InteractiveFly: GeneBrief

maternal gene required for meiosis: Biological Overview | References |

BIOLOGICAL OVERVIEW

Temporal patterning is a seminal method of expanding neuronal diversity. This study has unravel a mechanism decoding neural stem cell temporal gene expression and transforming it into discrete neuronal fates. This mechanism is characterized by hierarchical gene expression. First, Drosophila neuroblasts express opposing temporal gradients of RNA-binding proteins, Imp and Syp. These proteins promote or inhibit chinmo translation, yielding a descending neuronal gradient. Together, first and second-layer temporal factors define a temporal expression window of BTB-zinc finger nuclear protein, Mamo. The precise temporal induction of Mamo is achieved via both transcriptional and post-transcriptional regulation. Finally, Mamo is essential for the temporally defined, terminal identity of alpha'/beta' mushroom body neurons and identity maintenance. This study describes a straightforward paradigm of temporal fate specification where diverse neuronal fates are defined via integrating multiple layers of gene regulation. The neurodevelopmental roles of orthologous/related mammalian genes suggest a fundamental conservation of this mechanism in brain development (Liu, 2019).

The brain is a complicated organ which not only requires specific connections between neurons to form circuits, but also many neuronal types with variations in morphology, neurotransmitters and receptors. While mechanisms controlling neuronal diversity have not been globally examined, studying neural stem cells in the mouse and fruit fly have given insight into key aspects of neuronal specification. For example, in the mouse neocortex, radial glial progenitors (RGP) are multipotent-they produce a variety of neuron types organized sequentially into six layers, and then produce glia. In vivo lineage analysis demonstrated that after a stage of symmetric cell division, an individual neurogenic RGP produces an average of 8-9 progeny (range of 3-16) that can span all cortical layers. In Drosophila, clonal analysis has demonstrated a vast range of stem cell-specific lineage programs. On one extreme, lineage tracing of a single antennal lobe (AL) stem cell revealed a remarkable series of 40 morphologically distinct neuronal types generated sequentially. In light of these observations, a fundamental goal is to understand how distinct neuronal types correctly differentiate from a single progenitor. Despite a fundamental role for temporal patterning to create diverse neuronal lineages, understanding of neuronal temporal patterning is still limited. While scientists have discovered key temporal factors expressed in neural progenitors, much less is understood about how these signals are interpreted, that is what factors lie downstream of the specification signals to determine distinct neuronal temporal fates (Liu, 2019).

Despite its relatively small brain, Drosophila is leading the charge on studies of neuronal temporal fate specification. Many temporal transcription factors originally discovered in the fly have since been confirmed to have conserved roles in mouse retinal and cortical development. Moreover, temporal expression of an RNA binding protein, IGF-II mRNA-binding protein (Imp), that guides temporal patterning in the postembryonic fly brain is also implicated in mouse brain development. Drosophila brain development is an excellent model for studying neurogenesis; the neural stem cells, called neuroblasts (NB), are fixed in number, their modes of division are well characterized, and each NB produces a distinctive series of neurons which change fate based on birth order. Finally, the fruit fly is a genetically tractable system making it ideal for studying gene networks involved in cell fate decisions (Liu, 2019).

In Drosophila, cycling NBs express age-dependent genes that provide the serially derived newborn neurons with different temporal factors. In the embryonic ventral nerve cord and the optic lobe, the NBs express a rapidly changing series of four to six temporal transcription factors (tTF), some of which are directly inherited by the daughter neurons. Each tTF directly acts to specify a small number (two to four) of neuronal progeny. The neuronal progeny produced from one tTF window to the next can be quite different. The tTF series are intrinsically controlled, which ensures reliable production of all neuron types, but lacks the ability to adapt to complicated or changing conditions (Liu, 2019).

A separate mechanism is therefore required for adult brain development-both to produce very long series of related neuronal types and to coordinate with organism development. This can be accomplished utilizing protein gradients and hierarchical gene regulation, such as the mechanism used to pattern the fly's anterior/posterior (A/P) axis. In Drosophila A/P patterning, the embryo is progressively partitioned into smaller and smaller domains through layered gene regulation. This is initiated by asymmetric localization maternal mRNAs, bicoid (anterior) and nanos (posterior). The resulting opposing proteins gradients then act on maternal mRNA translation, and in the case of Bicoid, zygotic transcription. The embryo then progresses through expression of maternal morphogen gradients, then zygotic expression of gap genes to determine broad embryo regions, followed by progressive segmentation by the pair-rule and segment polarity genes, and finally specification by the homeotic selector genes (Liu, 2019).

Notably, in postembryonic brain development, two proteins in opposing temporal gradients expressed in NBs have been discovered. These proteins are Imp and Syncrip (Syp) RNA-binding proteins. Imp and Syp control neuronal temporal fate specification as well as the timing of NB termination (decommissioning). Imp promotes and Syp inhibits translation of the BTB-zinc finger nuclear protein, chinmo (chronologically inappropriate morphogenesis), so that protein levels in newborn neurons descend over time. The level of Chinmo correlates with the specification of multiple neuronal temporal fates. Discovering downstream layers in the Imp/Syp/Chinmo hierarchy is essential to fully comprehend the intricacies of temporal patterning in brain development (Liu, 2019).

Temporal regulation in the fly brain is easily studied in the relatively simple mushroom body (MB) neuronal lineages which are comprised of only three major cell types. These neuronal types are born in sequential order: beginning with γ neurons, followed by α'/β' neurons, and finally α/β neurons. Imp and Syp are expressed in relatively shallow, opposing temporal gradients in the MB NBs. Modulation of Imp or Syp expression results in shifts in the neuronal temporal fate. Imp and Syp post-transcriptionally control Chinmo so that it is expressed in a gradient in the first two temporal windows. γ neurons are produced in a high Chinmo window, α'/β' neurons are produced in a low Chinmo window, and α/β neurons are produced in a window absent of Chinmo expression. Moreover, altering Chinmo levels can shift the temporal fate of MB neurons accordingly, strongly implicating dose-dependent actions, similar to that of a morphogen. Despite its importance in temporal patterning, the mechanisms underlying the dosage-dependent effects of Chinmo on neuronal temporal identity is unknown (Liu, 2019).

