unkempt: Biological Overview | References
Gene name - unkempt
Cytological map position - 94E1-94E2
Function - unknown
Keywords - works downstream of insulin receptor/mTOR pathway to regulate temporal control of neuronal differentiation
Symbol - unk
FlyBase ID: FBgn0004395
Genetic map position - chr3R:18975270-18983904
Classification - zf-C3HC4_3: Zinc finger, C3HC4 type (RING finger)
Cellular location - unknown
Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. The insulin receptor (InR)/mTOR pathway has been identified as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. This pathway has also been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway transcriptional targets of mTOR were screened for neuronal differentiation phenotypes in photoreceptor neurons. The conserved gene unkempt (unk), which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. The gene headcase (hdc) was identified a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, Unkempt-like is expressed in the developing mouse retina and in neural stem/progenitor cells, suggesting that the role of Unk in neurogenesis may be conserved in mammals (Avet-Rochex, 2014).
Neural progenitors in the developing human brain generate up to 250,000 neurons per minute. After differentiating from these neural progenitors, neurons migrate and are then integrated into neural circuits. Temporal control of neurogenesis is therefore critical to produce a complete and fully functional nervous system. Loss of the precise temporal control of neuronal cell fate can lead to defects in cognitive development and to neurodevelopmental disorders such as epilepsy and autism (Avet-Rochex, 2014).
Mechanistic target of rapamycin (mTOR) signalling has recently emerged as a key regulator of neurogenesis. mTOR is a large serine/threonine kinase that forms two complexes, known as mTORC1 and mTORC2. mTORC1 is rapamycin sensitive and is regulated upstream by mitogen signalling, such as the insulin receptor (InR)/insulin like growth factor (IGF) pathway, amino acids, hypoxia, cellular stress and energy levels (Zoncu, 2011). mTORC1 positively regulates a large number of cellular processes including growth, autophagy, mitochondrial biogenesis and lipid biosynthesis and activation of mTOR has been linked to cancer. Hyperactivation of mTOR signalling in neurological disease is best understood in the dominant genetic disorder tuberous sclerosis complex (TSC), which causes epilepsy and autism. mTOR signalling has also been shown to be activated in animal models of epilepsy and in human cortical dysplasia (Avet-Rochex, 2014).
The control of neurogenesis by the InR/mTOR pathway was first discovered in the developing Drosophila melanogaster retina, where activation of the pathway caused precocious differentiation of photoreceptor neurons and inhibition caused delayed differentiation (Bateman, 2004; Bateman, 2006; McNeill, 2008). Subsequent in vitro studies demonstrated that insulin induces neurogenesis of neonatal telencephalonic neural precursor cells in an mTOR dependent manner and that Pten negatively regulates neuronal differentiation of embryonic olfactory bulb precursor cells. More recently, in vivo studies have shown that inhibition of mTOR suppresses neuronal differentiation in the developing neural tube. Furthermore, knock-down of the mTOR pathway negative regulator RTP801/REDD1 causes precocious differentiation of neural progenitors in the mouse embryonic subventricular zone (SVZ), while overexpression of RTP801/REDD1 delays neuronal differentiation. Loss of Pten, Tsc1, or overexpression of an activated form of Rheb, also cause premature differentiation of neurons in the SVZ. These studies have demonstrated that InR/mTOR signalling plays a conserved role in regulating neurogenesis in several different neural tissues. However, the downstream effectors of InR/mTOR signalling in neurogenesis are completely unknown (Avet-Rochex, 2014 and references therein).
To identify neurogenic downstream regulators of InR/mTOR signalling, genes were screened that were previously shown to be transcriptionally regulated by mTOR in tissue culture cells (Guertin, 2006), for in vivo neurogenic phenotypes in the developing Drosophila retina. From this screen the zinc finger/RING domain protein Unkempt (Unk) was identified as a negative regulator of photoreceptor differentiation. Loss of unk phenocopies the differentiation phenotype of InR/mTOR pathway activation and Unk expression is negatively regulated by InR/mTOR signalling. Importantly, unk does not regulate cell proliferation or cell size and so uncouples the function of InR/mTOR signalling in growth from its role in neurogenesis. The evolutionarily conserved basic protein Headcase (Hdc) was identified as a physical interactor of Unk, and it was shown that loss of hdc causes precocious differentiation of photoreceptors. Hdc expression is regulated by the InR/mTOR pathway and by unk, demonstrating that Hdc and Unk work together downstream of InR/mTOR signalling in neurogenesis. Unk also regulates the expression of and interacts with D-Pax2 (Shaven/Sparkling), suggesting a model for the regulation of neurogenesis by the InR/mTOR pathway. It was also shown that one of the mammalian homologs of Unk, Unkempt-like, is expressed in the developing mouse retina and in the early postnatal brain. This study has thus identified the Unk/Hdc complex as the first component of the InR/mTOR pathway that regulates the timing of neuronal differentiation (Avet-Rochex, 2014).
