly conserved from budding yeast to mammals, and preferentially acetylate histone H3 among the four core histones. The fly KAT6, Enok, has been shown to function as the major acetyltransferase for establishing the H3K23ac mark, which plays activating roles in transcription of genes. H3K23ac has been suggested to destabilize the interaction between H3K27me3 and the chromodomain of Polycomb, and therefore may contribute to transcription activation. In the ovarian germline, Enok is important for the maintenance of germline stem cells, and is required for proper polarization of oocytes by promoting expression of the actin nucleator spir. This study reports a novel role for Enok in the piRNA pathway. Mutating or knocking-down enok in the ovarian germline led to derepression of transposons and reduction in levels of piRNAs produced from a subset of piRNA clusters including the major piRNA source 42AB. Enok binds to and acetylates H3K23 in the 5' region of rhi, and is required for its normal expression levels in the ovary. It was further shown that Enok is also required for proper Rhi recruitment to a subset of piRNA clusters to promote their transcription. Therefore, Enok contributes to proper transposon silencing in the germline by promoting transcription of rhi and piRNA clusters (Tsai, 2021).

This paper reports a novel role for Enok in suppressing the activation of transposons in the germline. Loss of functional Enok in the ovarian germline resulted in activation of 7 transposon families. This amount of activated transposon families in enok mutant ovaries is comparable to the 17 families activated in the rhi mutant. RNA-seq analysis showed a ~75% reduction in the mRNA levels of rhi in enok mutant germline clone ovaries as compared with the WT control. Knocking down enok in the ovarian germline using two different UAS-shRNA-enok constructs also reduced the mRNA levels of rhi as compared with two different control fly lines. In addition, Enok ChIP-seq analysis revealed that Enok is localized to the 5' region of rhi, and the Enok-dependent enrichment of H3K23ac at the 5' end of rhi suggests that the Enok-mediated H3K23ac mark promotes rhi expression, contributing to proper piRNA production. Indeed, enok mutant germline clone ovaries showed decreased levels of piRNAs that mapped to Rhi-dependent source loci (RD-SL). However, not all RD-SL showed decreased piRNA levels in enok mutants. About 20% of the 6426 RD-SL showed reduced piRNA levels in enok mutants. Therefore, the remaining 25% of rhi levels in enok mutant ovaries may be sufficient to support transcription of the RD-SL that were not affected by loss of Enok. More strikingly, knocking down enok in the germline, without affecting the global protein levels of Rhi, reduced Rhi occupancies at Enok-dependent source loci (ED-SL) but not at Enok-independent source loci (EI-SL). This result suggests that Enok regulates Rhi recruitment specifically at ED-SL. The enok and rhi mutants show similar effects on the fold changes in transposon family expression and in antisense piRNAs. However, among the top 24 most highly overexpressed families in rhi, loss of Enok in the germline specifically activates 7 families. This specificity suggests that these 7 families may be more sensitive to reductions in Rhi recruitment to a subset of piRNA source loci. Taken together, Enok may contribute to fine-tuning transcription of piRNA clusters by modulating rhi expression and by regulating Rhi recruitment to Enok-dependent piRNA source loci (Tsai, 2021).

Three genome-wide RNAi screens have been reported before, but two of them were specifically performed in ovarian somatic cells. Knocking down enok in ovarian somatic cells using the tj-Gal4 driver did not activate the soma-dominant transposon, Gtwin, suggesting that Enok may be dispensable for transposon silencing in the soma. In the genome-wide screen in the germline, the enok RNAi construct (KK108400) is a long hairpin RNA. The efficiency of knocking down enok by long hairpin RNAs is lower than by short hairpin RNAs in the germline, even in the presence of additional Dicer-2. It has been shown that knocking down enok weakly activated the blood and Burdock transposons (z-scores of -0.5 and -0.74, respectively). However, this activation effect did not reach the threshold (z-score of -1.5 or lower) applied in the screen. This study used two different short hairpin RNA constructs against enok to deplete Enok in the germline, and therefore it was possible to detect stronger activation of transposons, possibly due to better knockdown efficiencies (Tsai, 2021).

Enok is the major enzyme responsible for the abundant H3K23ac mark. It was previously demonstrated that Enok is localized to the 5' end of its target genes, spir and mael, and promotes their expression by acetylating H3K23. This study further reports rhi and a subset of RD-SL (defined as ED-SL) as novel targets that are transcriptionally regulated by Enok. Intriguingly, while the 5' region of rhi is enriched with Enok and H3K23ac, Enok is not enriched at ED-SL relative to EI-SL. Also, knocking down enok in ovaries reduced the H3K23ac levels at the 5' end of rhi but not at piRNA clusters. These results suggest that Enok facilitates rhi expression by acetylating H3K23, but regulates the transcription of ED-SL through other mechanisms. Notably, knocking down enok in the ovarian germline severely reduced the Rhi occupancy to sites in 42AB, while global protein levels of Rhi and the H3K9me3 levels at 42AB were largely unaffected. Therefore, Enok is likely to promote transcription of ED-SL by regulating Rhi recruitment (Tsai, 2021).

