InteractiveFly: GeneBrief

insensitive: Biological Overview | References


Gene name - insensitive

Synonyms - CG3227

Cytological map position - FBgn0031434

Function - transcriptional regulator

Keywords - Notch pathway, peripheral nervous system, can antagonize Notch independently of Hairless

Symbol - insv

FlyBase ID: FBgn0031434

Genetic map position - chr2L:2575276-2577006

Classification - BEN domain protein

Cellular location - nuclear



NCBI link: EntrezGene
insv orthologs: Biolitmine
Recent literature
Fedotova, A., Aoki, T., Rossier, M., Mishra, R., Clendine, C., Kyrchanova, O., Wolle, D., Bonchuk, A., Maeda, R., Mutero, A., Cleard, F., Mogila, V., Karch, F., Georgiev, P. and Schedl, P. (2018). The BEN domain protein insensitive binds to the Fab-7 chromatin boundary to establish proper segmental identity in Drosophila. Genetics. PubMed ID: 30082280
Summary:
Boundaries (insulators) in the Drosophila bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. Tnis study shows that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the Fab-7 nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ~100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (~420 kD and ~265-290 kD, respectively) in nuclear extracts. Using a sensitized genetic background this study showed that the Insv protein is required for Fab-7 boundary function, and that PS11 identity is not properly established in insv mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.
Fedotova, A., Clendinen, C., Bonchuk, A., Mogila, V., Aoki, T., Georgiev, P. and Schedl, P. (2019). Functional dissection of the developmentally restricted BEN domain chromatin boundary factor Insensitive. Epigenetics Chromatin 12(1): 2. PubMed ID: 30602385
Summary:
Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. This study used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to an ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7. It is concluded that the subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. These studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.
Mukherjee, S., Calvi, B. R., Hundley, H. A. and Sokol, N. S. (2022). MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine. Cell Rep 41(3): 111495. PubMed ID: 36261011
Summary:
Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood. This study reports that commitment to the terminally differentiated enterocyte (EC) cell fate is under microRNA control. An intestinally enriched microRNA, miR-956, fine-tunes Notch signaling activity specifically in intermediate, enteroblast (EB) progenitor cells to control EC differentiation. This study further identified insensitive mRNA as a target of miR-956 that regulates EB/EC ratios by repressing Notch activity in EBs. In summary, this study highlights a post-transcriptional gene-regulatory mechanism for controlling differentiation in an adult intestinal stem cell lineage.
Mukherjee, S., Calvi, B. R., Hundley, H. A. and Sokol, N. S. (2022). MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine. Cell Rep 41(3): 111495. PubMed ID: 36261011
Summary:
Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood. This study reports that commitment to the terminally differentiated enterocyte (EC) cell fate is under microRNA control. An intestinally enriched microRNA, miR-956, fine-tunes Notch signaling activity specifically in intermediate, enteroblast (EB) progenitor cells to control EC differentiation. This study further identified insensitive mRNA as a target of miR-956 that regulates EB/EC ratios by repressing Notch activity in EBs. In summary, this study highlights a post-transcriptional gene-regulatory mechanism for controlling differentiation in an adult intestinal stem cell lineage.
Mukherjee, S., Calvi, B. R., Hundley, H. A. and Sokol, N. S. (2022). MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine. Cell Rep 41(3): 111495. PubMed ID: 36261011
Summary:
Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood. This study reports that commitment to the terminally differentiated enterocyte (EC) cell fate is under microRNA control. An intestinally enriched microRNA, miR-956, fine-tunes Notch signaling activity specifically in intermediate, enteroblast (EB) progenitor cells to control EC differentiation. This study further identified insensitive mRNA as a target of miR-956 that regulates EB/EC ratios by repressing Notch activity in EBs. In summary, this study highlights a post-transcriptional gene-regulatory mechanism for controlling differentiation in an adult intestinal stem cell lineage.
BIOLOGICAL OVERVIEW

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).

BEN-solo factors partition active chromatin to ensure proper gene activation in Drosophila

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein-Elba3-bridges Elba1 and Elba2 to form an ELBA complex. This paper reports comprehensive investigation of these proteins in Drosophila embryos. Common and distinct binding sites for Insv and ELBA and their genetic interdependencies are assessed. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. It was further demonstrated that ELBA collaborates with other insulators to regulate developmental patterning. Finally, this study found that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units (Ueberschar, 2019).