Several lines of evidence together demonstrate that unk and hdc act downstream of InR/mTOR signalling to negatively regulate the timing of photoreceptor cell fate. First, loss of either unk or hdc causes precocious differentiation of the same cells and to the same degree as activation of InR/mTOR signalling. Second, the expression of both Unk and Hdc are regulated by InR/mTOR signalling. Third, loss of unk suppresses the strong delay in photoreceptor differentiation caused by inhibition of the InR/mTOR pathway and combined overexpression of unk and hdc suppresses the precocious photoreceptor differentiation caused by loss of Tsc1. Fourth, although Unk has been shown to physically interact with mTOR (Glatter, 2011), neither unk nor hdc regulate cell or tissue growth. Taken together these data show that unk and hdc are novel downstream components of the InR/mTOR pathway that regulate the timing of neuronal differentiation (Avet-Rochex, 2014).
InR/mTOR signalling is a major regulator of cell growth. In Drosophila activation of InR/mTOR signalling by loss of either Tsc1, Tsc2, Pten, or overexpression of Rheb causes increased cell size and proliferation. In the genetic disease TSC, which is caused by mutations in Tsc1 or Tsc2, patients develop benign tumours in multiple organs including the brain. The previously identified components of the InR/mTOR pathway regulate both growth and neurogenesis in Drosophila and vertebrate model. unk and hdc therefore represent a branchpoint in the pathway where its function in neurogenesis bifurcates from that in growth control. Moreover, analysis of unk and hdc demonstrates that regulation of cell growth can be uncoupled from and is not required for the function of InR/mTOR signalling in the temporal control of neuronal differentiation (Avet-Rochex, 2014).
At the protein level this study shows that Unk and Hdc physically interact in S2 cells. Although this interaction remains to be demonstrated in vivo, the additional observations that they both regulate each other's expression and act synergistically in vivo strongly support the model that they physically interact (see A model for the regulation of the timing of neuronal differentiation by the Unk/Hdc complex acting downstream of InR/mTOR signalling). Moreover, Unk and Hdc have also previously been shown to physically interact by yeast-2-hybrid and co-immunoprecipitation. Unk and Hdc are both expressed in all developing photoreceptors and so it is hypothesised that they control the timing of differentiation through the regulation of neurogenic factors whose expression is restricted to R1/6/7 and cone cells. Loss of unk causes increased expression of D-Pax2, the main regulator of cone cell differentiation. hdc and Tsc1 mutant clones also cause a similar increase in D-Pax2 expression. Overexpression of D-Pax2 alone is insufficient to induce cone cell differentiation, which requires overexpression of both D-Pax2 and Tramtrack88 (TTK88). Thus, regulation of D-Pax2 expression by mTOR signalling may contribute to the rate of cone cell differentiation, while overall control would require the regulation of additional factors such as TTK88. Pax8, part of the Pax2/Pax5/Pax8 paired domain transcription factor subgroup that is homologous to D-Pax2, has been shown to physically interact with one of the two human homologs of Unkempt. This study found that Drosophila Unk physically interacts with D-Pax2, demonstrating that the physical interaction between Unk and this group of transcription factors is conserved. It is suggested that D-Pax2 may be one of several neurogenic factors regulated by InR/mTOR signalling, through a physical interaction with the Unk/Hdc complex, to control the timing of R1/6/7 and cone cell fate (Avet-Rochex, 2014).
Unk has been shown to physically interact with mTOR and the strength of this interaction is regulated by insulin (Glatter, 2011). This suggests the intriguing possibility that the inhibition of Unk activity by InR/mTOR signalling is dependent on the strength of the physical interaction between Unk and the mTORC1 complex. Unk was also identified as part of the mTOR-regulated phosphoproteome in both human and murine cells. Thus, Unk may potentially be regulated by mTOR through phosphorylation. Future studies will fully characterise the mechanism by which mTORC1 regulates Unk activity (Avet-Rochex, 2014).
This study represents the first demonstration of a role for unk in specific developmental processes. By contrast, hdc has previously been shown to regulate dendritic pruning during metamorphosis and to act as a branching inhibitor during tracheal developmen. A screen for genes affecting tracheal tube morphogenesis and branching recently identified Tsc1, suggesting that InR/mTOR also regulates tracheal development. Thus, hdc and unk may act repeatedly as downstream effectors of the InR/mTOR pathway during Drosophila development (Avet-Rochex, 2014).