This study describes Mamo (maternal gene required for meiosis, Mukai, 2007), a BTB zinc finger transcription factor critical for temporal specification of α'/β' neurons. Mamo is expressed in a low Chinmo temporal window and Mamo expression can be inhibited both by high Chinmo levels and loss of chinmo. Additionally, Mamo is post-transcriptionally regulated by the Syp RNA binding protein. This layered regulation, which is utilized in both MB and AL lineages results in a discrete window of Mamo expression in young, post-mitotic neurons. In the MB lineages, this window corresponds to the middle window of neurogenesis and it was establish that Mamo codes for middle temporal fate(s); α'/β' neuronal characteristics are lost when Mamo levels are reduced and ectopic Mamo drives an increase in α'/β' neuron production. The temporal fate determination paradigm described in this study utilizes multiple levels of gene regulation. Temporal fate specification begins in the stem cell and proceeds in a hierarchical manner in successive stages where top and second-tier factors work together to specify neuronal temporal fate. These data suggest that Mamo deciphers the upstream temporal specification code and acts as a terminal selector to determine neuronal fate (Liu, 2019).

Chinmo levels in newborn neurons correlate with adult neuron identity. Based on smFISH, mamo transcription is initiated in newborn MB neurons around 72 hr ALH, which corresponds to weak Chinmo expression. Moreover, Mamo is only expressed when Chinmo levels are low, as Mamo is not expressed after either eliminating or overexpressing Chinmo. Together these data indicate that low Chinmo levels activate mamo transcription in young/maturing neurons (Liu, 2019).

Transcription initiation is not the only requirement for Mamo protein expression; Syp is also required as discussed below. This could explain why no Mamo expression turn on is seen in γ neurons, even as they age and Chinmo levels decrease, becoming quite low around wandering larval stage. γ neurons begin to express Mamo later, around pupation, despite lacking Syp. It has not yet been tested whether weak Chinmo levels are required for later Mamo expression in γ neurons. It is therefore possible that Mamo expression is controlled at this stage by an additional factor(s) (Liu, 2019).

ChIP-chip performed in embryos found five Chinmo binding sites within the mamo gene, consistent with direct activation of mamo transcription. However, the nature of Chinmo's concentration dependent actions is still unclear. Some morphogens such as Bicoid bind different targets at increasing concentrations based on the affinity of binding to different sites as well as the chromatin accessibility of the binding sites. This may also be the case with Chinmo, but would not easily explain why Mamo expression is inhibited at higher Chinmo concentrations. The gap gene Krüppel, on the other hand, has concentration dependent activities at the same binding site. Krüppel acts as an activator at lower concentrations and as a repressor at high concentrations. Krüppels C-terminus has the ability to activate genes and is also the location for dimerization. Upon dimerization, the C-terminus can no longer activate genes and Krüppel transforms from an activator to a repressor. The current data suggests that low concentrations of Chinmo activate mamo. However, in the testis, Chinmo is suspected to function as a transcriptional repressor. It is feasible that Chinmo, like Krüppel, could switch from an activator to a repressor. The protein concentration would affect whether Chinmo is a monomer (in the presence of other BTB proteins, a heterodimer) or a homodimer, and thus potentially which cofactors are recruited (Liu, 2019).

The ascending Syp RNA binding protein temporal gradient regulates Mamo expression both indirectly via its inhibition of Chinmo and also presumably directly, interacting with the mamo transcript and promoting its expression. The bi-modal, transcriptional (Chinmo) and post-transcriptional (Syp), regulation of the Mamo terminal selector is extremely advantageous. Given the finding that Mamo expression is positively autoregulated and that Mamo continues to be expressed into adult neurons, it is particularly important to control the timing of Mamo's onset. The additional layer of post-transcriptional regulation adds an extra safeguard, helping to guarantee that neuronal temporal patterning is a robust system. Indeed, as brain development needs to adapt to environmental conditions such as nutrient deprivation, it is crucial to ensure that there is no loss of neuronal diversity (Liu, 2019).

Syp is a homolog of mammalian SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein) also known as hnRNP-Q. SYNCRIP is involved with multiple facets of mRNA regulation including mRNA splicing and maturation, mRNA localization and stabilization as well as inhibiting mRNA translation and miRNA-mediated repression via competition with Poly(A) binding proteins. The Drosophila ortholog seems to have corresponding functions. Drosophila Syp was isolated from the spliceosome B complex, indicating a conserved role in mRNA splicing. Syp has likewise been found to operate in mRNA localization and stabilization. Furthermore, it has clear roles in altering protein expression of its mRNA targets, both positively and negatively. The bidirectional influence on protein expression likely reflects different Syp modalities (Liu, 2019).

This study shows that Syp is required for Mamo protein expression in the MB and AL neuronal lineages. To determine the nature of this regulation, single molecule fluorescence in situ hybridization (smFISH) was performed. In the absence of Syp, mamo transcription was initiated prematurely in response to weak Chinmo levels, yet mature transcripts failed to accumulate. This leads to the belief that Syp directly binds mamo mRNA and aids in its splicing, maturation and/or stabilization. This is consistent with the finding that overexpressing a mamo cDNA (lacking 5' UTR, 3' UTR and introns) was able to promote cell fate changes despite repression of Syp (Liu, 2019).

Mamo is required to produce the α'/β' neurons in the middle temporal window of the MB lineages. Trio positive α'/β' neurons are clearly absent after RNAi depletion of Mamo during development. Cell production does not appear to be altered, as mamo-RNAi expressing MBs are a normal size. This begs the question of which, if any terminal fate the middle-born neurons adopt in the absence of Mamo. The limited markers for each MB cell type makes it difficult to determine whether the middle-born neurons undergo fate transformation or simply lack terminal fate. The presence of a Fas-II negative lobe hints that some middle-born neurons may not carry temporal fate information, but phenotypic analysis is complicated by defects in γ neuron maturation/remodeling. Removing the γ neurons with chinmo-RNAi eliminates this complication, but it is still unclear whether, without Mamo, the neurons are transformed to the α/β fate. The Fas-II positive, α/β lobe appears enlarged, but it is difficult to tell whether all axons are Fas-II positive or whether Fas-II negative axons are comingled with α/β axons. Without a cell type-specific, cell body marker for α/β neurons, it is ambiguous whether the middle-born cells are transformed to α/β or whether they simply lack α'/β' temporal fate. A transformation to α/β fate would suggest that either α/β is the default fate of MB neurons (requiring no additional terminal selector) or that Mamo expression inhibits α/β specific factors (Liu, 2019).