The transcription of dual-strand piRNA clusters utilizes noncanonical heterochromatin-dependent internal initiation. Transcription initiation at these clusters was proposed to take place by the H3K9me3-bound RDC complex recruiting the germline-specific paralog of TFIIA-L. This study shows that Enok is important for both Rhi and Pol II occupancies at a subset of RD-SL (defined as ED-SL), suggesting that Enok can facilitate transcription of these piRNA source loci. As Rhi is highly enriched across the entire 42AB cluster and no Enok peaks were detected within 42AB, Enok is unlikely to regulate the Rhi occupancy at 42AB by directly recruiting it. Also, the Co-IP assay failed to detect interaction between Enok and the overexpressed Rhi in ovaries. Interestingly, the HAT activity of Enok is critical for transcription of 42AB even when rhi is overexpressed. Therefore, it is possible that Enok may play a role in acetylating some factors that are required for Rhi recruitment, or it may have an indirect role in Rhi recruitment by promoting expression of other genes with yet unidentified functions in the piRNA pathway. Notably, while knocking down enok in the germline decreased the RNA levels transcribed from both genomic strands at cl1-A and from the sense strand at cl1-32, RNA levels transcribed from the antisense strand at cl1-32 was not affected by depletion of Enok. Thus, within dual-strand clusters, Enok may regulate the internal initiation in specific regions. Taken together, this study provides novel information regarding noncanonical transcription and transposon silencing in the germline (Tsai, 2021).

Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator

Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the specialization of cell types by maintaining differential gene expression patterns. This study aimed at discovering novel factors that elicit an anti-silencing effect to facilitate trxG-mediated gene activation. This study has developed a cell-based reporter system and performed a genome-wide RNAi screen to discover novel factors involved in trxG-mediated gene regulation in Drosophila. More than 200 genes were discovered affecting the reporter in a manner similar to trxG genes. From the list of top candidates, (Enoki mushroom), a known histone acetyltransferase, was characterized as an important regulator of trxG in Drosophila. Mutants of enok strongly suppressed extra sex comb phenotype of Pc mutants and enhanced homeotic transformations associated with trx mutations. Enok colocalizes with both TRX and PC at chromatin. Moreover, depletion of Enok specifically resulted in an increased enrichment of PC and consequently silencing of trxG targets. This downregulation of trxG targets was also accompanied by a decreased occupancy of RNA-Pol-II in the gene body, correlating with an increased stalling at the transcription start sites of these genes. It is proposed that Enok facilitates trxG-mediated maintenance of gene activation by specifically counteracting PcG-mediated repression. This ex vivo approach led to identification of new trxG candidate genes that warrant further investigation. Presence of chromatin modifiers as well as known members of trxG and their interactors in the genome-wide RNAi screen validated the reverse genetics approach. Genetic and molecular characterization of Enok revealed a hitherto unknown interplay between Enok and PcG/trxG system. It is concluded that histone acetylation by Enok positively impacts the maintenance of trxG-regulated gene activation by inhibiting PRC1-mediated transcriptional repression (Umer, 2019).

This study has developed an ex vivo approach that led to the discovery of several new genes regulating trxG-mediated gene activation. Using a well-characterized bxd-PRE-reporter, comprised of Ubx promoter and enhancers, a cell-based assay was developed, and a genome-wide RNAi screen in Drosophila was performed. Based on the Z scores of trx and ash1 knockdown, a stringent cut-off was defined and more than 200 genes affecting the reporter in a manner similar to trxG genes were identifed. Identification of known members of trxG and their interactors as well as chromatin modifiers in the genome-wide RNAi screen validated the reverse genetics approach and efficacy of the reporter system to discover new regulators of trxG. Moreover, presence of chromatin modifiers like members of TIP60 complex and proteins associated with RNA polymerase II, known to interact with trxG, further substantiates that regulators of gene activation were predominantly identified. Although only a subset of known trxG members were identified in the screen, failure to identify all can be attributed to the highly context-dependent working of PcG/trxG system. Since two specific enhancers of Ubx drive the expression of the reporter, it might be regulated by only a subset of trxG members, which could further explain the failure to identify all members of trxG. Interestingly, some of the top scoring candidates in the screen were also recently found to be a part of the interaction network of GAGA factor, a known trxG member (Umer, 2019).

TrxG-like behavior of Enok was characterized, and its genetic and molecular link with trxG was established. Although Drosophila Enok has previously been shown to interact with PC (Strubbe, 2011) and Ash1 (Kang, 2017), its physiological relevance with PcG/trxG or epigenetic cellular memory remains elusive. The current results demonstrate that enok behaves like a trxG gene, by antagonizing PcG, and is essential for maintaining active gene expression in Drosophila. Appearance of extra sex combs in Pc heterozygous males is a consequence of ectopic activation of homeotic genes which relies upon the trxG. However, depletion of trxG proteins counteracts the reduced dose of PC, restoring normal regulation of homeotic genes and suppressing the extra sex comb phenotype. Strong suppression of extra sex comb phenotype by two different mutants of enok illustrates that it acts as a trxG gene, consequently counteracting repression maintained by PcG. This finding is further supported by the fact that both mutant alleles of enok strongly enhance trx mutant phenotype, which also corroborates with drastic reduction in transcript levels of trxG target genes in embryos lacking functional enok. A significant overlap between Enok and TRX at chromatin further validates the genetic analysis. Since depletion of enok led to increased PC binding and enhanced H2AK118ub1 at trxG targets, it is suggested that Enok may specifically inhibit PRC1 and facilitate anti-silencing activity of trxG. In contrast, no change in enrichment of E(z) and its associated mark, H3K27me3, was observed at TSS of trxG targets in cells with reduced enok, indicating recruitment of PRC1 in a potentially H3K27me3-independent manner. Such PRC2-independent recruitment of PRC1 has also been reported previously (Umer, 2019).