The one previous study of either of the mammalian Unk homologs showed that Unkl binds specifically to an activated form of the Rac1 GTPase (Lores, 2010). If this function is conserved in Drosophila then the defects in photoreceptor apical membrane morphogenesis caused by activation of mTOR signalling or loss of unk/hdc may be mediated through Rac1 (Avet-Rochex, 2014).
The function of the two unk homologs, unk and unkl, in mammalian development is not known, but unk has been shown to be expressed in the mouse early postnatal mouse retina. This study found that Unkl is also expressed in the developing mouse retina, suggesting that Unk may play a conserved role in eye development in both flies and mammals. InR/mTOR signalling acts as a pro-survival pathway preventing retinal degeneration, but its role in mammalian eye development has not been characterised. By contrast InR/mTOR signalling has a well characterised role in NSC self-renewal and differentiation in the mouse SVZ. Loss of Tsc1 or expression of a constitutively active form of Rheb in neural progenitor cells in the postnatal mouse SVZ causes the formation of heterotopias, ectopic neurons and olfactory micronodules. Furthermore, individuals with TSC, which results in activated mTOR signalling, have aberrant cortical neurogenesis and develop benign cortical tumours during foetal development and throughout childhood. mTOR signalling has been shown to be active in proliferative NSCs and TAPs in the neonatal SVZ and inhibition of mTOR signalling prevents NSC differentiation. This study found that Unkl is expressed in both NSCs and TAPs in the early postnatal SVZ. Thus, Unkl may regulate NSC differentiation downstream of mTOR signalling in the mammalian brain. Unkempt may therefore play a conserved role in regulating the timing of neural cell fate downstream of mTOR signalling in both flies and mammals (Avet-Rochex, 2014).
The SWI/SNF chromatin remodelling complexes are important regulators of transcription; they consist of large multisubunit assemblies containing either Brm or Brg1 as the catalytic ATPase subunit and a variable subset of approximately 10 Brg/Brm-associated factors (BAF). Among these factors, BAF60 proteins (BAF60a, BAF60b or BAF60c), which are found in most complexes, are thought to bridge interactions between transcription factors and SWI/SNF complexes. This study reports on a Rac-dependent process leading to BAF60b ubiquitination. Using two-hybrid cloning procedures, this study identified a mammalian RING finger protein homologous to Drosophila Unkempt as a new partner of the activated form of RacGTPases; mammalian Unkempt specifically binds to BAF60b and promotes its ubiquitination in a Rac1-dependent manner. Immunofluorescence studies demonstrated that Unkempt is primarily localized in the cytoplasmic compartment, but has the ability to shuttle between the nucleus and the cytoplasm, suggesting that the Rac- and Unkempt-dependent process leading to BAF60b ubiquitination takes place in the nuclear compartment. Ubiquitinated forms of BAF60b were found to accumulate upon treatment with the proteasome inhibitor MG132, indicating that BAF60b ubiquitination is of the degradative type and could regulate the level of BAF60b in SWI/SNF complexes. These observations support the new idea of a direct connection between Rac signalling and chromatin remodelling (Lores, 2010).
Although the results reported above are consistent with BAF60b being ubiquitinated through a Rac- and Unkempt-dependent process, the molecular composition of the E3 ligase involved and the role of Unkempt RING finger remain uncertain. On the basis of the results of a mutational analysis, it appears that the RING finger of exogenously expressed Unkempt is not critically involved in the ubiquitination reaction. A possible explanation is that exogenously expressed mutants of Unkempt form dimers/oligomers with endogenous Unkempt and/or associates with other RING finger protein(s), resulting in active E3 ligase. As already mentioned, there are multiple examples of RING E3s, the activity of which critically depends on multiprotein complexes, including homo- or hetero-oligomers of RING finger proteins. Of note, interaction between RING finger proteins does not necessarily depend on the RING finger motif itself. Thus, yBRE1, a RING finger protein involved in H2B ubiquitination in budding yeast, forms a homomeric complex, possibly a tetramer, through multiple intermolecular interactions, implicating only minimally the C-terminal RING finger. Similarly, in human, the RING finger type paralogs hBRE1A and hBRE1B form a heterotetramer and are both required for H2B ubiquitination, but the hBRE1B RING finger is dispensable. Another interesting example is provided by Pirh2, a p53-induced RING finger E3 ligase promoting ubiquitination and degradation of p53; very recently, isoforms of Pirh2 with a disrupted RING finger motif have been found capable of promoting p53 ubiquitination, possibly through their ability to interact directly with MDM2, the principal E3 ligase for p53. The RING finger protein Unkempt may share similarities with these models. It was recently observed that UNK-C-ter is capable of forming homomeric complexes in GST pull-down experiments; however, it remains to be demonstrated that an E3 ligase activity is associated with Unkempt homomers (or with heteromers involving an unidentified RING finger protein) and whether and how RacGTP regulates this putative E3 ligase. To address these issues, in vitro studies aimed at analysing intrinsic E3 ligase activity of recombinant Unkempt will be required (Lores, 2010).
