InteractiveFly: GeneBrief

non-stop: Biological Overview | Developmental Biology | Effects of Mutation | References

Gene name - non-stop

Synonyms -

Cytological map position - 75D1--2

Function - protein degradation

Keywords - axon guidance, glia, SAGA complex

Symbol - not

FlyBase ID: FBgn0013717

Genetic map position - 3-

Classification - ubiquitin-specific protease

Cellular location - cytoplasmic

NCBI link: Entrez Gene

non-stop orthologs: Biolitmine
Recent literature
Ma, J., Brennan, K. J., D'Aloia, M. R., Pascuzzi, P. E. and Weake, V. M. (2016). Transcriptome profiling identifies Multiplexin as a target of SAGA deubiquitinase activity in glia required for precise axon guidance during Drosophila visual development. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27261002
The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2 and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration in SCA7 patients. Thus, this study sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, glia from wild-type, nonstop and sgf11 larval optic lobes were enriched using affinity-purification of KASH-EGFP tagged nuclei, and then examined each transcriptome using RNA-seq. The analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. It was further show, that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.

The visual system of Drosophila provides a powerful genetic system to analyze the cellular and molecular mechanisms that regulate axon target selection. The compound eye comprises ~750 ommatidia, each containing eight photoreceptor neurons (R cells; R1-R8). Each class of R cells forms specific connections with neurons in two different ganglia in the optic lobe, the lamina and medulla. R1-R6 axons terminate in the lamina, while R7 and R8 axons pass through the lamina and stop in the medulla. As R cell axons enter the lamina, they encounter both glial cells and neurons. non-stop (encoding a ubiquitin-specific protease) was isolated in a genetic screen for R cell projection defects (Martin, 1995). non-stop is required for glial cell development and hedgehog for neuronal development. Removal of glial cells but not neurons disrupts R1-R6 targeting. It is proposed that glial cells provide the initial stop signal promoting growth cone termination in the lamina. These findings uncover a novel function for neuron-glial interactions in regulating target specificity (Poeck, 2001).

Target layer selection occurs during larval development. At this stage, the lamina target area consists of glial cells and neurons. R1-R6 growth cones terminate between rows of epithelial and marginal glial cells. The cell bodies of lamina neurons form columns above the lamina plexus. These neurons represent the future synaptic partners of R1-R6 axons in the adult. Although R cell growth cones terminate within the lamina plexus in the larva, they do not form synapses until some four days later, during the second half of pupal development (Poeck, 2001).

The formation of the R cell projection pattern relies on complex bidirectional interactions between R cell axons and different populations of cells in the target. R cell axons provide anterograde signals (including the ligand Hedgehog and Spitz) to induce the proliferation and differentiation of lamina neurons as well as the differentiation and migration of glial cells. In turn, lamina neurons, glial cells, or both cell types may then provide retrograde signals acting as guidance cues for R cell axons. Genes encoding receptors, signaling molecules, and nuclear factors that act within R cells control target selection. Neither the targeting signals nor the cells that produce them in the lamina have been identified. While glial cells have been proposed to act as intermediate targets for R1-R6 growth cones, based on their characteristic positions in the lamina, this hypothesis has not been critically addressed. It has been shown that loss of glial cells but not of neurons at an early stage of lamina development results in R1-R6 mistargeting. These findings provide evidence that glial cells can regulate the target specificity of neuronal connections (Poeck, 2001).

Non-stop is expressed in the cytoplasm of cells in the optic lobe and central brain. Higher levels of expression, however, are observed in lamina precursor cells but not in differentiated lamina neurons. Non-stop is also expressed at higher levels in marginal, epithelial, and medulla glial cells adjacent to the lamina plexus. Based on the expression pattern, non-stop could function in the LPCs to control both R1-R6 targeting and glial cell migration. Alternatively, non-stop could function directly in lamina glial cells to promote their migration; failure to migrate leads to loss of intermediate targets and R1-R6 mistargeting. One research goal was to distinguish between these possibilities; two different genetic approaches were used. First, the ability of targeted expression of non-stop to glial cells was assessed, using the GAL4/UAS system to rescue the mutant phenotypes. Since basal expression of UAS-non-stop (i.e., in the absence of GAL4 driven in lamina glial cells) is sufficient to rescue both the glial cell migration and targeting defects, this approach to resolving the issue is not possible. Therefore the FLP/FRT system was used to generate clones of lamina glial cells or neurons (and their precursors) homozygous mutant for non-stop in an otherwise wild-type (not1/+) background. A heat shock-FLP (hsFLP) transgene provided recombinase in mitotically active cells in the target area. Animals carried a gene expressed in all cells, encoding the green fluorescent protein under the control of a ubiquitin promoter (Ub-GFP). As a result of mitotic recombination, non-stop homozygous mutant cells did not express GFP; heterozygous cells expressed moderate levels of GFP, since they carry a single copy of the marker gene; and wild-type clones, carrying two copies of Ub-GFP, were identified by their higher expression levels. non-stop homozygous mutant clones were compared to control clones created in the same manner by using a wild-type FRT in place of a not1 FRT chromosome (Poeck, 2001).

Eye-brain complexes were stained with mAb24B10 to visualize R cell axons and with anti-Repo to identify glial cells. Approximately 10% of all animals examined contained clones in the target area. In mosaic animals with clones in lamina precursors and lamina neurons, the R cell projection pattern appeared normal. Furthermore, normal rows of wild-type epithelial, marginal, and medulla glial cells formed beneath these clones. This indicates that non-stop is not required in lamina neurons to regulate either R1-R6 targeting or glial cell development (Poeck, 2001).

Glial cells are generated in the GPC areas and migrate to their positions adjacent to the lamina plexus. In about half of the wild-type control samples, glial cell clones were large, containing at least five epithelial or marginal glial cells. The GPC areas showed clones of variable sizes, with unlabeled cells next to groups of marked cells carrying one or two copies of GFP. The region adjacent to the dorsoventral midline close to the optic lobe surface appearsto give rise to especially large clones. Furthermore, it was observed that 21 of 23 clones consisted of both epithelial and marginal glial cells; two small clones contained epithelial glial cells only (Poeck, 2001).

The number of non-stop mutant epithelial and marginal glial cells bordering the lamina plexus was markedly reduced compared to control clones. In wild-type control clones, 203 unlabeled cells were counted in 23 clones. In contrast, in non-stop mutant clones, there were 125 cells in 39 clones examined. This represents about a 3-fold difference. Moreover, for marginal glial cells, this difference was 10-fold; in wild-type clones there were 71 cells in 23 clones, whereas there were 11 cells in 39 non-stop mutant clones. The non-stop mutant and control clones in the GPC area were similar in both size and frequency. In six cases, although homozygous non-stop clones were present in the GPC areas, there were no mutant glial cells in the target. In contrast, unlabeled epithelial and marginal glial cells were observed along the lamina plexus in all wild-type clones examined. Blocks of non-stop mutant glial cells were never observed adjacent to the lamina plexus. Presumably, in mosaic animals, wild-type cells migrate into these regions, effectively replacing the mutant glial cells and rescuing the R1-R6 targeting defect seen in non-stop mutants. These data support a model in which non-stop is required in glial cells, their precursors, or other cell types within the GPC area for the migration of epithelial and marginal glial cells from the GPC to the target area (Poeck, 2001).

While genetic mosaic studies established that non-stop is required in glial cells for their migration, it remained formally possible that non-stop was required in lamina neurons to mediate R1-R6 termination in the lamina. To critically assess this issue, attempts were made to remove lamina neurons from the target region. If non-stop were required in lamina neurons, then removing lamina neurons entirely should lead to R1-R6 mistargeting. hedgehog1 (hh1) is a regulatory mutation that specifically affects the visual system. In hh1, ~12 rows of R cell clusters are formed; R cell axons, however, lack Hh and fail to induce lamina neurons. Conversely, the migration and differentiation of glial cells do not depend on Hh signaling. A subset of R1-R6 axons was visualized in hh1 mutants, using the marker Ro-taulacZ. These axons stopped in the lamina, despite the absence of lamina neurons, as detected using an antibody to Dachshund. Labeling with mAb24B10 to visualize all R cell axons revealed that the array of R7 and R8 growth cones in the medulla was indistinguishable from wild type. These findings demonstrate that initial targeting of R1-R6 axons does not require lamina neurons (Poeck, 2001).

