insensitive: Biological Overview | References
Gene name - insensitive
Synonyms - CG3227
Cytological map position - FBgn0031434
Function - transcriptional regulator
Symbol - insv
FlyBase ID: FBgn0031434
Genetic map position - chr2L:2575276-2577006
Classification - BEN domain protein
Cellular location - nuclear
The Notch intracellular domain functions as a co-activator for the DNA-binding protein Suppressor of Hairless [Su(H)] to mediate myriad cell fate decisions. Notch pathway activity is balanced by transcriptional repression, mediated by Su(H) in concert with its Drosophila corepressor Hairless. This study demonstrates that the Drosophila neural BEN-solo protein Insensitive (Insv) is a nuclear factor that inhibits Notch signalling during multiple peripheral nervous system cell fate decisions. Endogenous Insv was particularly critical when repressor activity of Su(H) was compromised. Reciprocally, ectopic Insv generated several Notch loss-of-function phenotypes, repressed most Notch targets in the E(spl)-C, and opposed Notch-mediated activation of an E(spl)m3-luc reporter. A direct role for Insv in transcriptional repression was indicated by binding of Insv to Su(H), and by strong chromatin immunoprecipitation of endogenous Insv to most E(spl)-C loci. Strikingly, ectopic Insv fully rescued sensory organ precursors in Hairless null clones, indicating that Insv can antagonize Notch independently of Hairless. These data shed first light on the in vivo function for a BEN-solo protein as an Su(H) corepressor in the Notch pathway regulating neural development (Duan, 2011).
The peripheral nervous system (PNS) of Drosophila includes hundreds of mechanosensory organs arranged in characteristic patterns. Major aspects of the developmental progression of peripheral sensory organs are well understood. Within an initially undifferentiated ectodermal field, groups of cells termed proneural clusters (PNCs) selectively express basic helix-loop-helix (bHLH) activators, whose patterned activity defines territories of neural competence. Cell interactions among PNC cells, mediated by the Notch receptor and its associated signalling cascade, restrict neural potential to singular cells known as sensory organ precursors (SOPs); the remaining PNC cells eventually adopt an ordinary epidermal fate. At this stage, a loss of Notch signalling results in multiple SOPs emerging from a PNC, while a gain of Notch signalling extinguishes the SOP fate (Duan, 2011).
Once stably selected, each SOP executes a stereotyped series of asymmetric cell divisions. The first SOP division produces two cells termed pIIA and pIIB. pIIA generates socket and shaft cells, which are visible on the fly exterior. pIIB undergoes two sets of divisions yielding several internal cells, a glial cell, a sheath cell, and the neuron; the glial cell is apoptotic in mechanosensory organ lineages. Notch signalling operates at each division to guarantee the distinct developmental choices of each pair of daughter cells. The neuron escapes Notch activation throughout the sensory lineage, while the socket cell derives from cells that consistently activate the pathway. Consequently in Notch mutant clones, all cells of peripheral sensory lineages adopt the neural fate, while hyperactivation of Notch activity within the sensory lineage can yield mutant organs composed exclusively of sockets (Duan, 2011).
Upon activation by ligand, the Notch receptor undergoes a series of proteolytic cleavages, resulting in the release and nuclear translocation of its intracellular domain (NICD). This fragment binds directly to members of the CSL (for vertebrate CBF1, Drosophila Suppressor of Hairless (Su(H)), and nematode LAG-1) family of transcription factors, which mediate most if not all of the nuclear aspects of Notch signalling. Although originally recognized as a transcriptional repressor in cultured cells, CSL proteins were subsequently found to mediate activation of Notch target genes in vivo. These opposing activities have been reconciled by a 'switch; model in which CSL proteins repress target genes in the absence of signalling via associated corepressor molecules, but activate target genes via NICD and associated co-activator molecules (Duan, 2011).
The specific roles of CSL-mediated repression can be difficult to recognize owing to the massive and pleiotropic defects induced by loss of Notch signalling. Nevertheless, substantial mutant phenotypes have been observed in the appropriate genetic contexts. For example, Drosophila mutants of the dedicated Su(H) corepressor encoded by Hairless reveal many phenotypes in both inhibitory and inductive contexts of Notch signalling that reflect elevated Notch signalling. The asymmetry of pIIa division is particularly sensitive to Su(H) repressor function, since Hairless heterozygotes exhibit a number of double-socket organs that reflect Notch pathway gain-of-function (Duan, 2011).