Mamo's role in promoting α'/β' fate is further supported by Mamo overexpression phenotypes. Overexpression of Mamo in the MB is able to transform α/β and γ neurons to α'/β' neurons. In an otherwise wildtype scenario, overexpression of mamo did not transform every cell to α'/β' fate. Instead the α'/β' lobe was expanded and the other lobe seemed to be an amalgam of α and γ like lobes. This could be due to incomplete penetrance/low expression levels of the mamo transgene or it is possible that the α/β and γ cells retain their own terminal selector driven, cell-type specific gene expression, thus complicating the fate of the differentiated neuron. Mamo overexpression does not alter the specification factors Imp, Syp or Chinmo and presumably there are terminal selector genes expressed downstream of high Chinmo and possibly in Chinmo-absent cells. This seems a likely possibility when overexpressing Mamo in γ neurons. With Syp-RNAi, NBs are 'forever young' and divide into adulthood, persistently producing 'early-born' γ neurons. Interestingly when combining Syp-RNAi with the Mamo transgene, the newborn cells begin to take on a γ-like fate (expressing Abrupt) before a majority transform into an α'/β'-specific, strong Trio expression pattern and adopt α'/β'-like axon morphology. This suggests that Mamo functions downstream of the temporal fate specification genes, but is capable of overriding downstream signals in α/β and γ neurons to promote α'/β' terminal fate (Liu, 2019).

What this study describes about the BTB-ZF transcription factor, Mamo's role in α'/β' cell fate easily fits into the definition of a terminal selector gene, coined by Oliver Hobert (Hobert, 2008). Terminal selector genes are a category of 'master regulatory' transcription factors that control the specific terminal identity features of individual neuronal types. Key aspects of terminal selector genes are that they are expressed post-mitotically in neurons as they mature and they are continuously expressed (often via autoregulatory mechanisms) to maintain the terminal differentiated state of the neuron. Correspondingly, mamo transcription is initiated in newborn, post-mitotic neurons and Mamo protein expression is visible beginning in young/maturing neurons. After transcription initiation, Mamo positively regulates its own expression and continues to be expressed in α'/β' neurons into adulthood. The other quintessential feature of terminal selector genes is that they regulate a battery of terminal differentiation genes, so that removing a terminal selector gene results in a loss of the specific identity features of a neuron type and misexpression can drive those features in other neurons. Indeed, removing Mamo with RNAi results in the loss of α'/β' identity, both developmentally and into adulthood. Further, overexpressing Mamo in either α/β or γ MB neurons results in shift to α'/β' fate. Individual terminal selectors do not often function alone, but in combination with other terminal selectors. Therefore, there are likely terminal selectors downstream of the MB NB-specific genes that contribute to each of the MB neuron types. In this way, the lineage-specific and temporal patterning programs can combine to define individual neuron types. This feature enables the reutilization of terminal selector genes to create disparate neuron types when used in distinct combinations (Hobert, 2016). This further suggests that temporally expressed Mamo serves as a temporally defined terminal selector gene in other lineages, such as the AL lineages that are describe in this study (Liu, 2019).

Altering Chinmo levels via upstream RNA-binding proteins or miRNAs, or by reducing Chinmo with RNAi all result in shifts in the ratio of neurons with different neuronal temporal fates. This evidence suggests a mechanism where Chinmo acts in newborn neurons to promote temporal fate specification. A recent publication suggested that Chinmo affects temporal fate via a neuronal remodeling mechanism by controlling Ecdysone signaling (Marchetti, 2017). Marchetti demonstrates that Chinmo is required for EcR-B1 expression; however it remains unclear whether Chinmo directly affects EcR-B1 expression or if the Chinmo-dependent EcR-B1 expression is the sole mechanism for γ neuron temporal fate specification. Moreover, neuronal temporal fate is not accurately determined by neuronal morphology alone, particularly when ecdysone signaling has known effects on MB cell morphology and fate. Ecdysone receptor signaling is highly pleiotropic, including ligand-independent functions making dominant-negative and overexpression studies difficult to interpret. Therefore, further investigation is needed to clarify the roles of Ecdysone receptor signaling in MB neuronal temporal fate and remodeling. This will be addressed in a follow-up paper. This current manuscript strongly promotes the idea that Chinmo functions in newborn neurons to promote temporal fate as weak Chinmo expression directly precedes Mamo transcription and Mamo is essential for specification and maintenance of α'/β' fate (Liu, 2019).

This study describes a multilayered hierarchical system to define distinct neuronal temporal fate that culminates in the expression of a terminal selector gene. Analogous mechanisms likely underlie temporal patterning in mammalian brains. However, whether orthologous genes play equivalent roles in mammalian temporal patterning has not been fully investigated. The Imp and Syp RNA-binding proteins are evolutionarily conserved. Both homologs are highly expressed in the developing mouse brain and play vital roles in neural development and/or neuronal morphology. The opposing functions of Imp and Syp also appear to be conserved, as the murine orthologs IMP1 and SYNCRIP bind the identical RNA to either promote or repress axon growth, respectively. Moreover, IMP1 expression in fetal mouse neural stem cells plays important roles in stem cell maintenance and proper temporal progression of neurogenesis. It would likewise be very interesting to explore SYNCRIP in the context of temporal patterning (Liu, 2019).

While Chinmo and Mamo have no clear mammalian orthologs, they are both BTB-ZF (broad-complex, tram-track and bric-à-brac - zinc finger) transcription factors. The BTB domain is a protein interaction domain that can form homo or heterodimers and also binds transcriptional regulators such as repressors, activators and chromatin remodelers. The C2H2 (Krüppel-like) zinc fingers bind DNA-providing target specificity. BTB-ZF proteins have been found to be critical regulators of developmental processes, including neural development. Indeed, the BTB-zinc finger protein, Zbtb20, appears to be essential for early-to-late neuronal identity in the mouse cortex. Zbtb20 is temporally expressed in cortical progenitors and knockout results in cortical layering defects, as the inside-out layering of the cortex follows neuronal birth order. While mutations of other brain-expressed BTB-ZF proteins also show cortical layering phenotypes, potential roles in temporal patterning have not been explored (Liu, 2019).