The mechanisms controlling glial cell migration in the lamina and the molecular pathways involved are not known. That non-stop encodes a ubiquitin-specific protease suggests that protein degradation pathways may play an important role in this process. In the ubiquitin-proteasome pathway, proteins are targeted for degradation to the 26S proteasome after being 'tagged' with ubiquitin. Ubiquitin modification is reversible. Deubiquitination is catalyzed by two families of specific proteases, ubiquitin-C-terminal hydrolases and ubiquitin-specific proteases (UBPs). While these families are structurally distinct, they have overlapping functions. Non-stop is related to the second family because of two conserved consensus sequences within the catalytic domain, the Cys and His domains. UBPs have been shown to play diverse roles by either inhibiting or stimulating protein degradation. They can prevent protein degradation and reverse ubiquitin modification to 'proofread' mistakenly ubiquitinated proteins or to regulate protein stability by antagonizing proteasome activity. UBPs also have been found to stimulate protein degradation by editing the size of polyubiquitin chains, releasing ubiquitin after the protein has been targeted to the proteasome, or disassembling polyubiquitin chains to restore the cellular pool of free ubiquitin (Poeck, 2001).

The observation that additional ubiquitinated proteins accumulate in mutant larvae is consistent with Non-stop acting as a UBP. Furthermore, enhancement of targeting defects in non-stop mutants resulting from removing a single copy of a gene encoding a proteosome subunit suggests that Non-stop promotes protein degradation. Since the loss of non-stop results in a specific defect in the developing visual system, it may regulate the levels of specific substrates necessary for glial cell migration. Indeed, another Drosophila UBP, Ubiquitin-63E, controls border cell migration during oogenesis indirectly by stabilizing the transcription factor C/EBP (Rørth, 2000). There is also precedent for ubiquitin-dependent regulation of signaling proteins, such as cell surface receptors and cytoskeletal regulators, which may be directly involved in cell migration (Poeck, 2001).

non-stop mutations provide a key reagent for addressing the cellular requirement for R1-R6 targeting. Loss-of-function non-stop mutations selectively disrupt glial cell development at an early stage. During the time glial cells are generated in the precursor region, migration into the developing lamina is disrupted. As such, large regions of the lamina target are depleted of glial cells. Genetic mosaic analyses argue that R1-R6 mistargeting results from a loss of glial cells in the lamina target and not from a requirement for non-stop in R cell axons or lamina neurons. That lamina neurons are dispensable for R1-R6 targeting is further supported by the analysis of hedgehog1 mutants in which layer selection is normal in the absence of lamina neurons. The genetic analysis, however, does not allow for the exclusion of the formal (albeit unlikely) possibility that non-stop functions in glial cells in two distinct processes to regulate migration and R1-R6 targeting separately (Poeck, 2001).

The relative contributions of epithelial, marginal, and medulla glial cells in R1-R6 targeting could not be determined, since all three appear affected in non-stop mutants. Their morphology, however, suggests that they have different functions. R1-R6 growth cones in the lamina plexus are in intimate contact with a dense fringe of glial cell processes from both epithelial and marginal glia but not with processes from the medulla glia. It is likely that marginal glial cells issue the stop signals, since R1-R6 axons grow past epithelial glial cells but terminate along the distal face of the marginal glial cells. In addition, both epithelial and marginal glial cells may provide signals to R1-R6 growth cones, 'holding' them in place. Nitric oxide (see Drosophila Nitric oxide synthase) plays an important role in maintaining the position of R7 and R8 axons within medulla neuropil during early pupal development and this raises the intriguing possibility that a similar mechanism may keep R1-R6 neurons within the lamina (Poeck, 2001).

In summary, the requirement for non-stop in glial cell development, the failure of R1-R6 growth cones to terminate in the lamina in non-stop mutants, and the close association between lamina glial cells and R1-R6 growth cones in the lamina plexus argue that epithelial and marginal glial cells provide targeting signals for R1-R6 neurons (Poeck, 2001)

The existence of intermediate targets is essential for generating specific patterns of R cell connections. These targets provide a means of delaying the formation of neuronal connections until the future synaptic partners of R cells, the lamina neurons, have formed. R1-R6 axons project from the eye primordium in a sequential fashion to their target layer during larval development. Growth cones from R cells within the same ommatidium form a tight cluster nestled between the epithelial and marginal glial cells and pause within this region through early pupal development. Early arriving R cell axons wait for about 70 hr, while later arriving axons pause for about 36 hr. During this period, lamina neurons assemble into columns, adopt specific cell fates (i.e., L1-L5), and project axons through the lamina plexus and into the medulla. Maturation of lamina neurons is reflected by the expression of the molecular marker Elav. This marker is seen initially in single cells within columns closest to the lamina furrow and accumulates in all lamina neurons in the more mature columns at the posterior edge of the developing lamina. Thus, the precise array of lamina neurons develops long after R cell growth cones enter the target region (Poeck, 2001).

Moreover, the formation of connections between the retina and lamina requires the preassembly of a precisely organized target field. During pupal development, R1-R6 growth cones defasciculate from their original bundle. Each growth cone projects to different neighboring postsynaptic targets and develops into an extended terminal, forming many en passant synapses with a subset of lamina neurons. Synapse formation is complete by late pupal development. This complex reorganization of R cell axons within the target ensures that, in the adult, R cells 'looking' out from the eye at the same point in space connect to the same postsynaptic neurons in the lamina (Poeck, 2001).

The role of neurons as intermediate transient targets is well documented in the developing mammalian hippocampus and neocortex. As in the developing lamina, the final synaptic partners are not yet in place in these systems when the first afferent axons arrive. In this paper, it has been shown that glial cells also can act as intermediate targets. Indeed, this function for glial cells may be more widespread. In the developing olfactory system of the moth Manduca sexta, antennal axons interact with glial cells in the target area before establishing contacts with central target neurons. In animals rendered glial deficient by hydoxyurea treatment or gamma-irradiation, olfactory axons do not confine their projections to their normal targets, the olfactory glomeruli. This treatment also prevents the development of specific glial cells, which segregate axons into distinct fascicles as they enter the target region from the antennal nerve. Hence, it is not clear whether it is these glial cells or, alternatively, the neuropil-associated glial cells in the target area that are critical for regulating target specificity in this system. Glial cells also may function as intermediate targets in some regions of the mammalian nervous system. For instance, rodent olfactory axons interact with radial glial cells, prior to recruitment of mitral and periglomerular processes (e.g., the synaptic targets of olfactory neurons) into glomeruli. In the absence of mitral or periglomerular cells, olfactory axons select their appropriate glomerular targets. Based on these observations, it has been proposed that targeting of olfactory axons to specific glomeruli is regulated by glial cells. While the role for glial cells in target specification is new, glial cells have previously been shown to play key roles as 'guideposts' along axon trajectories to their targets. For instance, in the Drosophila embryonic central nervous system, midline glial cells provide a repellent signal, Slit, to prevent axons from inappropriate growth across the midline (Poeck, 2001 and references therein).

The identification of glial cells as intermediate targets for R1-R6 neurons provides an important step in the isolation of targeting signals regulating R1-R6 specificity. This may be achieved through molecular screens using GFP-labeled glial cells isolated from the developing optic lobe to identify genes encoding cell surface proteins selectively expressed in these cells. Alternatively, by targeting mitotic recombination to glial precursor cells using the FLP/FRT method, it may be possible to specifically isolate such genes required in glial cells to control target layer selection (Poeck, 2001).

A Non-stop identity complex (NIC) supervises enterocyte identity and protects from premature aging

A hallmark of aging is loss of differentiated cell identity. Aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, an RNAi screen targeting ubiquitin-related genes was performed in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging (Erez, 2021).

Differentiated cell states are actively established and maintained through action of 'identity supervisors'. Identity supervisors safeguard the expression of genes that enable differentiated cells to respond to environmental cues and perform required physiological tasks. Concomitantly, they ensure silencing/repression of previous fate and non-relevant gene programs and reduce transcriptional noise. Inability to safeguard cell identity is a hallmark of aging and results in diseases such as neurodegeneration, diabetes, and cancer. In many cases, transcription factors (TFs) together with chromatin regulators and architectural/scaffold proteins establish and maintain large-scale chromatin and nuclear organization that is unique to the differentiated state of the cell (Erez, 2021).

In adult Drosophila midgut epithelia, the transcription factor Hey [Hairy/E(spl)-related with YRPW motif], together with Drosophila nuclear type A lamin, Lamin C (LamC), co-supervise identity of fully differentiated enterocytes (ECs) (Monastirioti, 2010; Flint Brodsly, 2019). Highly similar to the vertebrate gut, Drosophila midgut epithelia intestinal stem cells (ISC) either self-renew or differentiate into progenitor cells that mature into enteroendocrine cells (EEs) or give rise to enteroblast (EB) progenitors. EBs mature into fully polyploid differentiated enterocytes (ECs) that carry out many critical physiological tasks of the intestine. Aging affects the entire midgut, and is associated with loss of EC identity, mis-differentiation of progenitors, pathological activation of the immune system, and loss of the physiological properties of the gut and its integrity. It also results in loss of intestinal compartmentalization, and microbiota-dysbiosis, all leading to reduced lifespan. During aging, the protein levels of identity supervisors such as Hey and LamC decline, resulting in inability to maintain EC-gene programs and ectopic expression of previous- and non-relevant gene programs. Indeed, continuous expression of Hey in aged ECs restores and protects EC identity, gut integrity, and tissue homeostasis (Flint Brodsly, 2019; Erez, 2021 and references therein).