Proper gene regulation requires coordinated activities of distinct classes of cis- and trans-regulators. Insulators (or boundary elements) are a special type of cis elements that constrain enhancer-promoter interactions and set chromatin boundaries. Historically, boundary or enhancer-blocking activities of newly identified insulators were mostly tested on a one-on-one basis in transgenic lines or genetically dissected for individual loci. Recent advances in genomics and chromatin structure capture techniques allowed more systematic identification of insulators and also assigned new properties to them in chromatin architecture organization (Ueberschar, 2019).

The activity of insulator elements depends upon their associated factors. The zinc-finger protein CTCF seems to be the only insulator protein conserved between vertebrates and invertebrates. In addition to its established roles as an insulator in chromatin organization, long-range regulatory element looping and enhancer segregating, several of the early studies on mammalian CTCF indicated that it functions in transcriptional repression. More than a dozen proteins have been shown to have insulator function in Drosophila. According to the combinatory co-occupancy patterns of the five insulator proteins CP190, BEAF-32, CTCF, Su(Hw), and Mod(mdg4), Drosophila insulators were divided into two classes. Class I insulators are mainly bound by CP190, BEAF-32, and CTCF in active chromatin regions proximal to promoters, while class II insulators are mostly bound by Su(Hw) located in distal intergenic loci. However, at the functional level, how these factors cooperate remains unclear (Ueberschar, 2019).

The BEN (BANP, E5R, and NAC1) domain is a recently recognized domain present in a variety of metazoan and viral proteins. Several BEN-containing proteins including mammalian BANP/SMAR1, NAC1, BEND3, and the C isoform of Drosophila Mod(mdg4) have chromatin-associated functions and have been linked to transcriptional silencing. The BEN domain has been shown to possesses an intrinsic sequence-specific DNA-binding activity. Mammalian RBB, a BEN and BTB domain protein, binds to and directly represses expression of the HDM2 oncogene through interaction with the nucleosome remodeling and deacetylase (NuRD) complex. Drosophila Insv binds to a palindromic motif, TCCAATTGGA and its variants (TCYAATHRGAA), and represses genes in the nervous system. Two other Drosophila BEN proteins, Elba1 and Elba2, along with the adaptor protein Elba3, are assembled in a heterotrimeric complex (ELBA) and associate with the asymmetric site 'CCAATAAG' in the Fab-7 insulator. elba1 and elba3 are closely linked in the genome and specifically expressed during the mid-blastula transition, which restricts ELBA activity to this early developmental window. Interestingly, the genes encoding Insv and Elba2 are also arranged next to each other in the genome, even though their gene products show different tissue specificity in later developmental stages (Ueberschar, 2019).

Most of the BEN-domain proteins contain other characterized motifs. However, Insv, Elba1, Elba2, and several mammalian homologs, such as BEND5 and BEND6, harbor only one BEN domain and lack other known functional domains. Thus, this sub-class is referred to as BEN-solo factors. Previous work demonstrated that Insv and ELBA BEN-solo factors share common properties, e.g., binding to the palindromic sites as homodimers and repressing reporter genes in cultured cells, but also display distinct activities, e.g., Insv being the only one that interacts with Notch signaling and its inability to bind to the asymmetric site. Interestingly, the Fab-7 insulator requires ELBA for its early boundary activity, but also needs Insv in later development (Ueberschar, 2019).

It remains to be determined how the ELBA factors regulate gene expression and embryogenesis. This study has comprehensively characterized the three Drosophila BEN-solo factors and the adapter protein Elba3, by analyzing DNA-binding preferences (symmetric versus asymmetric), chromatin binding inter-dependence (homodimers versus heterotrimeric complex) and mechanisms in gene regulation (repressor versus insulator). ChIP-seq (chromatin immunoprecipitation followed by deep sequencing) analyses show that ELBA and Insv bind many common and distinct genomic regions. Unexpectedly, Elba3 associates with chromatin even in the absence of its DNA-binding partners Elba1 and Elba2. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) assay distinguishes asymmetric heterotrimeric binding pattern of Elba1 and Elba2 from symmetric homodimer pattern of Insv. Although all four factors repress transcription, only the ELBA factors genetically interact with GAF and CP190 and are required for embryonic patterning. Finally, this study shows that adjacent genes separated by ELBA binding are less differentially expressed in the ELBA mutants. Insv-associated adjacent genes do not show such a global effect, despite individual loci relying on Insv insulation. And ELBA- and Insv-bound elements block enhancer-promoter interaction in transgenic reporters. Collectively, these findings indicate a role of ELBA and Insv as general insulators in partitioning transcription units in Drosophila (Ueberschar, 2019).