This study illustrates a fate specification process in which a layered series of temporal protein gradients guide the expression of terminal selector genes. The first-tier temporal gradients are expressed in neural stem cells, followed by a restricted expression window in newborn neurons to finally induce a terminal selector gene in a subset of neurons as they mature. This time-based subdivision of neuronal fate can likely be further partitioned, finally resulting in sequentially born neurons with distinct cell fates. This study demonstrates that Mamo, a BTB-ZF transcription factor, delineates α'/β' neurons, the middle temporal window of the MB lineages. Corresponding data in the AL lineages suggest that Mamo may serve as a temporally defined, terminal selector gene in a variety of lineages in the Drosophila brain. Mamo expression is regulated transcriptionally by the descending Chinmo BTB-ZF transcription factor gradient and post-transcriptionally by the Syp RNA binding protein. This multi-tiered, bimodal regulation ensures that only the progeny in a precise temporal window (those with both weak Chinmo and significant Syp levels) can effectively activate the terminal selector gene, mamo. This discovery attests to the power of gradients in creating diverse cells from a single progenitor. Utilizing layers of temporal gradients to define discrete temporal windows mirrors how in early embryos the spatial gradients of RNA-binding proteins and transcription factors specify the fly's A/P axis. This paradigm provides considerable complexity of gene network regulation, leading to abundant neural cell diversity (Liu, 2019).

A truncated form of a transcription factor Mamo activates vasa in Drosophila embryos

Expression of the vasa gene is associated with germline establishment. Therefore, identification of vasa activator(s) should provide insights into germline development. However, the genes sufficient for vasa activation remain unknown. Previous work showed that the BTB/POZ-Zn-finger protein Mamo is necessary for vasa expression in Drosophila. This study showed that the truncated Mamo lacking the BTB/POZ domain (MamoAF) is a potent vasa activator. Overexpression of MamoAF was sufficient to induce vasa expression in both primordial germ cells and brain. Indeed, Mamo mRNA encoding a truncated Mamo isoform, which is similar to MamoAF, was predominantly expressed in primordial germ cells. The results of these genetic and biochemical studies showed that MamoAF, together with CBP, epigenetically activates vasa expression. Furthermore, MamoAF and the germline transcriptional activator OvoB exhibited synergy in activating vasa transcription. It is proposed that a Mamo-mediated network of epigenetic and transcriptional regulators activates vasa expression (Nakamura, 2019).

Although it is well recognised that maternal translational and transcriptional repressors play essential roles in establishing PGCs in Drosophila, the genes sufficient for vas activation in PGCs remain unknown. This study identified two types of vas activators: full-length Mamo, a weak but specific inducer of vas, and Mamo short isoform, a potent inducer of vas activation. To clarify the molecular mechanisms of vas activation, biochemical and genetic analyses were conducted using MamoAF-induced vas expression; two cofactors of MamoAF, CBP and OvoB, are both involved in activation of vas in PGCs. Thus, MamoAF-induced vas expression is useful for identifying cofactors of vas activation in PGCs. MamoAF can induce vas expression in both PGCs and brain. In both cellular contexts, the transcriptional activator OvoB is necessary for MamoAF-induced vas expression. Moreover, overexpression of both MamoAF and OvoB is sufficient to induce vas expression in VNC. Thus, the Mamo-OvoB axis is essential for directing vas activation. These data revealed that MamoAF functions as a molecular hub: it collaborates with CBP to epigenetically activate the vas locus, and physically interacts with OvoB to stimulate vas transcription. Consistent with this notion, a reporter assay demonstrated that these factors worked together to stimulate transcription. It is concluded that the Mamo-mediated network of epigenetic and transcriptional regulators directs vas activation in Drosophila embryos (Nakamura, 2019).

MamoAF directly activates vas expression through the vas-A element in the first intron, which is essential for endogenous vas expression in PGCs. However, MamoAF could also activate vas transcription through other cis-elements that remain to be identified. It has been previously reported that Mamo has a role in the regulation of chromatin structure. Therefore, Mamo may regulate chromatin structure, in addition to transcriptional activation, to promote vas expression (Nakamura, 2019).

A previous study using reporter assays showed that a 40-bp element in the 5' flanking region of vas, the up-40 element, is sufficient to recapitulate germline-specific expression during oogenesis and embryogenesis. The up-40 element does not contain consensus sequences for either Mamo or Ovo. This implies that different transcriptional factors must control vas expression through the up-40 element. Because removal of the vas-A element decreases endogenous vas expression in PGCs, vas-A and up-40 elements may act in a partially redundant manner to upregulate vas expression in PGCs. Therefore, multiple enhancers may act in parallel to activate vas expression in germ cells. However, it remains unclear whether the up-40 element is necessary for endogenous vas expression (Nakamura, 2019).

This study focused on MamoAF to investigate the mechanisms of vas activation due to its potent activity for vas activation. Thus, it remains unclear how full-length Mamo activates vas expression. Understanding the mechanism requires the identification of factors regulating the nuclear localisation of full-length Mamo. Maternal full-length Mamo in the nuclei of early PGCs may interact with OvoB, which is maternally provided and enriched in PGC nuclei. However, it will be necessary to investigate the interaction between full-length Mamo and OvoB under conditions in which full-length Mamo is not converted into short derivatives (Nakamura, 2019).

Both full-length and short form Mamo mRNAs are expressed in germ cells in ovaries. However, full-length Mamo rescues the differentiation of mamo mutant germline clones more efficiently than MamoAF. It was found that full-length Mamo can be converted to truncated derivatives. Thus, full-length Mamo has the potential to complement the short isoform. By contrast, the short form of Mamo does not appear to complement the function of the full-length protein owing to the lack of the BTB/POZ domain. During oogenesis, the transcriptional activity of full-length Mamo may be regulated through interactions with epigenetic regulators via the BTB/POZ domain, as reported for other BTB/Zn-finger transcription factors. Full-length Mamo is enriched in the nuclei of the nurse cells in egg chambers after oogenic stage 6, but MamoAF is only weakly enriched. Thus, full-length Mamo may play a role in regulating gene expression in nurse cells and promoting the differentiation of oocytes into mature eggs (Nakamura, 2019).

It is proposed that Mamo short isoform is a potent vas activator. The C2H2 Zn-finger domains of Mamo short isoform are homologous to those of human Sp1;20 Sp1-related transcription factors regulate gene expression in germ cells in vertebrates. For example, chicken Sp1 promotes vas expression in PGCs. Moreover, Ovo has conserved roles in germline development in mouse and Drosophila. Accordingly, Sp1-related Zn-finger proteins and Ovo may also be key transcriptional activators of vas in other animals, including mouse and human. It is anticipated that these results will facilitate understanding of the molecular mechanisms that regulate germline development in animals (Nakamura, 2019).

Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development

In Drosophila, the expression of germline genes is initiated in primordial germ cells (PGCs) and its expression is known to be associated with germline establishment. However, the transcriptional regulation of germline genes remains elusive. Previous studies found that the BTB/POZ-Zn-finger protein, Mamo, is necessary for the expression of the germline gene, vasa, in PGCs. Moreover, the truncated Mamo lacking the BTB/POZ domain (MamoAF) is a potent vasa activator. This study investigated the genetic interaction between MamoAF and specific transcriptional regulators to gain insight into the transcriptional regulation of germline development. A general transcription factor, TATA box binding protein (TBP)-associated factor 3 (TAF3/BIP2), and a member of the TBP-like proteins, TBP-related factor 2 (TRF2), were found as new genetic modifiers of MamoAF. In contrast to TRF2, TBP was found to show no genetic interaction with MamoAF, suggesting that Trf2 has a selective function. Therefore, this study focused on Trf2 expression and investigated its function in germ cells. The Trf2 mRNA, rather than the Tbp mRNA, was found to be preferentially expressed in PGCs during embryogenesis. The depletion of TRF2 in PGCs resulted in decreased mRNA expression of vasa. RNA interference-mediated knockdown showed that while Trf2 is required for the maintenance of germ cells, Tbp is needed for their differentiation during oogenesis. Therefore, these results suggest that Trf2 and Tbp expression is differentially regulated in germ cells, and that these factors have distinct functions in Drosophila germline development (Nakamura, 2020).

During early embryogenesis in animals, primordial germ cells (PGCs) are specified, and the expression of germline genes is initiated in these cells. The vasa (vas) gene encodes a highly conserved DEAD-box RNA helicase, and its expression is a marker of germline establishment in many animal species. Additionally, recent transcriptome analyses have revealed more novel germline genes that are preferentially expressed in PGCs. Expression of germline genes implies that a network of transcriptional regulators exists in these germline cells. However, the underlying mechanism of transcriptional regulators that control germline gene expression remains elusive (Nakamura, 2020).

In Drosophila, maternal factors localized in the germ plasm are necessary for germ cell establishment. It was assumed that the regulation of germline gene expression requires the presence of maternal transcriptional regulators in the germ plasm. However, RNA interference (RNAi)-mediated knockdown experiments of germ plasm-enriched maternal mRNAs have demonstrated that several transcriptional regulators are required for the expression of germline genes in PGCs. Among these, the transcriptional activator, OvoB, is predominantly expressed in PGCs, and its function is essential for germline development (Nakamura, 2020).

Previous work has shown that Mamo, a PGC-enriched maternal factor, is necessary for the expression of vas in PGCs (Mukai, 2007). Mamo protein has been identified as a zinc-finger protein that contains both a BTB/POZ domain and C2H2 Zn-finger domains (Mukai, 2007). Subsequent biochemical analyses demonstrated that the C2H2 Zn-finger domains of Mamo directly bind to a specific DNA sequence in the first intron of the vas gene. Overexpression of N-terminal truncated Mamo (MamoAF) lacking the BTB/POZ domain, but having the C2H2 Zn-finger domains, strongly promotes vas expression. Furthermore, Mamo mRNA encoding a truncated Mamo isoform, which is similar to MamoAF, is predominantly expressed in PGCs (Nakamura, 2019). Therefore, these results suggest that the short Mamo lacking the BTB/POZ domain is a potent vas activator. Moreover, this study found that MamoAF collaborates with both an epigenetic regulator, CREB-binding protein (CBP), and the germline transcriptional activator, OvoB, to activate vas expression (Nakamura, 2019). These observations imply that MamoAF acts as a hub molecule to interact with transcriptional regulators (Nakamura, 2020).

Recently, core promoter factors and their homologs have been found to selectively regulate gene expression and control specific developmental processes. The TATA box-binding protein (TBP), which is a subunit of the TFIID complex, recognizes core promoters containing the TATA box to regulate gene expression (Haberle, 2018). TBP-associated factor 3 (TAF3/BIP2) is a general transcription factor, but exhibits selective physical interactions with transcriptional regulators such as Antennapedia, BAB1, and BAB2. TBP-related factor 2 (TRF2), a member of the TBP-like proteins, selectively regulates the expression of a subset of genes that differ from those regulated by TBP. TRF2 interacts with the transcriptional regulator, Fruitless, to masculinize neurobehavioral traits in Drosophila. Therefore, core promoter factors may mediate a new layer of transcriptional regulation that controls specific developmental processes (Nakamura, 2020).

In this study, a genetic experiment was conducted to identify the cofactors of MamoAF to provide insights into the role of transcriptional regulators in germ cells. The Drosophila compound eye is a precise structure that sensitively reflects genetic interactions. Genetic modifier screening using the compound eyes has been performed to identify cofactors for many genes. Genetic modifier screening has also succeeded in identifying the cofactors that play a role in germ cells. Therefore, a genetic experiment was conducted on compound eyes overexpressing MamoAF to identify the genetic modifiers of MamoAF. The core promoter factors, TAF3/BIP2 and TRF2, were identified as the genetic modifiers of MamoAF. Trf2 mRNA, rather than Tbp mRNA, was preferentially expressed in PGCs. Although Trf2 is required for maintenance of germ cells, Tbp is necessary for their differentiation during oogenesis. Therefore, these results suggest that the expression of Trf2 and Tbp is differentially regulated in germ cells and these factors have distinct functions in germline development in Drosophila (Nakamura, 2020).

Previous transcriptome analyses revealed that the genes encoding these Mamo interactors are expressed in the adult heads or the cell line derived from eye-antenna discs. Recently, Mamo has also been shown to play a role in neuronal fate specification and maintenance during brain development. Therefore, the transcriptional regulators that collaborate with Mamo can be expressed in neural cells. Moreover, genetic screening using adult compound eyes may be useful for the identification of Mamo interactors that act on germ cells (Nakamura, 2020).

As several regulators of histone modifications are essential for germline development, this study found that both CBP and HDAC1 preferentially interacted with MamoAF. Additionally, since CBP is responsible for H3K27ac and HDAC1 is required for the deacetylation of H3K27ac, it can be said that proper regulation of H3K27ac levels may play an essential role in germline development. Therefore, future studies will focus on identifying the target genes that are epigenetically regulated by H3K27ac in germ cells (Nakamura, 2020).