Regulation of EC identity requires signaling to the nucleus to communicate physiological changes in the gut environment. An important mechanism involves changes in post-transcriptional modifications (PTMs) which may propagate, amplify, or conduct signals, ultimately leading to differential gene regulation. One type of PTM is the covalent attachment of ubiquitin or ubiquitin-like (Ub/UbL) molecules, that affect protein stability, function, localization, as well as modulatex chromatin structure. Recent works suggest an intimate link between ubiquitin, proteostasis, and aging. A RNAi screen was performed in this study to search for Ub/UbL-related genes within ECs that supervise identity. Screening of 362 genes, 17 were identified whose conditional elimination in fully differentiated ECs resulted in loss of EC identity. Further analysis revealed one of them, the deubiquitinating isopeptidase (DUB) Non-stop (Non-stop/dUSP22) is a key EC identity supervisor. Purification and proteomic analysis identified Non-stop as part of a CP190/Nup98/Sgf11/e(y)2/mdg4 protein complex, termed Non-stop identity complex (NIC), that is essential for maintenance of EC identity. In part, Non-stop protects NIC proteins from age-dependent decline, safeguarding the EC-gene expression signature, as well as large-scale nuclear organization in these cells, preventing premature aging. Over lifespan, Non-stop protein levels in ECs declined, leading to loss of NIC subunits. This decline is associated with loss of gut identity and physiology at the cellular and tissue levels. Maintaining youthful levels of Non-stop prevented loss of the NIC and prevented aging of the gut (Erez, 2021).

Based on cell-specific secondary tests, this study identified three categories of supervisors; 1. EC-specific identity regulators 2. Genes that are required for differentiated cell identity (both EEs and ECs) 3. Genes that are required for identity of all cell types (general identity regulators). Of specific interest were a group of genes (CG1490; CG2926; CG4080) whose elimination in EEs resulted in a loss of EC, but not EE, identity, acting as inductive identity regulators. Likely their effect on ECs involves cell~cell communication via diffusible factors. While loss of each individual gene identified in the screen resulted in loss of EC identity, the molecular and genetic connections between different identity regulators requires further studies (Erez, 2021).

The observation that Ub/UbL-related genes protect the differentiated identity is conserved across species. Screens in mammalian systems identified enzymes within the SUMO and ubiquitin pathways acting as a barrier against forced reprogramming of differentiated cell. Among these genes were Ubc9 (Drosophila Lesswright), the sole SUMO conjugating enzyme, that was also identified in this screen and the isopeptidase Psmd14. In addition to Non-stop, the screen identified the iso-isopeptidase UTO6-like (CG7857), Usp7, and Rpn11 as regulators of EC identity. Rpn11 is part of the lid particle of the 26S proteasome, involved in deubiquitinating proteins undergoing proteasomal degradation (Erez, 2021).

Genes regulating identity are likely to serve as a barrier to tumorigenesis having a tumor suppressive function. The human ortholog of Non-stop, USP22 has mixed oncogenic and tumor-suppressive functions. Relevant to the current study is the observation that USP22 has tumor suppressive functions in colon cancer by reducing mTor activity. Along this line it is interesting to note that 10/17 of human orthologs to genes discovered in the screen are either mutated or silenced in cancer. Thus, future studies of these human orthologs may identify potent tumor suppressors in cancer (Erez, 2021).

The proteomic, biochemical, and genetic analyses established that to maintain identity Non-stop acts as part of the NIC protein complex. Like SAGA, NIC contained the entire DUB module including Non-stop, E(y)two and Sgf11. Within SAGA, Sgf11 is critical for Non-stop activity. However, loss of Sgf11 did not result in loss of EC identity. While this may reflect intrinsic differences between the complexes it may also be possible that knock-down of Sgf11 transcripts in ECs did not reduce Sgf11 protein levels sufficiently enough to result in a phenotype (Erez, 2021).

The BTB domain of Mod(mdg4) interacts with the M domain of Cp190, and together they enable the recruitment of NIC to the promoter/enhancer regions. There are more than 30 Mod(mdg4) isoforms with different C-ends, each of which interacts with different DNA-binding TF. Cp190 can also be able to recruit different TFs and interacts with Chromo and Zinc-finger 4, that are co-localized with Nup98-96. The Mod(mdg4)/CP190 sub-complex is bound to promoters through interactions with many promoter specific C2H2 proteins including dCTCF, Su(Hw), and possibly via other TFs enriched in the ATAC-seq analysis (Erez, 2021).

Prominent phenotypes of loss of Non-stop were the reduced protein level of Nup98 and its miss-localization from the nuclear periphery to an intranuclear punctate distribution. Nup98 was shown to recruit Set1~COMPASS to enhance histone H3K4me2-3 methylations in hematopoietic progenitors. Thus, a possible function of the NIC may be the recruitment of the H3K4me2/3 COMPASS methylases to catalyze H3K9 di- and tri-methylation at enhancers and promotors, which are fundamental for gene activation. Members of the NIC complex were previously shown to form a Nuclear pore complex that enhances transcriptional memory upon exposure to ecdysone (Pascual-Garcia, 2017), raising the possibility that NIC is required for enhancing transcriptional memory promoting the transcription of EC-related genes and prevents Polycomb-dependent transcriptional repression (Erez, 2021).

In addition to NIC subunits the complex described by Pascual-Garcia (2017) contained Trithorax-like protein (Trl)/GAF and the boundary/architectural proteins CTCF, Suppressor of Hairy Wing (SuHw) and Mtor, but not Non-stop or E(y)2. Interestingly, the DNA binding site of Trl/GAF/ was enriched in EC genes that their expression required Non-stop for maintaining chromatin accessibility. However, Trl, CTCF, SuHw, or Mtor that interact with Cp190 were not detected as Non-stop bound proteins in the proteomic analysis, and RNAi-mediated elimination of Trl/GAF, or SuHw from ECs did not result in loss of EC identity. Thus, suggesting that these are likely separate regulatory complexes with shared subunits (Erez, 2021).

Non-stop was found to be required for the stability of NIC in ECs. RNA-seq analysis established that Non-stop did not regulate the mRNA level of NIC subunits. Suggesting that Nonstop acts by deubiquitinating NIC subunits in ECs. In this regard, it is possible that the stabilization of NIC subunits may also require the EC-related lamin, LamC. It was noticed that Lamin C protein levels (but not mRNA) decline upon loss of Non-stop. Moreover, LamC expression partially restored the protein levels of NIC subunits and their intranuclear localization, potentially by serving as a scaffold for NIC at the nuclear periphery. Therefore, additional experiments are required to establish whether NIC subunits are directly deubiquitinated and stabilized by Non-stop iso-peptidase activity and the potential contribution of LamC to stabilization of NIC subunits (Erez, 2021).

Thus, Non-stop may function at two levels; One is a direct role in transcription safeguarding chromatin accessibility at EC genes, while a second function is the stabilization of identity supervisors including NIC subunits and LamC (Erez, 2021).

Both Non-stop and the transcription factor Hey are bona-fide regulators of EC identity required for the expression of EC-related genes. A significant number of EC-related genes required both Non-stop and Hey for their expression, suggesting that Hey and Non-stop may co-regulate these genes. However, functional and epistatic tests suggest that Hey also acts upstream or in additional pathways to Non-stop. Hey binds to enhancers in lamin genes repressing the expression of the ISC-related lamin, LamDm0 and enhances the expression of LamC. In contrast, Non-stop does not regulate the accessibility, or expression of either LamDm0 or LamC at mRNA level. However, Non-stop is required for maintaining the levels of LamC protein. Therefore, loss of Non-stop results in a decline in LamC but not in the ectopic expression of LamDm0, which is observed upon acute loss of Hey or aging. This discrepancy may be due to the presence of Hey on its repressed targets in young ECs where Non-stop is targeted, and directly repressing their expression as maybe in the case of LamDm0. Moreover, EC-specific expression of Non-stop did not suppress the phenotypes associated with acute loss of Hey in young ECs further supporting for Hey-dependent, but Non-stop independent functions. However, the ECs-specific expression of either Non-stop or Hey in aging midguts restores expression of LamC and repressed ectopic LamDm0 expression (Erez, 2021).