In light of these results, it is proposed that Enok counteracts PRC1-mediated block of transcription, evident in the form of stalled Pol-II at the TSS of pnr and pnt in cells with depleted enok. Molecular interaction of Enok with PRC1 on developmental genes in flies and humans (Kang, 2017) further supports the notion that Enok facilitates trxG by inhibiting PRC1. In mice, MOZ (homolog of Enok) is known to play an antagonistic role to PcG member BMI1 in regulating Hox genes (Sheikh, 2015). In agreement with the finding that PC chromodomain binding to H3K27me3 requires an unmodified H3K23, the data suggest that Enok-mediated H3K23ac inhibits binding of PC to its target genes. It is proposed that in the presence of Enok at active loci, acetylated H3K23 inhibits binding of PRC1 leading to increased transcriptional activity of Pol-II. In contrast, loss of Enok leads to decreased H3K23ac, thus allowing PRC1 binding and consequent stalling of Pol-II at TSS. Since Enok was also found to associate with silent loci (bxd, Dfd, iab-7) and interact with PRC1, it is suggested that Enok is kept in an inactive state on these loci by PC in a manner similar to the inhibitory interaction between PC and CBP. Further molecular and biochemical characterization of this intricate relationship between PcG and Enok will help discover how trxG maintains dynamic gene expression patterns during development (Umer, 2019).

In summary, this study has developed a cell-based assay for an ex vivo genome-wide RNAi screen to identify potential trxG regulators in Drosophila. The RNAi screen led to the discovery of more than 200 genes that perturbed the luciferase-based reporter in a manner similar to known trxG members. This study has also provided evidence that Enok, a top trxG candidate in the screen, contributes to anti-silencing action of trxG by counteracting PcG proteins. It is proposed that H3K23 acetylation by Enok counteracts PcG-mediated suppression by inhibiting PRC1 recruitment, contributing to gene activation. Genetic and molecular evidence obtained suggests that Enok interacts with trxG and as a result with their major developmental regulatory targets, thus providing a possible molecular link through which it could influence epigenetic cell memory (Umer, 2019).

Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila

Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, this study found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. This study confirmed PRC1-Br140 and PRC1-Fs(1)h interactions and identified their genomic binding sites. PRC1-Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. It is proposed that identification of PRC1-Br140 'bivalent complexes' in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. It is further speculated that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during developments (Kang, 2017).

Inappropriate activation and/or repression of gene expression underlies many human diseases, yet the mechanisms that execute transitions in developmental gene expression remain poorly defined. How are genes chosen to be initially active or repressed, and how are transitions in gene activity managed with fidelity? Transcription factors clearly regulate these changes, but how can this regulation occur with such specificity when their consensus binding sites and genomic occupancy appear so promiscuous? Together, proteomic and ChIP-seq analyses suggest a model in which PRC1 and MOZ/MORF function to create a poised regulatory state during development (see Model for the role of bivalent complexes in developmental transitions of transcriptional state). As cells differentiate, bivalent protein complexes may eventually be diminished locally, as most loci resolve into either an active or silent state. It is speculated that the choice of activation may occur via increased acetylation, influenced by nearby transcription factors, and subsequent enrichment of Fs(1)h and TrxG proteins such as Ash1, which was specifically recovered in a Br140 pull-down. A transition toward silencing may involve deacetylation and a decrease in TrxG (Kang, 2017).

The retention of some bivalency after initial transcriptional choices are made in embryogenesis is likely to allow critical reversibility for subsequent gene expression programming. However, if transcriptional state is not dictated strictly by the occupancy of bivalent components, how are these states manifested? It is speculated that local post-translational modifications (PTMs) may be critical for the specification of transcriptional state and for reversibility. For example, the Enok subunit of dMOZ/MORF is known to acetylate H3K23, while this mark is incompatible with Pc chromodomain binding to H3K27me3 on the same histone tail. Interestingly, enrichment of H3K23ac from modENCODE data sets on the set of potentially bivalent genes was not observed, but further analysis will be required to investigate the significance of this finding. Competition between the cognate enzymatic activities within bivalent complexes and their interactors may be central to their ability to act as reversible switches of transcriptional state. Future studies to address this hypothesis will require improved approaches to comprehensive PTM detection as well as in vitro reconstitution of key interactions and biochemical activities of bivalent complexes containing the appropriately modified subunits (Kang, 2017).

The results are consistent with recent studies in which PRC1 is found on active genes in many systems, and PRC1 targeting is largely independent of PRC2 (Kahn, 2016). Most exciting is the likely conservation in zebrafish (Laue, 2008) and mice (Sheikh, 2015), based on the opposing genetic activities of PRC1 and MOZ/MORF complexes in regulation of the Hox genes. The reliance on a universal transducer of transcription factor activity in developmental decisions would be an elegant solution to the problem of widespread binding of sequence-specific regulators, as, in the model (see Model for the role of bivalent complexes in developmental transitions of transcriptional state), only local interactions with preset bivalency will result in functional consequences (Kang, 2017).