Some TFIID subunits vary with target genes and cell types. The Drosophila TAF-TRF2 complex has been proposed to perform distinct functions in regulating neural stem cell identity (Neves, 2019). Moreover, previous biochemical studies have shown that both TAF3/BIP2 and TRF2 interact with TAF6 (Weake, 2009). This study also found that MamoAF preferentially interacted with both Taf3 and Trf2. Therefore, future studies investigating the physical interaction between Mamo and these general transcription factors will provide insights into the mechanism through which Mamo collaborates with the complex containing both TAF3/BIP2 and TRF2 (Nakamura, 2020).

Core promoter factors are considered to be universally required in all eukaryotic cells. However, recent studies suggested that some core promoter factors are developmentally regulated. This study showed that Trf2 and Tbp expression was differentially regulated in germ cells. A previous report showed that TBP protein is highly stable in insect TN-368 cells. Consistent with this report, TBP protein appeared to be stable in these experiments, as it was detected in PGCs and germline cysts where Tbp mRNA was not detected. Therefore, the stability of TBP protein may ensure the robustness of transcriptional regulation. In contrast, Trf2 mRNA was continuously expressed in germline cells, and TRF2 protein in PGCs was found to be sensitive to Trf2-RNAi. Therefore, as compared with Tbp, Trf2 activity can be transcriptionally regulated in germline cells (Nakamura, 2020).

Trf2 may regulate the expression of target genes that differ from those of Tbp during germline development. TRF2, rather than TBP, specifically mediates the transcription of ribosomal protein genes. TRF2 selectively regulates the TATA-less histone H1 gene promoter. Moreover, TRF2 is required for piRNA expression. Combined with the current data, TRF2 may selectively support the transcription of target genes in order to maintain germline cells. This study also found that the Trf2 RNAi (BL64561)-mediated knockdown affected the formation of egg chambers. Therefore, the expression of the genes that control germ cell differentiation may be regulated by TRF2 (Nakamura, 2020).

Drosophila Trf2 encodes TRF2S and TRF2L. TRF2S is conserved between Drosophila and mammals because TRF2S appears to be more closely related to the human TRF2 protein, which lacks the long N-terminal extension present in TRF2L. TRF2S is known to act on the DNA replication-related element, downstream promoter element (DPE), and TCT motifs. In this study, Trf2 activity was found to be required for vas expression in PGCs. The vas gene contains a DPE-like motif (+25 to +29) near the transcription start site. Whether TRF2S acts on this DPE motif is unclear, but it supports the idea that TRF2S is involved in the transcriptional regulation of vas. In contrast to Trf2 RNAi (V10443), the overexpression of TbpRNAi did not decrease vas expression. However, as it was not possible to knock down TBP in PGCs, it could not be definitively excluded that TBP mediates the transcription of vas. TBP binds to the TATA box in promoters to regulate TATA-dependent transcription. However, the vas promoter does not appear to contain a TATA box near the transcription start site. Therefore, it is concluded that vas transcription is regulated by TRF2S rather than by TBP. TRF2L may also regulate vas transcription because TRF2L can activate both DPE-dependent and TATA-dependent promoters (Nakamura, 2020).

Previous studies using a weak Trf2 allele, polyhomeoticP1 Trf2P1, in which TRF2 expression in germ cells is decreased but TRF2 is not depleted from germ cells, have shown that Trf2 is required for germ cell differentiation and that TRF2L and TRF2S can rescue the mutant phenotypes individually. This shows that TRF2S and TRF2L have redundant functions in germ cell differentiation. This study also showed that Trf2 RNAi (V10443)-mediated knockdown resulted in an agametic phenotype and that the overexpression of Trf2L rescued the agametic phenotype, thereby suggesting that germ cell maintenance may require a TRF2L function. The Trf2L cDNA used in the rescue experiment may produce both TRF2S and TRF2L because of the presence of the IRES sequence in the Trf2L cDNA. However, this study found that Trf2S expression alone did not rescue the agametic phenotype induced by Trf2 RNAi, thereby confirming that TRF2L is required for germ cell maintenance (Nakamura, 2020).

Previous reporter assays have shown that TRF2S preferentially activates DPE-dependent promoters, whereas TRF2L activates both DPE-dependent and TATA-dependent promoters TRF2L may regulate the expression of distinct target genes to maintain germ cells. Previous biochemical studies have shown that both TRF2S and TRF2L are present in the same protein complex that contains ISWI ATPase. However, some TRF2L proteins exhibit different chromatographic properties from those of TRF2S, thereby suggesting that TRF2L cofactors may differ from those of TRF2S. Moreover, the coiled-coil domains in the long N-terminal domains of TRF2L may mediate their interaction. Therefore, future studies on the identification of TRF2L target genes and TRF2L cofactors may provide insights into the mechanisms through which TRF2L regulates germ cell maintenance (Nakamura, 2020).

The core promoter factors that mediate a new layer of transcriptional regulation may be involved in germline development in Drosophila. Moreover, the homologs of core promoter factors have been found to selectively regulate transcriptional programs and control specific developmental processes in Drosophila and mouse models. This study found that TRF2 and TBP had distinct functions in germline development. As Trf2 is conserved in bilaterian organisms and is required for spermiogenesis in mice, it is speculated that Trf2 might play a role in germline development in other animals as well. Hence, it is expected that the current results will facilitate the understanding of the transcriptional regulation network that controls germline development in animals (Nakamura, 2020).

Binding of Drosophila maternal Mamo protein to chromatin and specific DNA sequences

Alterations in chromatin structure dynamically occur during germline development in Drosophila and are essential for the production of functional gametes. The maternal factor Mamo, which contains both a BTB/POZ domain and C(2)H(2) zinc-finger domains and is enriched in primordial germ cells (PGCs), is required for the regulation of meiotic chromatin structure and the production of functional gametes. However, the molecular mechanisms by which Mamo regulates germline development remained unclear. To evaluate the molecular function of Mamo protein, this study has investigated the binding of Mamo to chromatin and DNA sequences. The data show that Mamo binds to chromatin and specific DNA sequences, particularly the polytene chromosomes of salivary gland cells. Overexpression of Mamo affected the organization of polytene chromosomes. Reduction in maternal Mamo levels impaired the formation of germline-specific chromatin structures in PGCs. Furthermore, it was found that the zinc-finger domains of Mamo directly bind to specific DNA sequences. These results suggest that Mamo plays a role in regulating chromatin structure in PGCs (Hira, 2013).