Changes in large-scale nuclear organization are hallmarks of aging. Expression of identity supervisors can prevent age-related distortion of the nucleus EC identity and protect overall the epithelial tissue). However, to accomplish this, Non-stop or Hey were continuously expressed in ECs and temporal expression of Hey or Non-stop in already aged ECs was not sufficient to suppress aging phenotypes. Thus, if the levels of identity supervisors are kept at youthful levels, they can continue to maintain cell identity and prevent signs of aging, effectively keeping the gut organization and structure similar to young tissue (Erez, 2021).

Furthermore, it is not clear how expression of a single regulator like Non-stop has an extensive impact on the entire nucleus. Recent studies suggest that Non-stop functions in additional multiprotein complexes that may regulate large-scale cellular organization. For example, Non-stop is part of an Arp2/3 and WAVE regulatory (WRC) actin-cytoskeleton organization complex where it deubiquitinates the subunit SCAR (Cloud, 2019). In this regard, a nuclear actin organizing complex, WASH, interacted with nuclear Lamin and was required for large scale nuclear organization. Thus, it is tempting to suggest that such complexes are required to maintain cell identity, and that subunits within these complexes are deubiquitinated by Non-stop (Erez, 2021).

In this regard many nuclear proteins are extremely long-lived proteins (LLPs) among them are nuclear pore complex proteins (NPCs) and core histones. The extended stability of LLPs may originate from intrinsic properties of LLPs, or due to sequestration and evading degradation. However, increased stability may be also actively maintained by constitutive de-ubiquitination. Indeed, post-translational modification by ubiquitin and SUMO were shown to regulate lamin stability and their intranuclear localization. Specifically, type-A lamin and its splice variant Progerin, the cause of Hutchinson Gilford progeria syndrome (HGPS), a premature aging syndrome, are degraded by the HECT-type E3 ligase Smurf2 via ubiquitin-dependent autophagy. The elimination of Progerin by expression of Smurf2 in HGSP-fibroblasts reduced the deformation observed in these cells. Thus, it is possible that enhancing Progerin degradation by inhibiting the human ortholog of Non-stop, USP22, will restore nuclear architecture, and suppress the premature aging phenotypes observed in HGPS cells (Erez, 2021).

Drosophila USP22/nonstop polarizes the actin cytoskeleton during collective border cell migration

Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. This study identified, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop (not) function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA's histone acetyltransferase module, which does not bind to Expanded or Merlin, is dispensable for migration. Taken together, these results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, that may help guide studies in other systems where USP22 is necessary for cell motility and invasion (Badmos, 2021).

This study reports that Drosophila USP22, encoded by not, is necessary for F-actin polarity and collective cell migration of invasive BCs. Collective BC migration requires actomyosin polymerization and contraction at the cortex around the cluster as it moves over the nurse cell substrate; F-actin is effectively excluded from the center of the cluster where polarity determinants acting via the Hippo complex block the activity of the F-actin regulator Enabled. Not has been reported to regulate the actin cytoskeleton directly by promoting the stability of Scar/WAVE. However, this study did not observe a reduction in Scar levels in not mutant clones, and scar loss of function did not disrupt F-actin polarity. Furthermore, no significant change was observed in the number of actin protrusions following not loss of function. This might be expected if Scar were a target in BCs. Interestingly, scar RNAi weakly suppressed not loss of function, suggesting that accumulation of branched actin, mediated by Scar at BC-BC junctions, may contribute to disrupted cell polarity and impaired migration. The data suggest that not regulates inside-out F-actin polarity by regulating the expression of Hippo signaling components ex and mer, that are direct Not targets, in a yki-independent manner. Reanalysis of ChIP-Seq data from embryos indicates that Not and Ada2b bind other core Hippo pathway components, so expression of multiple components may be affected by loss of SAGA components. However, ex and mer are targets for Not, but not Ada2b, which is largely dispensable for migration. Notably, this study found that overexpression of ex suppressed not1-induced F-actin accumulation at inner BC junctions, consistent with partial restoration of Hippo function and inhibition of Enabled function. It was also observed that cpb overexpression rescued loss of not, again consistent with disruption of Enabled function due to competitive binding of Cpb to F-actin barbed ends and the inhibition of F-actin polymerization at inner BC junctions. Incomplete rescue of not1 with overexpressed ex or cpb means that other parallel downstream targets that contribute to not function may exist. Interestingly, the data suggest that not is dispensable in polar cells for BC migration. It will be interesting to examine whether the requirement for not in Hippo pathway function is limited to situations where the Hippo complex acts in a yki-independent fashion. The nature of putative noncell autonomous signaling mediated by not controlling polar cell number remains to be elucidated, but altered signaling may be an indirect consequence of changes in polarity or via direct changes in the expression of affected signaling molecules (Badmos, 2021).

A striking effect of not loss of function in BCs is the redistribution of Crb from inner to outer BC junctions. When possible effects of this on other polarity determinants were examined, it was found that localization of aPKC to the inside apical junction between BCs was disrupted, consistent with studies showing that Crb, acting together with the Par complex and endocytic recycling machinery, is necessary for ensuring its correct distribution. Mislocalized aPKC generates protrusions at the side and back of BCs, just as were seen in not1 clusters. Why is Crb mislocalized to the cortex of the BC complex? Complementation experiments suggest that this is partially accounted for by loss of expression of the FERM domain proteins Ex and Mer, which in follicle cells act together with Moe to recruit Crb to the apical surface. Moe stabilizes Crb at the apical membrane of epithelia by linking Crb to cortical actin. Although the physical interaction between Moe and Crb may be weak, Moe is an important regulator of dynamic Crb localization because it acts to antagonize interactions between Crb and aPKC at the marginal zone of the apical membrane domain while stabilizing interactions between Crb and the apical surface. Importantly, in BCs, Moe is cortically localized where it organizes a supercellular actin cytoskeleton network and promotes cortical stiffness. An attractive hypothesis, therefore, is that Moe, along with other proteins, is a sink for Crb at the cortex of the BC cluster following loss of Ex and Mer at inner BC junctions in not mutants. When ex was overexpressed, the normal pattern of Crb localization was partially restored in support of there being competitive binding. Interestingly, weak rescue of Crb localization was also observed following Cpb overexpression. This might be because Moe, or other proteins that tether Crb on the outer membrane, is only accessible in the absence of a strong supercellular F-actin cortex and that restoration of cortical F-actin in not1 cpb+ cells displaces Crb. In WT BCs, Crb needs to be constantly moved from the outside membrane in a dynamin- and Rab5-dependent manner. Another possibility therefore, which is not mutually exclusive from the first, is that polarization of the F-actin cytoskeleton is important for correct trafficking of Crb in BCs as it is in follicle cells (Badmos, 2021).

The growth, specification, and migration of cells during tissue development requires precisely regulated patterns of gene expression that depend on numerous cues for temporal and spatial gene activation involving crosstalk with multiple signaling pathways. Strikingly, it has emerged that factors once considered to be ubiquitous regulators of transcription, including the SAGA chromatin-modifying complex, can have specific roles in discrete developmental processes. Although it has been suggested that SAGA is required for all transcribed genes in some contexts, numerous studies have shown that loss of SAGA components affects the expression of only a subset of genes and that different components modulate distinct and overlapping subsets. These differences in expression are likely to explain their different physiological roles; for instance, during female germline development in Drosophila, ada2B affects the expression of many genes and is required for oogenesis, whereas not affects relatively few and is dispensable. Genome-wide ChIP studies indicate that even though both DUB and HAT modules bind the same genes, many of the targets do not require the DUB module for expression, explaining the observed dependencies. These experiments also revealed nonoverlapping sites of chromatin occupancy for the DUB and HAT modules of SAGA in Drosophila, but the significance of differences in transcriptional targeting for cell function had not been established. Notably, in this respect, this study found that the requirement for not in BC migration is not matched by a requirement for HAT components, including ada2b or gcn5. Furthermore, Ada2b has not been found to bind the ex and mer promoters, providing a molecular explanation for not's SAGA-independent role. Importantly, these findings challenge the perceived view that transcriptional roles for not/USP22 are mediated solely by SAGA. This may have broader relevance to situations where USP22, but not other members of SAGA, is associated with human disease states, particularly where cell polarity is frequently disrupted, such as cancer. Current efforts are directed at identifying SAGA-independent factors that facilitate Not's chromatin binding and function (Badmos, 2021).

Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization

Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7, but heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR (Cloud, 2019).

Purification of Atxn7-containing complexes indicated that Atxn7 functions predominantly as a member of SAGA. In yeast, the Atxn7 orthologue, Sgf73, can be separated from SAGA along with the deubiquitinase module by the proteasome regulatory particle. Without Sgf73, the yeast deubiquitinase module is inactive. In higher eukaryotes, Atxn7 increases, but is not necessary for Non-stop/USP22 enzymatic activity in vitro. In Drosophila, loss of Atxn7 leads to a Non-stop over activity phenotype, with reduced levels of ubiquitinated H2B observed (Mohan, 2014). In this study, purification of Non-stop revealed the active SAGA DUBm associates with multi-protein complexes including WRC and Arp2/3 complexes separate from SAGA. SCAR was previously described to be regulated by a constant ubiquitination/deubiquitination mechanism. SCAR protein levels increased upon knockdown of Atxn7 and decreased upon knockdown of non-stop. Decreases in SCAR protein levels in the absence of non-stop required a functional proteasome (Cloud, 2019).