The results also raise the questions of the physiological relevance and significance of BAF60b ubiquitination. Unfortunately, using available antibodies to BAF60b, no ubiquitinated forms of endogenous BAF60b were detected. However, in HeLa cells expressing exogenous BAF60b, it was found that BAF60b is significantly ubiquitinated, even in the absence of exogenous Unkempt; in addition, the ubiquitinated forms of BAF60b strongly accumulated in the presence of MG132, suggesting that the fate of ubiquitinated BAF60b is proteasomal degradation. Thus, it may be that ubiquitination results in degradation of an excess of BAF60b subunits, thereby allowing the stoichiometry of SWI/SNF complexes to be maintained. Another interesting possibility would be that BAF60b, alone or in complex with Unkempt, interacts with other unidentified substrates of Unkempt-dependent E3 ligase. As previously mentioned, BAF60 proteins are thought to bridge interactions between transcription factors and SWI/SNF complexes; therefore, candidate substrates include other constituents of SWI/SNF complexes, some of which have been found to be regulated by proteasomal degradation, and transcription factors targeted by BAF60b that remain to be defined (Lores, 2010).
Whatever the precise mechanisms are, Unkempt may be importantly linked to the physiological control of the SWI/SNF complexes, thus opening up a direct connection between Rac signalling and chromatin remodelling (Lores, 2010).
The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys3His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development (Mohler, 1992).
Search PubMed for articles about Drosophila Unkempt
Avet-Rochex, A., Carvajal, N., Christoforou, C. P., Yeung, K., Maierbrugger, K. T., Hobbs, C., Lalli, G., Cagin, U., Plachot, C., McNeill, H., Bateman, J. M. (2014). Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control. PLoS Genet 10: e1004624. PubMed ID: 25210733
Bateman, J. M. and McNeill, H. (2004). Temporal control of differentiation by the insulin receptor/tor pathway in Drosophila. Cell 119: 87-96. PubMed ID: 15454083
Bateman, J. M. and McNeill, H. (2006). Insulin/IGF signalling in neurogenesis. Cell Mol Life Sci 63: 1701-1705. PubMed ID: 16786222
Glatter, T., Schittenhelm, R. B., Rinner, O., Roguska, K., Wepf, A., Junger, M. A., Kohler, K., Jevtov, I., Choi, H., Schmidt, A., Nesvizhskii, A. I., Stocker, H., Hafen, E., Aebersold, R. and Gstaiger, M. (2011). Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome. Mol Syst Biol 7: 547. PubMed ID: 22068330
Guertin, D. A., Guntur, K. V., Bell, G. W., Thoreen, C. C. and Sabatini, D. M. (2006). Functional genomics identifies TOR-regulated genes that control growth and division. Curr Biol 16: 958-970. PubMed ID: 16713952
Lores, P., Visvikis, O., Luna, R., Lemichez, E. and Gacon, G. (2010). The SWI/SNF protein BAF60b is ubiquitinated through a signalling process involving Rac GTPase and the RING finger protein Unkempt. FEBS J 277: 1453-1464. PubMed ID: 20148946
McNeill, H., Craig, G. M. and Bateman, J. M. (2008). Regulation of neurogenesis and epidermal growth factor receptor signaling by the insulin receptor/target of rapamycin pathway in Drosophila. Genetics 179: 843-853. PubMed ID: 18505882
Mohler, J., Weiss, N., Murli, S., Mohammadi, S., Vani, K., Vasilakis, G., Song, C. H., Epstein, A., Kuang, T., English, J. and et al. (1992). The embryonically active gene, unkempt, of Drosophila encodes a Cys3His finger protein. Genetics 131: 377-388. PubMed ID: 1339381
Zoncu, R., Efeyan, A. and Sabatini, D. M. (2011). mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12: 21-35. PubMed ID: 21157483
date revised: 5 October 2014
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