This study characterizes Drosophila insensitive (insv) that encodes a novel protein containing a BEN domain. Null mutants of insv were earlier reported to be lethal and to exhibit Notch gain-of-function phenotypes in notum clones (Reeves, 2005). These phenotypes were confounded by simultaneous loss of the Notch antagonist lethal giant larvae from available alleles (Roegiers, 2009). Nevertheless, upon cleaning of these stocks, viable insv mutant animals maintained detectable Notch gain-of-function PNS phenotypes that were fully rescued by insv genomic DNA. Detailed genetic interaction analysis revealed the endogenous role of Insv to restrain Notch signalling during multiple cell fate decisions, including SOP specification, pIIA-pIIB decision, and socket-shaft decision. The nuclear localization of Insv suggested that it might regulate Notch target gene expression. Consistent with this hypothesis, ectopic Insv generated multiple Notch loss-of-function phenotypes, strongly repressed the expression of an array of Notch target genes across the Enhancer of split-Complex, and suppressed Notch-mediated activation of an E(spl)m3-luc reporter in cultured cells (Duan, 2011).
It was determined that Insv is a direct corepressor for Su(H), as revealed by protein-protein interactions in vitro and strong binding of endogenous Insv to multiple Su(H) target genes by chromatin immunoprecipitation (ChIP). While both Insv and Hairless bind Su(H), ectopic Insv supported SOP specification in null clones of Hairless, and could in fact generate a lateral inhibition defect in Hairless clones, as in wild-type. Therefore, Insv is capable of inhibiting Notch signalling independently of Hairless. Altogether, these findings shed first light on a member of the BEN-solo protein family as an Su(H) corepressor that regulates multiple Notch-mediated cell fate decisions during neural development (Duan, 2011).
Insensitive (insv) is an SOP-specific gene product of novel structure, containing only the domain of unknown function 1172 (DUF1172; Reeves, 2005). An extended version of DUF1172 was recently recognized across a set of >100 animal and viral proteins, and renamed the BEN domain (Abhiman, 2008). BEN domains are often found in association with other domains with chromatin-relevant functions (e.g., POZ, SCML1, or MCAF N-terminal domains). However, Insv belongs to a family of invertebrate and vertebrate proteins containing only the BEN domain ('BEN-solo' proteins), which have been little studied to date (Duan, 2011).
Insv, as detected using an antiserum, accumulates in pupal SOP/pI cells at 14 h after puparium formation (APF), colocalizing with the nuclear transcription factor Senseless (Sens). Notably, Insv appears exclusively nuclear, potentially reflecting a chromatin-associated role (Duan, 2011).
Insv expression was traced through the bristle lineage. Insv was detected in both pIIA and pIIB, and was later seen in their daughters at the 4-cell stage. However, Insv was strongly downregulated in all but one of the lineage cells. Insv was extinguished in this cell before expression of typical markers of terminal PNS cell fates, such as Prospero (marking the sheath cell) or Elav (marking the neuron). However, weak co-expression of Insv and Elav was seen at a number of positions, while Insv never colocalized with Pros. This identified the last Insv+ cell in the microchaete lineage as the neuron. The accumulation of Insv in SOPs and nascent neurons is analogous in the sense that neither of these cells activates Notch signalling during PNS development (Duan, 2011).
Default repression by members of the conserved CSL transcription factor family is critical for proper cell fate decisions mediated by Notch signalling. Curiously, while activation of Notch target genes involves a conserved N[ICD]-Mastermind-CSL complex, a diversity of corepressor complexes have been defined in invertebrate and vertebrate systems. The major corepressor for the Drosophila CSL protein Su(H) is Hairless, an adaptor protein that recruits both CtBP and Groucho repressor complexes. Mammalian Hairless proteins have not been identified; however, it should be noted that Hairless is extremely rapidly evolving and not trivial to identify even in other insects. Therefore, the absence of mammalian proteins aligning to Hairless is not necessarily conclusive. On the other hand, mammalian SHARP and CIR were reported to bind the mammalian CSL protein CBF1, and recruit SMRT/N-CoR and HDAC repressor complexes (Hsieh, 1999: Oswald, 2005). Recently, the histone demethylase KDM5A/Lid was reported to be a direct partner of both CBF1 and Su(H) corepressor complexes (Moshkin, 2009: Liefke, 2010), although KDM5A/Lid is also documented to have pleiotropic functions involving diverse DNA binding partners such as Rb, Myc, and PRC2 (Duan, 2011 and references therein).