Conversely, overexpression of Non-stop in cells led to increased SCAR protein levels and this increased SCAR protein colocalized to subcellular compartments where Non-stop was found. Nuclear Arp2/3 and WRC have been linked to nuclear reprogramming during early development, immune system function, and general regulation of gene expression. Distortions of nuclear shape alter chromatin domain location within the nucleus, resulting in changes in gene expression. Nuclear pore stability is compromised in SAGA DUBm mutants, resulting in deficient mRNA export. Similarly, mutants of F-actin regulatory proteins, such as Wash, show nuclear pore loss. Non-stop may contribute to nuclear pore stability and mRNA export through multiple mechanisms (Cloud, 2019).

When the basis for this unexpected regulatory mechanism was examined, a series was uncovered of WIRS motifs conserved in number and distribution between flies and mammals. These sequences functionally modulate Non-stop ability to increase SCAR protein levels. Point mutants of each WIRS resulted in less SCAR protein per increase in Non-stop protein. WIRS mutant Non-stop retained the ability to incorporate into SAGA, indicating these are separation of function mutants (Cloud, 2019).

Overall, these findings suggest that the cell maintains a pool of Non-stop that can be made available to act distally from the larger SAGA complex to modulate SCAR protein levels (see Non-stop regulates SCAR protein levels and location). In yeast, the proteasome regulatory particle removes the DUBm from SAGA. In higher eukaryotes, caspase-7 cleaves Atxn7 at residues which would be expected to release the DUBm, although this remains to be shown explicitly. The mechanisms orchestrating entry and exit of the DUBm from SAGA remain to be explored (Cloud, 2019).

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

dDsk2 (Ubqln2/Ubiquilin) is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. This study reports that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1 (monoubiquitylated H2B), a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications (Kessler, 2015).

This study reports that the extraproteasomal ubiquitin receptor dDsk2 interacts with the dHP1c complex, localizes at promoters and is required for transcription. Binding sites of the dHP1c complex are marked by H2Bub1 and the results suggest that, through the UBA domain, dDsk2 binds H2Bub1. However, reducing H2Bub1 levels affects binding of the complex only weakly, indicating that the interaction with H2Bub1 has only a minor contribution to recruitment. Actually, dDsk2 contains a single ubiquitin-binding site of low affinity (Kd~400 μM), which is in contrast to most ubiquitin receptors that contain several ubiquitin-binding sites that act synergistically to provide high-affinity binding. Similarly, the interaction of dDsk2 with H2Bub1 is of low specificity since selective recognition of ubiquitylated substrates is largely based on the recognition of the linkage type, length and anchoring site of a polyubiquitin chain, and, consequently, requires the presence of several ubiquitin-binding sites. In fact, the UBA domain of dDsk2 recognizes a monoubiquitylation in PTEN with a similar affinity as in H2B. On the other hand, binding of the dHP1c complex likely involves the recognition of specific DNA sequences since it depends on the zinc-finger proteins WOC and ROW. Noteworthy, dHP1c complex-binding sites are significantly enriched in a specific DNA sequence motif. However, although the interaction with H2Bub1 is weak, binding of the complex unexpectedly depends on dDsk2. Besides, dHP1c is dispensable for WOC and ROW binding, as well as for dDsk2 binding). These effects do not appear to be the consequence of changes in gene expression levels since dDsk2 mRNA levels do not significantly change on WOC, ROW or dHP1c depletion. Furthermore, dDsk2 depletion upregulates ROW and weakly downregulates dHP1c mRNA levels. Finally, it was reported that dHP1c interacts with RNA pol II, suggesting that binding of the dHP1c complex might depend on RNA pol II. However, arguing against this possibility, it was observed that binding of the complex at promoters is resistant to treatment with Actinomycin D. Altogether, these results suggest that WOC, ROW and dDsk2 constitute the actual binding module of the complex, being fully interdependent for binding to chromatin and required for binding of dHP1c. In this regard, the slight reduction of ROW and dHP1c protein levels observed on dDsk2 depletion is most likely due to their inability to bind chromatin, as described previously for dHP1c in ROW and WOC knockdowns, as well as for other chromatin-associated proteins when their binding to chromatin is impaired (Kessler, 2015).

These results suggest that the main function of dDsk2 in the dHP1c complex is to prevent H2Bub1 deubiquitylation by dUbp8/Nonstop, as H2Bub1 levels are strongly reduced on dDsk2 depletion and recovered after dUbp8/Nonstop co-depletion. Protection against deubiquitylation has also been reported for Rad23 and appears to be a common feature of many ubiquitin receptors. Simultaneous dDsk2 and dUbp8/Nonstop depletion also restores expression of target genes, whereas it has no effect on recruitment of the complex at promoters. Furthermore, overexpression of the ΔUBA–dDsk2 construct, which misses the UBA domain that mediates interaction with H2Bub1 in vitro, reduces H2Bub1 levels at promoters and downregulates expression of target genes. Altogether, these results strongly suggest that the contribution of dDsk2 to transcription regulation is mainly based on this protective function (Kessler, 2015).

dHP1c complex target genes show features associated with strong RNA pol II pausing. The results support a contribution of dDsk2 to pausing since its depletion reduces RNA pol II occupancy preferentially at TSS and strongly decreases NELF-E levels. dDsk2 depletion downregulates expression and, in good agreement, total RNA pol II occupancy across target genes is reduced. Interestingly, the majority of NELF target genes are also downregulated on NELF depletion in S2 cells51, and NELF potentiates gene expression in the Drosophila embryo. Actually, ~80% of dHP1c complex target genes that change expression in NELF knockdown conditions are downregulated. Furthermore, NELF-E depletion shows a similar reduction of total RNA pol II occupancy across target genes. Altogether, these observations suggest that disrupting RNA pol II pausing does not generally increase productive transcription, but results in reduced total RNA pol II occupancy and decreased expression. It is possible that premature pause release interferes with RNA pol II activation into elongation, resulting in abortive transcription that, in turn, could affect RNA pol II recruitment and/or re-initiation. Notice, however, that dDsk2 depletion does not affect the extent of histone acetylation detected at target promoters, suggesting that they retain the transcriptional active chromatin state (Kessler, 2015).

Notably, co-depletion of dUbp8/Nonstop, which rescues H2Bub1, also rescues the pausing defect caused by dDsk2 depletion and expression levels are restored, suggesting that dynamic regulation of H2Bub1 levels at promoters of dHP1c target genes plays a role in RNA pol II pausing. In this regard, work performed in yeast suggested that transcriptional activation involves sequential cycles of H2B ubiquitylation and deubiquitylation and that Ubp8 promotes Ctk1-dependent phosphorylation of Ser2 in the CTD33, a modification that is required for activation into the elongating Pol IIoser2 form. Nevertheless, H2Bub1 deubiquitylation at TSS does not appear to be sufficient by itself to induce pause release since dBre1 depletion, which also reduces H2Bub1 at TSS, has no significant effect in pausing. In this regard, it must be noted that dBre1 travels with the elongating RNA pol II along coding regions to induce H2Bub1, which stimulates Facilitates-Chromatin-Transcription (FACT) activity and, thus, facilitates elongation. On the other hand, dUbp8/Nonstop activity is mainly restricted to promoters since its depletion in dBre1-deficient cells has little effect in H2Bub1 levels at coding regions. On the contrary, dUbp8/Nonstop depletion in dDsk2-deficient cells strongly rescues H2Bub1 levels at coding regions. Interestingly, whereas dUbp8/Nonstop depletion restores expression of target genes in dDsk2-depleted cells, it has only a slight effect in dBre1-deficient cells. Altogether, these results suggest that dBre1 depletion impairs elongation and, thus, might prevent the release of paused RNA pol II by disturbing its actual engagement into elongation. Further work is required to better understand the mechanisms that regulate RNA pol II pausing, the actual contribution of dDsk2 and whether it involves H2Bub1 and/or additional factors also targeted by ubiquitylation (Kessler, 2015).