Key fundamental questions remain. In particular, how are PRC1 and MOZ/MORF targeted in the first place? PREs are cis-acting regulatory elements that can recruit PRC1 and PRC2 to target genes in Drosophila. PREs lack universal consensus sequences but contain combinations of motifs for many DNA-binding proteins. Therefore, diverse protein-protein interactions with the PcG could be critical for initial binding, as postulated from classical genetics. A speculative alternative is that the 5′ TSSs of developmentally regulated genes may remain epigenetically marked throughout the life cycle of the organism to specify the initial association of bivalent complexes. Both BRD4 and BRPF1 have been identified as 'bookmarking proteins' that may retain vital information throughout the cell cycle, based on their ability to remain at their chromosomal binding sites through mitosis (Dey, 2003; Laue, 2008). Furthermore, Fs(1)h and Enok are essential for oogenesis, and genic acetylation is detected very early in embryogenesis (Li, 2014). Finally, the importance of maternal E(z) suggests that H3K27me3 could be at least part of such an inherited mark for developmental genes (Kang, 2017).

In summary, the results provide evidence for bivalent protein complexes that may correspond to a bivalent transcriptional state in Drosophila embryos and mammalian stem cells. Beyond identification of these intriguing protein interactions in flies, it is speculated that their identity reveals a likely role for acetylation in the resolution of bivalency. It is envisioned that the choice toward activation may be triggered and maintained by cell-specific transcription factors that drive the acetylated state, favoring MOZ/MORF and BRD4 bromodomain-dependent association with chromatin. Cell type decisions may be governed by a constant assessment of the amount of acetylation at each TSS, consistent with the enrichment of deacetylases on even very active genes. Deacetylation would favor loss of bromodomain-acetyl interactions and ultimately the loss of coactivators, leading toward the establishment of a stably silenced state. The ability to regulate genes while only partially resolving bivalent complexes is likely to be critical for reversibility in response to changes in cell type-specific transcription factor expression and binding. It is proposed that regulatory elements possess the intrinsic ability to switch fate dependent on this local balance, with de novo targeting rarely required (Kang, 2017).

The Enok acetyltransferase complex interacts with Elg1 and negatively regulates PCNA unloading to promote the G1/S transition

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and are involved in cell cycle regulation. However, information regarding their roles in regulating cell cycle progression is limited. This study reports the identification of subunits of the Drosophila Enok complex and demonstrates that all subunits are important for its HAT activity. A novel interaction is reported between the Enok complex and the Elg1 proliferating cell nuclear antigen (PCNA)-unloader complex. Depletion of Enok in S2 cells resulted in a G1/S cell cycle block, and this block can be partially relieved by depleting Elg1. Furthermore, depletion of Enok reduced the chromatin-bound levels of PCNA in both S2 cells and early embryos, suggesting that the Enok complex may interact with the Elg1 complex and down-regulate its PCNA-unloading function to promote the G1/S transition. Supporting this hypothesis, depletion of Enok also partially rescued the endoreplication defects in Elg1-depleted nurse cells. Taken together, this study provides novel insights into the roles of KAT6 HATs in cell cycle regulation through modulating PCNA levels on chromatin (Huang, 2016).

This study reports that Enok forms a complex homologous to the human MOZ complex and that all four subunits contribute to its HAT function in vivo. Notably, in addition to stimulating the HAT activity of Enok toward H3K23, Br140 also expanded its substrate specificity to include H3K14 in vitro (Huang, 2014). This result suggests that Br140 plays a role in regulating the enzymatic specificity of the Enok complex, which is consistent with the recent study showing that the human homolog of Br140, BRPF1, switches the substrate specificity of the HBO1 HAT complex to histone H3 (Lalonde et al. 2013). However, although BRPF1 interacts with both MOZ and HBO1 in human cells, the Drosophila homolog of HBO1, Chameau, was not detected in Br140 purification, indicating that Br140 may be an Enok complex-specific component in flies (Huang, 2016).

This study has also revealed a novel physical and functional interaction between the Enok HAT complex and the Elg1 PCNA-unloader complex, suggesting a role for Enok in modulating PCNA levels on chromatin during cell cycle progression. The physical interaction between Enok and Elg1 complexes is also supported by a recent large-scale study on protein-protein interactions. This study reported the interacting partners of 459 Drosophila transcription-related factors, and four subunits of the Elg1 complex (Elg1, Rfc4, Rfc38, and Rfc3) were identified by affinity purification using Br140 as the bait. Interestingly, instead of Elg1, the largest component of the PCNA-loader complex (Rfc1) copurified with the yeast Sas3-containing NuA3 complex using Pdp3 as the bait protein. This difference in interacting partners between the Enok and Sas3 complexes may be one of the reasons that Enok-depleted S2 cells accumulate at the G1 phase but that populations with a ploidy ≥2C (G2/M) accumulate when SAS3 is deleted in gcn5Δ yeast cells. These results also raise the possibility that the roles of KAT6 HATs in regulating PCNA levels on the chromatin may be evolutionarily adapted by switching their interacting partners between different RFC/RFC-like complexes. The human MOZ complex has been implicated in playing a role in DNA replication via interacting with the MCM helicase and has been shown to regulate cell cycle arrest at the G1 phase by promoting p21 expression. Given that MOZ is a critical regulator of proliferation of hematopoietic precursors and is involved in leukemia, it may advance knowledge of hematopoiesis to investigate whether the MOZ complex also interacts with an RFC/RFC-like complex and regulates PCNA loading/unloading (Huang, 2016).