Genetic and biochemical studies show that Insv is a neural nuclear protein that functions as a direct Su(H) partner to antagonize Notch pathway activity during multiple steps of Drosophila peripheral neurogenesis. These data shed first light on the in vivo function for a BEN-solo protein as a neural corepressor in the Notch pathway. Although the phenotypes of insv mutants are mild, they were seen in multiple allelic combinations and were fully rescued by insv genomic DNA. More substantially, insv mutants exhibited strong genetic interactions with several Notch pathway alterations. This genetic situation is not unique to insv, as other critical components of the Notch pathway exhibit redundancy in the nervous system (e.g., multiple E(spl)bHLH-encoding genes must be removed to reveal strong neurogenic defect, and both Notch ligands must be removed to reveal PNS lineage defects. Perhaps most striking is the fact that shaft cell specification completely fails in insv mutants where Su(H) corepressor function is reduced by heterozygosity of Hairless, the major direct Su(H) corepressor identified to date. Reciprocally, elevation of Insv level completely compensates for the null condition of Hairless during SOP specification, and can partially rescue the specification of internal cells including neurons. In fact, ectopic Insv can still generate a Notch loss-of-function lateral inhibition defect without Hairless. These data do not rule out the possibility of a trimeric Su(H)-Hairless-Insv repression complex, but they indicate that Insv does not require Hairless to mediate in vivo repression by Su(H). Preliminary tests indicate that Insv may not bind directly to Groucho, as shown for Hairless. However, now that a molecular function has been assigned to Insv, future studies can be aimed at understanding how it interfaces with other silencing proteins and perhaps eventually to chromatin modifying enzymes (Duan, 2011).
The Drosophila genome contains other loci encoding BEN domains (Abhiman, 2008), including other BEN-solo factors (CG9883 and CG12205) and mod(mdg4), a highly alternatively spliced locus that encodes proteins with BEN and POZ domains. It remains to be seen whether the Drosophila BEN proteins exhibit any functional overlap. More generally, the data shed light on the in vivo function for a BEN-solo protein as a corepressor in the Notch pathway. Other BEN domain proteins containing BTB/POZ domains have been linked to transcriptional repression (vertebrate NAC1) and enhancer blocking [Drosophila mod(mdg4)] activities, and the mammalian BEN-solo protein SMAR1/BANP recruits the SIN3/HDAC1 repressor complex. The data add to a growing theme for BEN factor involvement in transcriptional repression. While there are not clear mammalian orthologues of Insv, they do express several BEN-solo proteins (Abhiman, 2008). In light of the relatively specific effects of Insv in Notch-mediated cell fate decisions in both endogenous and ectopic contexts, these studies generate hypotheses to direct the study of mammalian BEN-solo proteins (Duan, 2011).
Finally, it is noted that BEN domains are also encoded by viral genomes, including the BEN-solo protein Chordopox E5R_VVC_137623. Viral proteins such as Epstein Barr viral oncoprotein EBNA2 and the adenoviral oncoprotein 13S E1A bind CBF1 and function as NICD mimics. This elucidation of a BEN-solo protein as a CSL corepressor raises the possibility that viruses may have co-opted cellular proteins to dominantly repress Notch signalling (Duan, 2011).
Search PubMed for articles about Drosophila Insensitive
Abhiman, S., Iyer, L. M. and Aravind, L. (2008). BEN: a novel domain in chromatin factors and DNA viral proteins. Bioinformatics 24: 458-461. PubMed ID: 18203771
Duan, H., et al. (2011). Insensitive is a corepressor for Suppressor of Hairless and regulates Notch signalling during neural development. EMBO J. 30(15): 3120-33. PubMed ID: 21765394
Hsieh, J. J., et al. (1999). CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proc. Natl. Acad. Sci. 96: 23-28. PubMed ID: 9874765
Liefke, R., et al. (2010). Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex. Genes Dev. 24: 590-601. PubMed ID: 20231316
Moshkin, Y. M., et al. (2009). Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing. Mol. Cell 35: 782-793. PubMed ID: 19782028
Oswald, F., et al. (2005) RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes. Mol. Cell Biol. 25: 10379-10390. PubMed ID: 16287852
Reeves, N. and Posakony, J. W. (2005). Genetic programs activated by proneural proteins in the developing Drosophila PNS. Dev. Cell 8: 413-425. PubMed ID: 15737936
Roegiers, F., et al. (2009). Frequent unanticipated alleles of lethal giant larvae in Drosophila second chromosome stocks. Genetics 182: 407-410. PubMed ID: 9279324
date revised: 20 November 2011
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