The dHP1c complex appears to have a particularly important contribution to nervous system development and function since target genes are enriched in related functions and knockdown conditions preferentially affect gene expression in the nervous system. Actually, WOC and ROW are highly expressed in the nervous system during embryo and larval development, and mutant larvae show brain defects. Furthermore, in humans, the WOC homologue DXS6673E/ZNF261 has been implicated in X-linked mental retardation. Interestingly, mutations in the human Dsk2 homologues (Ubqln-1/2) have been associated with Alzheimer's disease as well as other neurodegenerative diseases. Noteworthy, Ubqln-1/2 are detected in both the nucleus and the cytoplasm, and the development and progression of neurofibrillary tangles in Alzheimer's disease brains associate with an altered nuclear Ubqln-1 content. Whether the role of dDsk2 in transcription regulation is conserved in humans and contributes to disease remains to be determined (Kessler, 2015).

In summary, these results indicate that the ubiquitin receptor dDsk2 plays a nonproteolytic function in the regulation of H2Bub1 and RNA pol II pausing at promoters of dHP1c complex target genes. Ubiquitin receptors have been previously reported to play nonproteolytic functions in DNA repair and transcription elongation. Furthermore, in response to DNA damage, human Rad23B was found to interact with ubiquitylated p53, localize at chromatin and accumulate at the p21 promoter. In addition, in mouse embryonic stem cells, several components of the NER complex, including Rad23B, have been shown to act as an Oct4/Sox2 co-activator complex that associates with chromatin and is required for stem cell maintenance. Recruitment of NER factors to active promoters has also been reported in HeLa cells in the absence of DNA damage. However, in these cases, the precise function of the ubiquitin receptor has not been elucidated. In this regard, these results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications and transcription initiation. It must also be noted that ubiquitylation participates in the regulation of multiple genomic functions and that the number of proteins containing ubiquitin-binding domains is large, ~100 in humans. Therefore, a role of ubiquitin-binding proteins as epigenetic regulators of chromatin emerges as a distinct possibility (Kessler, 2015).


The non-stop expression pattern was examined in the target area as a first step toward assessing in which cells non-stop may function. In situ hybridization labeling of eye-brain complexes with non-stop antisense mRNA probes revealed weak expression in the optic lobe and higher expression in the lamina precursor cells. Since the resolution of in situ hybridization in the optic lobe is not high, protein expression was examined by placing a c-myc tag at the C terminus of the non-stop open reading frame in a genomic clone and the transgene was introduced into flies. Since this transgene rescues the projection phenotype and lethality associated with non-stop mutations and expression is similar in two independently derived transgenic lines, it is concluded that the transgene is expressed in a pattern similar to wild type. Anti-myc labeling reveals ubiquitous expression in the cytoplasm of cells in the optic lobe and central brain. Higher levels of expression, however, are observed in lamina precursor cells but not in differentiated lamina neurons. Non-stop is also expressed at higher levels in marginal, epithelial, and medulla glial cells adjacent to the lamina plexus (Poeck, 2001).

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).

The expression of the non-stop (not) gene decreased 3.9 fold from 24 to 40 hrs. Mutations in not result in photoreceptor neurons projecting through the lamina instead of terminating there. The mutations also result in approximately 20% of ommatidia being misoriented -- a planar polarity phenotype. Strong alleles of not die as prepupae so not clones were examined in both adult and pupal wings. Large numbers of clones were induced. Perhaps 25% of wing cells are found in clones. All adult wings of this genotype had regions where there were cells that failed to form hairs or that had very small hairs. These were found only in proximal medial regions on the ventral wing surface. All such wings also had subtle polarity abnormalities; small groups of hairs with slightly abnormal polarity in all regions of the wing. Consistently finding such defects leads to the conclusion that these were due to not clones. Of 47 such wings examined 27 also contained multiple hair cells and a further 10 contained regions with planar polarity defects reminiscent of genes such as fz and dsh. When marked not clones were examined in pupal wings most, but not all, showed cells where hair differentiation was delayed or absent. Such clones were seen in all wing regions. It is suggested that all not clones have delayed hair formation. When the clones are located in wing regions where hairs normally form first (distal or peripheral regions) the hairs form later than normal but still have enough time to reach a relatively normal length. In contrast, when clones are located in regions where hair formation is normally late (proximal and medial regions on the ventral wing surface) not enough time remains prior to cuticle deposition to produce a normal hair. The not gene encodes a ubiquitin carboxyterminal hydrolase likely to function in the removal of ubiquitin from proteins during protein degradation (Ren, 2005).

Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. This study analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5' end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression (Weake, 2011).

SAGA has been purified from Drosophila and mammalian cells and was found to contain homologs of most of the yeast SAGA subunits, including the Gcn5 (see Drosophila Pcaf) and Ubp8 catalytic subunits. In metazoans, SAGA may have roles in both normal development and cancer (Koutelou, 2010). Individual loss of the SAGA subunits Gcn5, Ada2b, Ada3, WDA, Sgf11, and SAF6 results in developmental defects and larval lethality in flies (Weake, 2009 and references therein). Similarly, Gcn5 deletion in mice leads to defects in mesoderm development and embryonic lethality (Xu, 2000). However, catalytic site mutations in Gcn5 survive longer but suffer neural tube closure defects and exencephaly (Bu, 2007). Furthermore, loss of the ubiquitin protease in Drosophila SAGA (Nonstop) leads to defects in photoreceptor axon targeting followed by lethality at late larval stages (Weake, 2011).

A system was designed in which SAGA could be isolated from different cell types in Drosophila embryos so that its composition and localization pattern could be determined in different tissues. To this end, the GAL4/UAS system was used to express a Flag-HA tagged version of the SAGA-specific protein Ada2b (Ada2bH1F2) in muscle or neuronal cells using the mef2-GAL4 and elav-GAL4 drivers, respectively. Expression of Ada2bH1F2 under the control of its genomic enhancer sequences rescues viability of the lethal ada2b1 allele. Whereas mef2 is expressed in committed mesoderm, the somatic and visceral musculature, and cardiac progenitors, elav is expressed prominently in neuronal cells and transiently in glial cells of the embryonic CNS. Ada2bH1F2 is expressed at levels similar to those of endogenous Ada2b using this system. To enrich for cell populations of interest that express tagged Ada2b, muscle and neuronal cells were labelled using GFP, and these cells were isolated using fluorescence-activated cell sorting (FACS). To determine whether the purified cells exhibit the characteristic gene expression profiles of each cell type, GFP-labeled neuronal and muscle cells were isolated from late stage embryos by FACS. RNA isolated from these tissues was compared with RNA extracted from whole embryos using cDNA microarrays, and genes were identified that are differentially expressed in muscle or neurons using significance analysis of microarrays. The differentially expressed genes identified using this approach were compared with ImaGO terms that describe the expression pattern of individual genes during Drosophila embryogenesis as determined by in situ hybridization. Genes identified as being expressed preferentially in muscle relative to neurons were enriched for ImaGO terms including embryonic/larval muscle system and dorsal prothoracic pharyngeal muscle. In contrast, genes identified as being expressed preferentially in neurons were enriched for ImaGO terms such as ventral nerve cord and dorsal/lateral sensory complexes. It is concluded that cells isolated using the FACS approach are enriched for the cell types of interest (Weake, 2011).

This study examined SAGA composition and localization in muscle and neuronal cells of late stage Drosophila embryos. Surprisingly, extensive colocalization of SAGA with Pol II was observed at both promoters and coding regions in muscle cells. Notably, genes at which SAGA was not detected in this assay have low levels of Pol II bound. It is suggested that SAGA might be important for recruitment and/or retention of high levels of Pol II at the promoter-proximal pause site in flies, and perhaps, therefore, more generally in higher eukaryotes. SAGA has been previously observed on the coding sequence of a small number of individual transcribed genes in yeast. Recently, low levels of Ada2b were detected on the 3' region of several different genes during larval development (Zsindely, 2009). It is noted that although some SAGA is present across the coding region of many genes, the peak of acetylated H3-Lys9 is restricted to the 5' region of the two genes that were examined: wupA and exba. A similar 5' bias of acetylated H3-Lys9 has been observed previously in genome-wide studies of histone modifications. It is speculated that the acetylation activity of SAGA in the 3' region of the gene is counteracted by histone deacetylases such as Rpd3S that have been shown to associate with the elongating form of Pol II (Weake, 2011).

SAGA localizes to different genes in muscle and neurons of late stage Drosophila embryos, and the number of genes bound by the complex in each tissue correlates with the number of transcription factors associated with the complex. These findings indicate that the differential localization of SAGA may be regulated by its association with different transcription factors in different cell types. A number of studies have found that transcription factor-binding sites tend to be clustered within the fly genome. This observed colocalization of transcription factors, together with the current data showing the association of SAGA with a large number of different transcription factors, indicates that multiple transcription factors might be involved in recruiting SAGA to its target genes (Weake, 2011).