Reducing enok expression levels by dsRNA increased the rate of G2/M progression. While this faster G2/M progression is not dependent on Elg1, an ~40% increase was also detected in the mRNA levels of the Drosophila CDC25 phosphatase that activates the mitotic kinase Cdk1, string (stg), in Enok-depleted cells. As it has been reported previously that Enok plays a positive role in transcriptional activation by acetylating H3K23 (Huang, 2014), Enok may promote transcription of some repressor genes that down-regulate stg expression. Alternatively, Enok might repress transcription at a subset of gene loci, including stg, in a context-dependent manner, and, last, the possibility cannot be excluded that Enok may directly interact with other protein machinery to regulate G2/M progression (Huang, 2016).

Depletion of Enok also resulted in a block at the G1/S transition that is partially dependent on Elg1. This partial Elg1 dependence indicates that Enok has other roles in regulating the G1/S transition. While it is conceivable that Enok may regulate the expression of genes involved in cell cycle regulation, no significant changes were detected in the mRNA levels of Cyclin A, Cyclin B, Cyclin D, Cyclin E, Cyclin G, Cdk2, E2f1, Rbf (Rb), dap (p21/p27), Dp, or Myc in Enok-depleted S2 cells as compared with cells treated with control LacZ dsRNA. Nevertheless, further genome-wide analysis of gene expression levels in Enok-depleted cells may provide more information on the transcriptional roles of Enok in cell cycle regulation (Huang, 2016).

The model proposed in this paper that the Enok complex interacts with Elg1 via Br140 and down-regulates the PCNA-unloading function of Elg1. This hypothesis is supported by the findings that Br140 interacts with Elg1 in vivo and in vitro and that knocking down enok decreased the PCNA levels on chromatin. The Elg1 dependence of the G1/S block in Enok-depleted S2 cells and the genetic interaction between enok and elg1 in germline cells also agree well with the model, further supporting the negative role of the Enok complex in regulating Elg1 activity. Interestingly, small decreases were often observed in the Elg1 protein levels in Enok-depleted S2 cells or germline cells compared with the controls, while the elg1 mRNA levels remained largely unaffected by Enok depletion. This observation suggests that, in addition to regulating the PCNA-unloading function of the Elg1 complex, Enok may be involved in maintaining Elg1 protein levels or that the protein level and the PCNA-unloading activity of Elg1 may be inversely coregulated. Taken together, the physical and functional interactions between Enok and Elg1 provide a novel insight into the mechanisms underlying regulation of cell proliferation by KAT6 HATs (Huang, 2016).

Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. This study demonstrates that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. These results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification (Huang, 2014).

This study reveals a previously unknown transcriptional role for Enok in regulating the polarized localization of Osk during oogenesis through promoting the expression of spir and mael. Spir and Mael are required for the properly polarized MT network in oocytes from stages 8 to 10A. However, protein levels of both decreased at later stages of oogenesis, allowing reorganization of the MT network and fast ooplasmic streaming. The persistent presence of Spir extending into stage 11 led to loss of ooplasmic streaming and resulted in female infertility. These findings suggest that the temporal regulation of spir expression is crucial for oogenesis, and, interestingly, Enok protein levels were also reduced in egg chambers during stages 10-13 compared with stages 1-9. While the stability of Spir or the translation of spir mRNA may also be a target for regulation, the results suggest that Enok is involved in the dynamic modulation of spir transcript. Furthermore, the results demonstrate the importance of Enok for expression of spir and mael in both ovaries and S2 cells, suggesting that Enok may play a similar role in other Spir- or Mael-dependent processes such as heart development (Huang, 2014).

Notably, Mael is also important for the piRNA-mediated silencing of transposons in germline cells. Mutations in genes involved in the piRNA pathway, including aub and armitage (armi), result in axis specification defects in oocytes as well as persistent DNA damage and checkpoint activation in germline cells. The activation of DNA damage signaling is suggested to cause axis specification defects in oocytes, as the disruption of Osk localization in piRNA pathway mutants can be suppressed by mutations in mei-41 or mnk, which encode ATR or checkpoint kinase 2, respectively. However, mutation in mnk cannot suppress the loss of posteriorly localized Osk in the mael mutant oocyte, indicating that the oocyte polarization defect in the mael mutant is independent of DNA damage signaling. Therefore, although the possibility that the piRNA pathway is affected in enok mutants due to down-regulation of mael cannot be excluded, the Osk localization defect in the enok mutant oocyte is likely independent of mei-41 and mnk (Huang, 2014).