This study found that Mamo protein is predominantly localized to the nuclei of salivary gland cells and binds to polytene chromosomes. Overexpression of Mamo in salivary gland cells affects the organization of the polytene chromosome. Reduction in maternal Mamo function impairs the formation of germline-specific chromatin structure in PGCs. Furthermore, this study found that MZD-FLAG, which contains the C2H2 zinc-finger domains but lacks the BTB/POZ domain, directly binds to specific DNA sequences. These findings support the roles of maternal Mamo protein in the regulation of chromatin structure in PGCs. Thus, Mamo constitutes a maternal protein that is enriched in PGCs and binds to chromatin and specific DNA sequences (Hira, 2013).

Mamo protein accumulates in the nuclei of salivary gland cells. Proteins localized to the nucleus usually contain classical nuclear localization signals (cNLS), i.e., monopartite or bipartite cNLSs, which are recognized by the importin α-mediated nuclear transport system. To determine whether Mamo contains cNLSs, the Mamo primary structure was analyzed by using the search algorithm from PSORT II. No NLS was found, Thus, the nuclear import of Mamo may be mediated by other systems. Signal transducer and activator of transcription (STAT) 1 does not contain cNLSs but is recognized and imported to the nucleus via an importin-&alpha mediated-system. It is therefore plausible that nuclear localization of Mamo may be mediated by importin α. In contrast to the results for salivary gland cells, a considerable amount of Mamo was also present in the cytoplasm of PGCs. Thus, the nuclear localization of Mamo may be regulated by a germline-specific mechanism (Hira, 2013).

This study has shown that Mamo is required for the formation of germline-specific chromatin structures in PGCs. A similar germline-specific chromosome condensation in PGCs has been reported in Caenorhabditis elegans, which indicates that the chromatin architecture in PGCs is conserved and may be essential for germline development. The oocytes derived from the PGCs with reduced maternal Mamo activity fail to form meiosis-specific chromosomal configurations. Thus, Mamo may have several functions in the regulation of chromatin structure during germline development. Because BTB/POZ-zinc finger proteins are known to form complexes with chromatin regulators to exert their function, Mamo may recruit chromatin factors to condense chromatin in PGCs. It is also possible that Mamo is involved in the regulation of gene expression of chromatin factors in PGCs (Hira, 2013).

The GST-MZD fusion protein binds specific DNA sequences that are rich for guanine. These data are consistent with previous reports showing that the consensus binding sites for C2H2 zinc-finger proteins contain guanine-rich sequence. Mamo may regulate chromatin structure in PGCs through direct binding to specific DNA sequences and/or target genes (Hira, 2013).

MAMO, a maternal BTB/POZ-Zn-finger protein enriched in germline progenitors is required for the production of functional eggs in Drosophila

A hallmark of germline cells throughout the animal kingdom is their ability to execute meiosis. However, despite its prime importance, little is known about how germline progenitors acquire this ability. In Drosophila, the primordial germ cells (PGCs) are characterized by the inheritance of germ plasm, which contains maternal factors that have sufficient ability to direct germline development. This study shows that a novel maternal factor, MAMO, is autonomously required in PGCs to produce functional gametes. MAMO protein which contains both a BTB/POZ (Broad Complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) domain and C(2)H(2) zinc finger motifs is enriched in PGCs during embryogenesis. The PGCs with reduced maternal MAMO activity are able to undergo oogenesis, but fail to execute meiosis properly. In the resulting oocytes, meiosis-specific chromosomal configurations are impaired. It was additionally shown that the decondensation of fertilized sperm nuclei is also affected in the eggs. It is proposed that maternal MAMO activates downstream genes to promote specialized morphological changes of both female meiotic chromosomes and the sperm nucleus, which are critical in zygote formation (Mukai, 2007).

This study has identified a novel maternal gene, mamo, which encodes a putative transcription factor enriched in pole cells. The maternal MAMO protein is required in the germline to promote specialized morphological changes of both female meiotic chromosomes and the sperm nucleus later during oogenesis and fertilization. This protein is also required for proper expression of the genes involved in these cellular events. MAMO is the first reported example of a maternal protein that regulates these germline-specific events that are critical in zygote formation (Mukai, 2007).

Pole cells lacking maternal mamo activity were transplanted into agametic hosts to examine its autonomous function in the germline. Because pole cell transplantation was carried out without discriminating the sex of donors and hosts, approximately half of the host animals are expected to receive heterosexual pole cells. However, it is unlikely that this heterosexual transplantation of pole cells is a simple cause of the observed mamo phenotype, because this phenotype is distinct from that of male pole cells in ovaries. It has been reported that male pole cells differentiate as spermatocytes in ovaries and never contribute to oogenesis. In contrast, mamo pole cells are able to contribute to egg production, but the resulting eggs are nonfunctional due to the defects in meiosis and decondensation of sperm nuclei (Mukai, 2007).

It is concluded that mamo function is encoded by CG32611, as the following reasons. First, mRNAs with aberrant lengths of 5'-UTRs were transcribed from CG32611 in ovaries containing mamo-homozygous germline clones, although no nucleotide change is evident in its ORF region. Second, the data also suggest that these alterations in 5'-UTR length cause a reduction in the production of normal CG32611 protein. In pole cells of the embryos derived from mamo-homozygous germline clones, the level of CG32611 protein was significantly reduced. Finally, the defects in meiotic chromosome configurations and decondensation of sperm nuclei in the germ cells of the progenies derived from mamo germline clones is fully rescued by expression of CG32611 cDNA in the germline clones. Thus, the observed mamo phenotype is caused by a reduction of CG32611 activity, rather than by a second site mutation, although only one mutant allele of mamo was used in this study (Mukai, 2007).

MAMO is a member of the BTB/POZ-zinc finger proteins, which are evolutionarily conserved among a wide variety of animals, including humans. These proteins are involved in various fundamental biological processes such as embryonic development, cell-cycle regulation and oncogenesis. Many BTB/POZ-zinc finger proteins regulate transcription of downstream genes by altering chromatin structure. For example, the GAGA factor encoded by Trithorax-like activates and maintains transcription by counteracting chromatin repression. This study showed that maternal mamo mutation affects gene expression in the ovaries. Reduction of maternal MAMO activity in the pole cells downregulates CG15636 and Hira expression in their descendants within the ovaries. In contrast, these downstream genes were normally expressed in mamo-homozygous germline clones during oogenesis, suggesting that zygotic MAMO activity is dispensable for their expression. It is speculated that maternal MAMO acts in an indirect manner to regulate CG15636 and Hira expression in the ovaries. Considering the molecular nature of MAMO, it is likely that maternal MAMO establishes a germline-specific epigenetic state that permits and maintains the expression of the downstream genes, although interaction of MAMO with chromatin remains to be investigated (Mukai, 2007).