SAGA is present at the promoter-proximal pause site together with Pol II at genes that are stalled or infrequently transcribed. The presence of SAGA together with paused Pol II is consistent with a role for SAGA in post-initiation deubiquitination of H2B, which has been shown in yeast to be important for phosphorylation of Ser-2 of the Pol II CTD and its subsequent transition into transcription elongation. In flies, phosphorylation of Ser-2 of the Pol II CTD by P-TEFb is also required for release of the paused polymerase into transcription elongation. Hence, the strong colocalization of SAGA with polymerase that has initiated transcription but is paused prior to elongation suggests a prominent function for SAGA in regulating tissue-specific gene expression at a step occurring post-initiation in metazoans. Consistent with the possibility, it was observe that the SAGA-bound genes that are most dependent on its ubiquitin protease activity for full expression are preferentially expressed in a specific tissue (Weake, 2011).


Biochemical and genetic studies support a role for Non-stop acting as a ubiquitin-specific protease. Three ubiquitinated proteins of 29, 55, and 200 kDa accumulated in P-element insert mutations not1 and not2 extracts of third instar larval tissue, as assessed on Western blots probed with an anti-Ubiquitin antibody. Both the non-stop targeting defect and prepupal lethality are dominantly enhanced by loss of one copy of a proteasome subunit encoded by l(3)73Ai. This suggests that Non-stop functions to regulate the degradation of a substrate via a ubiquitin-dependent pathway (Poeck, 2001).

It was necessary to show that non-stop is not required in the eye to regulate R cell target selection. To examine the genetic requirement of non-stop in targeting, the FLP/FRT system was used to induce eye-specific mitotic recombination and the marker Rh1-taulacZ was used to assess R1-R6 projections in adult cryostat sections. The eyeless-FLP (eyFLP) transgene was used to provide FLP recombinase activity in dividing cells of eye imaginal discs. To increase the size and number of clones in the eye, the FRT chromosome carried either a recessive cell lethal mutation (rfc) or a Minute mutation [M(3)RpS174]. Using an eye pigment marker, it was estimated that about 60% (with Rfc) to 80% (with RpS) of the cells in mosaic eyes were homozygous mutant. As in wild type, all axons of Rh1-taulacZ-expressing R cells in non-stop mutant eye tissue terminate in the lamina. Minor defects in ommatidial polarity and cell number were observed in non-stop mosaic eyes. However, because the projections are normal, these defects cannot account for the R1-R6 targeting errors. In further support of this finding, the projections of non-stop mutant R cell axons into a wild-type target in third instar larvae, as visualized by mAb24B10, were largely indistinguishable from wild type. Thus, it is concluded that non-stop function is required in the target but not in R cell axons for normal layer selection (Poeck, 2001).

If non-stop were to act in the target tissue, then it could do so in two different ways. It could be directly required for the production of a targeting signal. Alternatively, it could be required for the development of the cells expressing targeting signals. To distinguish between these possibilities, the development of lamina neurons and glial cells was examined in the target area in non-stop mutants. A subpopulation of neuroblasts in the outer proliferation center, adjacent to the developing lamina, generates lamina precursor cells (LPCs) that, in response to inductive signals from R cell axons, give rise to neurons. Older R cell axon bundles are associated with columns of lamina neurons and are shifted posteriorly as additional R cell axon bundles enter the target region. Lamina neuron development in non-stop mutants was assessed using the early neuronal differentiation marker Dachshund, the late neuronal differentiation marker Elav, and an antibody to a Brain-specific homeobox (Bsh) protein. In addition, the organization of lamina neurons into columns was examined. As in wild type, Dachshund is expressed in LPCs and was maintained in differentiating lamina neurons in non-stop mutants. Elav is expressed in mature lamina neurons L1-L5, and anti-Bsh is restricted to L5. Lamina neurons in non-stop mutants form columns largely as in wild type. Hence, non-stop is not required for lamina neuron differentiation (Poeck, 2001).

R cell axons encounter different glial cell types in the eye disc and in the optic stalk as they project into the optic lobe. Glial cells in the larval eye-brain complex can be visualized using the nuclear glial cell-specific marker anti-Repo. R cell axons grow past subretinal glial cells at the base of the optic stalk as well as satellite glial cells, which are interspersed among lamina neurons. R1-R6 growth cones terminate between rows of epithelial (distal) and marginal (proximal) glial cells. A third row of glial cells (medulla glial cells) lies beneath the marginal glial cells, demarcating the boundary between the developing lamina and medulla. Analysis of glial cell morphology using specific Gal4 drivers to express cytoplasmic ß-galactosidase has shown that epithelial and marginal glial cells have a unique morphology in comparison with the other glial cell types in the larval visual system. In contrast to the long ensheathing processes of eye disc, satellite, and medulla glial cells, epithelial and marginal glial cells assume a cuboidal shape and elaborate numerous fine processes, especially from the surface juxtaposing R1-R6 growth cones within the lamina plexus. The close contact between these glial cells and R1-R6 growth cones is consistent with a role as intermediate target cells (Poeck, 2001).

The development of glial cells in non-stop homozygous mutant eye-brain complexes was assessed using the marker anti-Repo. While glial cells found in the eye imaginal disc, optic stalk, and the entrance to the optic lobe appear normal in non-stop mutants, the rows of epithelial, marginal, and medulla glial cells are severely disrupted. The number of Repo-positive glial cells surrounding the lamina plexus is reduced in non-stop when compared to wild type. Whereas an average of 55 epithelial, marginal, and medulla glial cells is found in optical sections of third instar larval optic lobes in wild type, only about 20 glial cells are observed in non-stop mutants. The number of glial cells in wild type was determined using single optical sections, while the number of these cells in non-stop mutants was assessed using a merged image comprising between 4 and 11 1 µm thick optical sections. Hence, this difference is an underestimate (Poeck, 2001).

Glial cells in the target area originate from glial precursor cell areas that are located at the most dorsal and ventral edges of the R cell projection field on the surface of the optic lobe. Glial cells migrate to their characteristic positions along the lamina plexus. In wild type, a small number of migrating glial cells express Repo as they enter the R cell projection field. In non-stop mutants, however, a marked increase (57%; wild type, n = 13; non-stop, n = 31) is observed in the number of Repo-expressing glial cells in this region. This indicates that non-stop, either directly or indirectly, affects the migration of epithelial, marginal, and medulla glial cells into the target region (Poeck, 2001).

SAGA-mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system

Nonstop, which has previously been shown to have homology to ubiquitin proteases, is required for proper termination of axons R1-R6 in the optic lobe of the developing Drosophila eye. This study establish that Nonstop actually functions as an ubiquitin protease to control the levels of ubiquitinated histone H2B in flies. Nonstop is the functional homolog of yeast Ubp8, and can substitute for Ubp8 function in yeast cells. In yeast, Ubp8 activity requires Sgf11. In Drosophila, loss of Sgf11 function causes similar photoreceptor axon-targeting defects as loss of Nonstop. Ubp8 and Sgf11 are components of the yeast SAGA complex, suggesting that Nonstop function might be mediated through the Drosophila SAGA complex. Indeed, it was found that Nonstop does associate with SAGA components in flies, and mutants in other SAGA subunits display nonstop phenotypes, indicating that SAGA complex is required for accurate axon guidance in the optic lobe. Candidate genes regulated by SAGA that may be required for correct axon targeting were identified by microarray analysis of gene expression in SAGA mutants (Weake, 2008).

This study has identified a novel role for the coactivator complex dSAGA in Drosophila neural development. The Gcn5 (KAT2) HAT acts as the catalytic subunit of the yeast SAGA, SLIK, ADA and A2 multi-subunit protein complexes. Although many studies in multicellular organisms have focused on the HAT activity of the Gcn5 (KAT2) complexes, SAGA itself possesses a second catalytic activity. In addition to its HAT activity, yeast SAGA contains an H2B deubiquitinating enzyme, Ubp8. Ubp8 functions as part of a modular subunit domain within yeast SAGA that contains two additional proteins, Sgf11 and Sus1. Previous studies on histone deubiquitination have focused on its role in transcription in yeast. This study has characterized a role for histone deubiquitination in gene regulation in Drosophila (Weake, 2008).

Nonstop and Sgf11 constitute the H2B deubiquitination module within dSAGA. Moreover, both Nonstop and Sgf11 are required for correct axon targeting in the developing visual system. As in yeast, the two catalytic functions of SAGA are separable. However, mutations that differentially affect the two catalytic activities of dSAGA have overlapping but distinct effects on gene expression. Despite the differences in activity, both catalytic modules of dSAGA are required for correct axon targeting in the optic lobe. This implicates dSAGA in the regulation of pathways essential for neural development in higher eukaryotes (Weake, 2008).