In addition to the osk mRNA localization defect, both spir and mael mutants affect dorsal-ventral (D/V) axis formation in oocytes. However, no defects in the D/V patterning were observed in the eggshells of enok mutant germline clone embryos. Interestingly, among the spir mutant alleles that disrupt formation of germ plasm, only strong alleles result in dorsalized eggshells and embryos, while females with weak alleles produce eggs with normal D/V patterning. Since the enok¹ and enok² ovaries still express ~25% of the wild-type levels of spir mRNA, enok mutants may behave like weak spir mutants. Similarly, the ~40% reduction in mael mRNA levels in enok mutants as compared with the wild-type control may not have significant effects on the D/V axis specification (Huang, 2014).

Redundancy in HAT functions has been reported for both Moz and Sas3, the mammalian and yeast homologs of Enok, respectively. In yeast, deletion of either GCN5 (encoding the catalytic subunit of ADA and SAGA HAT complexes) or SAS3 is viable. However, simultaneously deleting GCN5 and SAS3 is lethal due to loss of the HAT activity of the two proteins, suggesting that Gcn5 and Sas3 can compensate for each other in acetylating histone residues. Indeed, while deleting SAS3 alone had no effect on the global levels of H3K9Ac and H3K14Ac, disrupting the HAT activity of Sas3 in the gcn5Δ background greatly reduced the bulk levels of H3K9Ac and H3K14Ac in yeast. Also, mammalian Moz targets H3K9 in vivo and regulates the expression of Hox genes, but the global H3K9Ac levels are not significantly affected in the homozygous Moz mutant, indicating that other HATs have overlapping substrate specificity with Moz. In flies, a previous study had reported that the H3K23Ac levels were reduced 35% in nejire (nej) mutant embryos, which lack functional CBP/p300 . However, knocking down nej by dsRNA in S2 cells severely reduced levels of H3K27Ac but had no obvious effect on global levels of H3K23Ac. This study showed that the global H3K23Ac levels decreased 85% upon enok dsRNA treatment in S2 cells. This study also showed that the H3K23Ac levels are highly dependent on Enok in early and late embryos, larvae, adult follicle cells and nurse cells, and mature oocytes. Therefore, although Nej may also contribute to the acetylation of H3K23, the results indicate that, in contrast to its mammalian and yeast homologs, Enok uniquely functions as the major HAT for establishing the H3K23Ac mark in vivo (Huang, 2014).

The H3K23 residue has been shown to stabilize the interaction between H3K27me3 and the chromodomain of Polycomb. Therefore, acetylation of H3K23 may affect the recognition of H3K27me3 by the Polycomb complex. Another study showed that the plant homeodomain (PHD)-bromodomain of TRIM24, a coactivator for estrogen receptor α in humans, binds to unmodified H3K4 and acetylated H3K23 within the same H3 tail. Also, the levels of H3K23Ac at two ecdysone-inducible genes, Eip74EF and Eip75B, have been shown to correlate with the transcriptional activity of these two genes at the pupal stage, suggesting the involvement of H3K23Ac in ecdysone-induced transcriptional activation. This study further provided evidence for the activating role of the Enok-mediated H3K23Ac mark in transcriptional regulation (Huang, 2014).

In mammals, MOZ functions as a key regulator of hematopoiesis. Interestingly, one of the genes encoding mammalian homologs of Spir, spir-1, is expressed in the fetal liver and adult spleen, indicating the expression of spir-1 in hematopoietic cells. Thus, it will be intriguing to investigate whether the Drosophila Enok-Spir pathway is conserved in mammals and whether Spir-1 functions in hematopoiesis. Taken together, the results demonstrate that Enok functions as an H3K23 acetyltransferase and regulates Osk localization, linking polarization of the oocyte to histone modification (Huang, 2014).

The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche

Maintenance of adult stem cells is largely dependent on the balance between their self-renewal and differentiation. The Drosophila ovarian germline stem cells (GSCs) provide a powerful in vivo system for studying stem cell fate regulation. It has been shown that maintaining the GSC population involves both genetic and epigenetic mechanisms. Although the role of epigenetic regulation in this process is evident, the underlying mechanisms remain to be further explored. This study found that Enoki mushroom (Enok), a Drosophila putative MYST family histone acetyltransferase controls GSC maintenance in the ovary at multiple levels. Removal or knockdown of Enok in the germline causes a GSC maintenance defect. Further studies show that the cell-autonomous role of Enok in maintaining GSCs is not dependent on the BMP/Bam pathway. Interestingly, molecular studies reveal an ectopic expression of Bruno, an RNA binding protein, in the GSCs and their differentiating daughter cells elicited by the germline Enok deficiency. Misexpression of Bruno in GSCs and their immediate descendants results in a GSC loss that can be exacerbated by incorporating one copy of enok mutant allele. These data suggest a role for Bruno in Enok-controlled GSC maintenance. In addition, it was observed that Enok is required for maintaining GSCs non-autonomously, functioning in cap cells. Compromised expression of enok in the niche (cap) cells (CpC) impairs the niche maintenance and BMP signal output, thereby causing defective GSC maintenance. This is the first demonstration that the niche size control requires an epigenetic mechanism. Taken together, studies in this paper provide new insights into the GSC fate regulation (Xin, 2013).