CG15636 (Heterochromatin protein 6) is downregulated in mamo ovaries, and its mutation causes defects in meiotic chromosome configurations similar to that observed in mamo oocytes. It is plausible that CG15636 acts downstream of MAMO in the pathway regulating meiotic chromosome structures. Alternatively, it is also possible that CG15636 may act separately from MAMO, because the phenotype of the CG15636 mutation had a lower penetrance than that of the maternal mamo mutation. However, it is speculated that this could be due to the presence of multiple genes, including CG15636, which act downstream of MAMO in a partially redundant manner. For example, the expression of HmgZ gene is also reduced in mamo ovaries. The DNA binding activity of an HMG protein is required to recruit HP1 onto chromatin , and HP1 is a component of karyosome. CG15636 may act redundantly with HP1 to establish highly compact chromosomes. HmgZ may recruit these proteins onto the meiotic chromosomes. In addition, a mutation in nucleosomal histone kinase-1 (nhk-1; ballchen) exhibits a phenotype nearly identical to the maternal mamo mutation. Although nhk-1 expression was unaffected by the mamo mutation in a microarray analysis, it is speculated that maternal MAMO regulates the expression of downstream gene(s) to activate NHK-1 function, which in turn alters histone modifications and recruits heterochromatin proteins to the meiotic chromosomes, as predicted by the histone code hypothesis (Mukai, 2007).

This study further showed that the decondensation of sperm nuclei is impaired in mamo eggs and Hira expression is downregulated in mamo ovaries. The reduction of Hira activity impairs the decondensation of the fertilizing sperm nuclei in eggs. These results argue convincingly that Hira acts as a downstream effector of MAMO to regulate proper decondensation of sperm nuclei, although genetic interactions between mamo and Hira remain to be investigated (Mukai, 2007).

This study presents evidences that maternal MAMO acts autonomously in the germline to regulate morphological changes of both female meiotic chromosomes and the sperm nucleus. Furthermore, mamo is also functional in somatic cells; its mutation causes zygotic and maternal lethal phenotypes. Compatible with this phenotype, maternal mamo transcript and its protein product are weakly detectable in the somatic region of early embryos, and its zygotic expression is also detectable in several somatic tissues. It has been reported that some genes essential for germline development are also functional in somatic cells. For example, maternal Nos is autonomously required in the germline for their proper development, and is also involved in abdominal patterning in the somatic cells. Nos fulfills these distinct functions via regulating different target molecules through its interaction with different cofactors. By analogy, it is possible that mamo also play distinct roles in the germline and somatic cells through interacting with different cofactors. Hence, it will be important to identify cofactors of MAMO and downstream genes that are directly regulated by them (Mukai, 2007).

MAMO is a member of the widely conserved BTB/POZ-zinc finger proteins. Furthermore, other members of the BTB/POZ-zinc finger protein family, BCL6 and PLZF, are also required for germline development in mouse . In addition, Hira function is conserved between vertebrate and invertebrate. It is anticipated that these results will facilitate better understanding of the molecular mechanisms that regulate sexual reproduction (Mukai, 2007).

Search PubMed for articles about Drosophila Mamo

Haberle, V. and Stark, A. (2018). Eukaryotic core promoters and the functional basis of transcription initiation. Nat Rev Mol Cell Biol 19(10): 621-637. PubMed ID: 29946135

Hira, S., Okamoto, T., Fujiwara, M., Kita, H., Kobayashi, S. and Mukai, M. (2013). Binding of Drosophila maternal Mamo protein to chromatin and specific DNA sequences. Biochem Biophys Res Commun 438(1): 156-160. PubMed ID: 23876313

Hobert, O. (2008). Regulatory logic of neuronal diversity: terminal selector genes and selector motifs. Proc Natl Acad Sci U S A 105(51): 20067-20071. PubMed ID: 19104055

Hobert, O. (2016). A map of terminal regulators of neuronal identity in Caenorhabditis elegans. Wiley Interdiscip Rev Dev Biol 5(4): 474-498. PubMed ID: 27136279

Liu, L. Y., Long, X., Yang, C. P., Miyares, R. L., Sugino, K., Singer, R. H. and Lee, T. (2019). Mamo decodes hierarchical temporal gradients into terminal neuronal fate. Elife 8. PubMed ID: 31545163

Marchetti, G. and Tavosanis, G. (2017). Steroid Hormone Ecdysone Signaling Specifies Mushroom Body Neuron Sequential Fate via Chinmo. Curr Biol 27(19): 3017-3024 e3014. PubMed ID: 28966087

Mukai, M., Hayashi, Y., Kitadate, Y., Shigenobu, S., Arita, K. and Kobayashi, S. (2007). MAMO, a maternal BTB/POZ-Zn-finger protein enriched in germline progenitors is required for the production of functional eggs in Drosophila. Mech Dev 124(7-8): 570-583. PubMed ID: 17600690

Nakamura, S., Hira, S., Fujiwara, M., Miyagata, N., Tsuji, T., Kondo, A., Kimura, H., Shinozuka, Y., Hayashi, M., Kobayashi, S. and Mukai, M. (2019). A truncated form of a transcription factor Mamo activates vasa in Drosophila embryos. Commun Biol 2: 422. PubMed ID: 31799425

Nakamura, S., Hira, S., Kojima, M., Kondo, A. and Mukai, M. (2020). Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development. Dev Growth Differ. PubMed ID: 33219538

Neves, A. and Eisenman, R. N. (2019). Distinct gene-selective roles for a network of core promoter factors in Drosophila neural stem cell identity. Biol Open 8(4). PubMed ID: 30948355

Weake, V. M., et al. (2009). A novel histone fold domain-containing protein that replaces TAF6 in Drosophila SAGA is required for SAGA-dependent gene expression. Genes Dev. 23: 2818-2823. PubMed ID: 20008933

date revised: 15 April 2021

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