This analysis of the axon-targeting defects in the nonstop, sgf11 and ada2b mutants indicates that the R-cell misprojection phenotype is associated in all three cases with a loss of glial cells from the lamina region of the optic lobe, and an increase in the number of glial cells at the dorsal and ventral margins of the lamina. Clonal analysis indicates that nonstop glial cells fail to migrate from these dorsal and ventral regions into the lamina plexus. This suggests that dSAGA may be required within glial cells to regulate pathways important for their migration. It is possible that the requirement of dSAGA may be due to its role in the ecdysone response as indicated by microarray analysis of genes downregulated in nonstop, sgf11 and ada2b mutant larvae. However, further studies on the role of dSAGA in these glial cells will be required to determine which pathway(s) are primarily responsible for the axon-targeting defect observed in these dSAGA mutants (Weake, 2008).

Mutations that affect the deubiquitination activity of dSAGA, such as nonstop and sgf11, appear to result in a more severe axon-targeting defect than those that affect the acetylation activity, such as ada2b. However, there is some variability in the degree of expressivity of the phenotype in all three mutant genotypes, and some ada2b mutants show phenotypes very similar to nonstop and sgf11. It is evident from studies on yeast SAGA that both enzymatic activities are required for optimal transcription upon gene induction. The variability in the ada2b mutant may be due to the large maternal contribution of Ada2b, and it is notable that the ada2b mutant larvae show considerably less developmental delay in comparison to nonstop, sgf11 and gcn5. Thus far, mutations in other components of dSAGA, such as wda and nipped-A, have not been examined for this phenotype because these do not reach the third instar larval stage of development (Weake, 2008).

Although in this study a specific effect was observed of the dSAGA deubiquitination module on histone H2B, it remains likely Nonstop and Sgf11 are also required for deubiquitination of other target proteins within the cell. Previous studies observed an increase in the level of three ubiquitinated proteins of 29, 55 and 200 kDa in extracts from nonstop third instar larval tissue relative to wild-type extracts. The smallest of these may correspond to ubiquitinated H2B, but the others may correspond to as yet unidentified potential targets of the deubiquitination module within dSAGA (Weake, 2008).

It is likely that the role of SAGA and H2B deubiquitination in neural development may be conserved in mammalian systems, as there is a striking degree of similarity between Sgf11 and ATXN7L3/ATXN7 in humans. ATXN7 is a subunit of the human STAGA and TFTC complexes. Polyglutamine expansions in the Spinocerebellar ataxia type 7 (sca7) gene, encoding ATXN7, result in a dominant neurodegenerative disorder that affects the retina. There is increased recruitment of STAGA/TFTC containing this polyQ-expanded ATXN7 at certain promoters in a mouse SCA7 model, resulting in hyperacetylation of H3. Interestingly, despite this increased promoter recruitment and hyperacetylation, these genes show decreased levels of transcription. However, in other studies incorporation of polyQ-expanded ATXN7 into STAGA reduces the acetyltransferase activity of the complex. The effect of the polyQ expansions in ATXN7 on the putative deubiquitination activity of mammalian STAGA/TFTC has not yet been examined (Weake, 2008).

Van der Knaap (2005) has demonstrated that another ubiquitin protease in flies, USP7, specifically deubiquitinates Histone H2B in vitro. However, RNAi of USP7 had little effect on global levels of ubiquitinated H2B, and it was found that USP7 cannot functionally replace UBP8 in yeast. Yeast Ubp15, the putative homolog of USP7, also deubiquitinates H2B in vitro, but the biological role of this activity is presently unknown. In addition to Ubp8 in yeast, Ubp10 is also required for deubiquitination of H2B at the telomeres and at the rDNA locus. It is unknown whether the putative homolog of Ubp10 in flies, CG15817, is also required for H2B deubiquitination and gene silencing. It is intriguing that both Ubp10 and USP7 (via an interaction with the Polycomb complex) function in regulating heterochromatin structure, while Ubp8/Nonstop are required for active transcription. It remains to be determined whether alternative mechanisms for regulating histone ubiquitination at distinct chromatin environments exist within the genomes of higher eukaryotes (Weake, 2008).

Taken together, these results advance the understanding of the role of histone deubiquitination in transcription, while demonstrating a novel role for SAGA in regulating neural development in the visual system of Drosophila. These findings have implications for the use of Drosophila as a model system to understand the underlying mechanisms of neurodegenerative diseases (Weake, 2008).


Search PubMed for articles about Drosophila non-stop

Badmos, H., Cobbe, N., Campbell, A., Jackson, R. and Bennett, D. (2021). Drosophila USP22/nonstop polarizes the actin cytoskeleton during collective border cell migration. J Cell Biol 220(7). PubMed ID: 33988679

Bu, P., Evrard, Y. A., Lozano, G. and Dent, S. Y. (2007). Loss of Gcn5 acetyltransferase activity leads to neural tube closure defects and exencephaly in mouse embryos. Mol. Cell Biol. 27: 3405-3416. PubMed Citation: 17325035

Cloud, V., Thapa, A., Morales-Sosa, P., Miller, T. M., Miller, S. A., Holsapple, D., Gerhart, P. M., Momtahan, E., Jack, J. L., Leiva, E., Rapp, S. R., Shelton, L. G., Pierce, R. A., Martin-Brown, S., Florens, L., Washburn, M. P. and Mohan, R. D. (2019). Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization. Elife 8. PubMed ID: 31348003

Erez, N., Israitel, L., Bitman-Lotan, E., Wong, W. H., Raz, G., Cornelio-Parra, D. V., Danial, S., Flint Brodsly, N., Belova, E., Maksimenko, O., Georgiev, P., Druley, T., Mohan, R. D. and Orian, A. (2021). A Non-stop identity complex (NIC) supervises enterocyte identity and protects from premature aging. Elife 10. PubMed ID: 33629655

Flint Brodsly, N., Bitman-Lotan, E., Boico, O., Shafat, A., Monastirioti, M., Gessler, M., Delidakis, C., Rincon-Arano, H. and Orian, A. (2019). The transcription factor Hey and nuclear lamins specify and maintain cell identity. Elife 8. PubMed ID: 31310235

Kessler, R., Tisserand, J., Font-Burgada, J., Reina, O., Coch, L., Attolini, C. S., Garcia-Bassets, I. and Azorin, F. (2015). dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes. Nat Commun 6: 7049. PubMed ID: 25916810

Koutelou, E., Hirsch, C. L. and Dent, S. Y. (2010). Multiple faces of the SAGA complex. Curr. Opin. Cell Biol. 22: 374-382. PubMed Citation: 20363118

Martin, K. A., Poeck, B., Roth, H., Ebens, A. J., Conley Ballard, L. and Zipursky, S. L. (1995). Mutations disrupting neuronal connectivity in the Drosophila visual system. Neuron 14: 229-240. 7857635

Mohan, R. D., Dialynas, G., Weake, V. M., Liu, J., Martin-Brown, S., Florens, L., Washburn, M. P., Workman, J. L. and Abmayr, S. M. (2014). Loss of Drosophila Ataxin-7, a SAGA subunit, reduces H2B ubiquitination and leads to neural and retinal degeneration. Genes Dev 28(3): 259-272. PubMed ID: 24493646

Monastirioti, M., Giagtzoglou, N., Koumbanakis, K. A., Zacharioudaki, E., Deligiannaki, M., Wech, I., Almeida, M., Preiss, A., Bray, S. and Delidakis, C. (2010). Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis. Development 137(2): 191-201. PubMed ID: 20040486

Pascual-Garcia, P., Debo, B., Aleman, J. R., Talamas, J. A., Lan, Y., Nguyen, N. H., Won, K. J. and Capelson, M. (2017). Metazoan nuclear pores provide a scaffold for poised genes and mediate induced enhancer-promoter contacts. Mol Cell 66(1): 63-76. PubMed ID: 28366641 28366641

Poeck, B., Fischer, S., Gunning, D., Zipursky, S. L. and Salecker, I. (2001). Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila. Neuron 29: 99-113. 11182084

Ren, N., Zhu, C., Lee, H. and Adler, P. N. (2005). Gene expression during Drosophila wing morphogenesis and differentiation. Genetics 171(2): 625-38. 15998724

Rørth, P., Szabo, K., and Texido, G. (2000). The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation. Mol. Cell 6: 23-30. PubMed Citation: 10949024

van der Knaap, J. A., et al. (2005). GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 17: 695-707. PubMed Citation: 15749019

Weake, V. M., et al. (2008). SAGA-mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system. EMBO J. 27(2): 394-405. PubMed Citation: 18188155

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 Citation: 20008933

Weake, V. M., et al. (2011). Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation. Genes Dev. 25(14): 1499-509. PubMed Citation: 21764853

Xu, W., et al. (2000). Loss of Gcn5l2 leads to increased apoptosis and mesodermal defects during mouse development. Nat. Genet. 26: 229-232. PubMed Citation: 11017084

Zsindely, N., et al. (2009). The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes. Nucleic Acids Res. 37: 6665-6680. PubMed Citation: 19740772

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date revised: 25 August 2021

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