As a Drosophila putative histone acetyltransferase of the MYST family, Enok has been shown to be essential for neuroblast proliferation in the mushroom body (Scott, 2001). This paper presents evidence that Enok is required intrinsically and extrinsically for maintaining GSCs in the ovary. In the case of intrinsic mechanisms, Bruno was identified as an intermediate factor for Enok-controlled GSC maintenance. Molecular and genetic studies revealed that enok mutations in the germline lead to ectopic expression of Bruno in the GSCs, thereby inducing GSC loss probably via promoting cell differentiation. Meanwhile, Enok was also shown a having a non-cell autonomous role in controlling GSC self-renewal through regulating the niche maintenance and niche-derived BMP signaling output. Thus, this study unraveled a novel regulatory mechanism governing the GSC maintenance mediated by a putative epigenetic regulator in Drosophila. Since Moz and Qkf, the mammalian homologs of Enok, are involved in controlling self-renewal of adult stem cells such as hematopoietic and neural stem cells, the new findings in this paper will help to address how the adult stem cell fate regulation occurs in higher organisms (Xin, 2013).

Numerous studies have shown that GSC maintenance in the Drosophila ovary depends on at least three intrinsic machineries: the BMP/Bam pathway, the Nos/Pum complex and the miRNA pathway. The present study observed that Enok in the germline controls GSC self-renewal independently of BMP/Bam pathway. In the meantime, it was found that loss of enok function does not intrinsically alter the expression pattern of either Nos or Pum in the GSCs, and that enok displays no genetic interactions with either nos or pum in GSCs maintenance. Hence, the results exclude the possibility that the Nos/Pum complex is implicated in Enok-controlled GSC maintenance. Intriguingly, the molecular studies identified Bruno as a potential target of Enok involved in the GSC maintenance. Further genetic analyses suggest that increased expression of Bruno in the GSCs mutant for enok contributes to the GSC loss. bruno encodes an RNA-Recognition-Motifs-containing RNA binding protein that targets a number of mRNAs for their translational repression in the ovary and early embryo. Early on, Bruno was shown to function in patterning the embryo along the AP and DV axis by regulating the translation of oskar and gurken mRNA during late oogenesis. Later, it was reported that Bruno plays a pivotal role in CB differentiation and germline cyst formation at early oogenesis via targeting the Sex-lethal (Sxl) gene. This study has defined a novel function for Bruno in mediating the intrinsic requirements of Enok for maintaining GSCs (Xin, 2013).

Misexpression of Bruno in the germline causes a derepression of PGC differentiation in the gonads from the late third instar larvae. This precocious differentiation phenotype further suggests that bruno gain-of-function in the enok mutants promotes GSC differentiation, thereby eliciting a stem cell loss (Xin, 2013).

To better understand how enok mutation-induced ectopic expression of Bruno promotes the GSC differentiation, it is necessary to identify the potential mRNA target(s) of this RNA-binding protein in the GSCs and their immediate descendants that may function as the differentiation-inhibiting factor in this context. Of all known target genes of Bruno, only Sxl is dynamically expressed in early germ cells including GSCs and CBs, and essential for the GSC/CB fate switch. Preliminary data show that the expression pattern of Sxl remains unchanged in the mutant GSC or CB clones homozygous for the enok allele, ruling out a possible role of Sxl in Enok/Bruno-mediated differentiation control process. Given that the Bruno Response Element (BRE) consensus sequences located in the 3'UTR of the target mRNAs is important for Bruno binding, target candidates from the ovarian mRNAs that contain putative BRE sequences will be sought, based on bioinformatics approaches. However, it is noteworthy that Bruno can also regulate the expression of its target mRNA in a BRE-independent manner. Thus, high-throughput screens such as microarray analysis for differentially expressed genes in the enok mutant ovaries may give more clues for unraveling the mystery (Xin, 2013).

It has been shown that mammalian Moz can acetylate histones H3 and H4 at a number of specific lysine residues. In particular, this MYST family histone acetyltransferase is required for H3K9 acetylation at Hox gene clusters, thus for correct body segment patterning in mice. As the Drosophila homolog of Moz, Enok possesses a conserved MYST histone acetyltransferase (HAT) domain, as well as two PHD fingers and a shared N-terminal domain. Previous studies showed that a point mutation in the MYST HAT domain of Enok causes an arrest in neuroblast proliferation of mushroom body as a null allele (Scott, 2001). Combined with the observation in this paper that the same mutation (enok²) gives defective GSC maintenance phenotype, it is proposed that the HAT activity is implicated in Enok's function during the indicated developmental processes. To further test this scenario, studies will attempt to determine whether the expression of Bruno in the early germ cells could be under the epigenetic control of Enok by examining a possible binding of Enok to bruno gene using chromatin immunoprecipitation (ChIP). In this case, high-throughput screens based on a combination of ChIP-seq and microarray analysis may lead to identification of more target genes of Enok that could mediate the GSC fate regulation controlled by this putative epigenetic factor (Xin, 2013).

The GSC niche plays a key role in controlling GSC self-renewal in the ovary. Although the niche regulation itself is less understood, recent studies showed that systemic factors such as insulin signaling control the niche size, and consequently GSC maintenance at adulthood. Specifically, systemic insulin-like signals maintain the cap cell (CpC) population via modulating Notch signaling. The present study provides the first evidence that the niche maintenance also requires a putative epigenetic factor, and that decrease in the CpC number induced by enok knockdown in the niche is attributable to impaired Notch signaling. Thus, identification and functional characterization of the targets of Enok in controlling the niche size would provide more insights towards understanding how the niche is maintained. Given that insulin signaling is required for controlling the normal decline of both CpCs and GSCs in the aging process, and that epigenetic regulation is important for aging stem cells in mammals, it is assumed that Enok-mediated niche maintenance via Notch signaling has implications in both niche and GSC aging. If this is the case, Enok activity in the niche should display an age-dependent decline. Furthermore, increasing Enok activity could significantly attenuate the age-dependent decrease in the number of both CpCs and GSCs (Xin, 2013).

In conclusion this paper shows that Enok controls GSC maintenance in the Drosophila ovary at multiple levels. In the case of a cell-autonomous control of GSC self-renewal, Enok acts in a BMP/ Bam-independent manner. Instead, activation of Bruno expression in the GSCs and their differentiating progeny links enok mutations in the germline to the GSC loss. In parallel, Enok plays a non-autonomous role in maintaining the GSC population via regulating the niche size and niche-derived BMP signal output from cap cells. Collectively, these results reveal a novel mechanism underlying a putative epigenetic factor-controlled GSC fate regulation (Xin, 2013).

Search PubMed for articles about Drosophila Enok

Cho, H. I., Kim, M. S. and Jang, Y. K. (2016). The BRPF2/BRD1-MOZ complex is involved in retinoic acid-induced differentiation of embryonic stem cells. Exp Cell Res 346(1): 30-39. PubMed ID: 27256846

Dey, A., Chitsaz, F., Abbasi, A., Misteli, T. and Ozato, K. (2003). The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc Natl Acad Sci U S A 100(15): 8758-8763. PubMed ID: 12840145

Huang, F., Paulson, A., Dutta, A., Venkatesh, S., Smolle, M., Abmayr, S. M. and Workman, J. L. (2014). Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire. Genes Dev 28: 2750-2763. PubMed ID: 25512562

Huang, F., Saraf, A., Florens, L., Kusch, T., Swanson, S. K., Szerszen, L. T., Li, G., Dutta, A., Washburn, M. P., Abmayr, S. M. and Workman, J. L. (2016). The Enok acetyltransferase complex interacts with Elg1 and negatively regulates PCNA unloading to promote the G1/S transition. Genes Dev 30: 1198-1210. PubMed ID: 27198229

Kang, H., Jung, Y. L., McElroy, K. A., Zee, B. M., Wallace, H. A., Woolnough, J. L., Park, P. J. and Kuroda, M. I. (2017). Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila. Genes Dev 31(19): 1988-2002. PubMed ID: 29070704

Laue, K., Daujat, S., Crump, J. G., Plaster, N., Roehl, H. H., Tubingen Screen, C., Kimmel, C. B., Schneider, R. and Hammerschmidt, M. (2008). The multidomain protein Brpf1 binds histones and is required for Hox gene expression and segmental identity. Development 135(11): 1935-1946. PubMed ID: 18469222

Li, X. Y., Harrison, M. M., Villalta, J. E., Kaplan, T. and Eisen, M. B. (2014). Establishment of regions of genomic activity during the Drosophila maternal to zygotic transition. Elife 3. PubMed ID: 25313869

Scott, E. K., Lee, T. and Luo, L. (2001). enok encodes a Drosophila putative histone acetyltransferase required for mushroom body neuroblast proliferation. Curr. Biol. 11: 99-104. PubMed ID: 11231125

Sheikh, B. N., Downer, N. L., Phipson, B., Vanyai, H. K., Kueh, A. J., McCarthy, D. J., Smyth, G. K., Thomas, T. and Voss, A. K. (2015). MOZ and BMI1 play opposing roles during Hox gene activation in ES cells and in body segment identity specification in vivo. Proc Natl Acad Sci U S A 112(17): 5437-5442. PubMed ID: 25922517

Strubbe, G., Popp, C., Schmidt, A., Pauli, A., Ringrose, L., Beisel, C. and Paro, R. (2011). Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners. Proc Natl Acad Sci U S A 108(14): 5572-5577. PubMed ID: 21415365

Tsai, S. Y. and Huang, F. (2021). Acetyltransferase Enok regulates transposon silencing and piRNA cluster transcription. PLoS Genet 17(2): e1009349. PubMed ID: 33524038

Umer, Z., Akhtar, J., Khan, M. H. F., Shaheen, N., Haseeb, M. A., Mazhar, K., Mithani, A., Anwar, S. and Tariq, M. (2019). Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator. Epigenetics Chromatin 12(1): 55. PubMed ID: 31547845

Vanyai, H. K., Garnham, A., May, R. E., McRae, H. M., Collin, C., Wilcox, S., Smyth, G. K., Thomas, T. and Voss, A. K. (2019). MOZ directs the distal-less homeobox gene expression program during craniofacial development. Development 146(14). PubMed ID: 31340933

Xin, T., Xuan, T., Tan, J., Li, M., Zhao, G. and Li, M. (2013). The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche. Dev Biol 384: 1-12. PubMed ID: 24120347

date revised: 8 September 2021

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.