The BEN domains of Elba1, Elba2, and Insv share similar amino acid sequences and identical protein-DNA interaction sites. However, their DNA-binding activities seem to be complex. When expressed in cultured cells, all of these factors display high affinity to the palindromic site, while only the ELBA complex is able to bind the asymmetric site. In vitro translated proteins of Elba1 and Elba2 can bind to both types of motifs when additional bridging factor is present. ChIP-seq analyses confirm that in vivo Elba1 and Elba2 target the genome only through forming a heterotrimeric complex with Elba3. This suggests that the affinity of Elba1 and Elba2 binding to DNA is weak and needs to be enhanced by additional factors. The ChIP-seq analyses also demonstrate that Elba3 shows broader binding to the genome and is able to target many genomic loci in the absence of Elba1 and Elba2. The Elba3 protein does not have any known functional motif and not even a predictable DNA-binding domain. One potential factor that can bring Elba3 to chromatin is Insv. Indeed, the Elba3 peaks that are independent of Elba1/2 overlap more with Insv peaks. However, Insv is unlikely the only co-factor, as many of the Elba1/2-independent peaks do not overlap with Insv sites. Other insulator proteins with DNA-binding property, such as CP190 and GAF, are candidates that can bring Elba3 to the genome given that these factors co-occupy many genomic loci and display genetic interaction (Ueberschar, 2019).

This study used a high-resolution ChIP-nexus approach and confirmed that Insv and the ELBA factors associate with both types of DNA motifs. The ChIP-nexus analyses also provided evidence that Elba1 and Elba2 in the ELBA complex bind to DNA in an asymmetric configuration. Intriguingly, at some of the loci, such as the asymmetric sites in Fab-7 and Parp, Elba1 and Elba2 show '+' versus '-' strand preference. The genomic loci with asymmetric binding are expected to represent weak association of ELBA with DNA, as strong DNA binding would allow equal pull down of the subunits with the antibody against any of the three components. There are many other loci showing symmetric read distribution for Elba1 and Elba2. These sites either mediate strong binding of the complex or symmetric binding of Elba1 and Elba2 (e.g., as homodimers). Insv binding is always symmetric, suggesting that it binds to the sites as homodimers. This evidence shows ChIP-nexus can be a powerful tool to resolve binding symmetry by a heterotrimeric complex. It will be of interest to understand how the BEN domains have evolved in DNA-binding affinity and sequence specificity across species. Previous and current work well exemplifies the approach to determine the molecular properties of a less studied DNA-binding protein family (Ueberschar, 2019).

The activity of ELBA in the early embryo was examined by RNAi knockdown experiments, where it was shown to influence early boundary activity of the HS1 element. However, the effect of complete loss of ELBA in embryonic development has not been investigated. Loss-of-function mutants for the ELBA genes were generated, and the genes were found to be dispensable for viability. This is not surprising as other chromatin insulator proteins, such as dCTCF35 and BEAF-3236, are not required for viability. One possibility is that Drosophila utilizes multiple backup mechanisms to ensure boundary fidelity. Indeed, when one copy of CP190 or GAF is removed, loss of ELBA led to drastic developmental consequences in this sensitized background. Despite both ELBA and Insv associating with known insulator proteins, such as CP190, BEAF-32, and GAF, ELBA showed strong genetic interactions with CP190 and GAF in viability and early embryonic patterning, while insv did not. It is possible that Insv is less needed during the embryonic stage and/or that another unknown factor compensates for its joint function with CP190 and GAF. In support of the first possibility, insv is required for maintaining segmentation of adult flies when the GAF sites are mutated from Fab-725 (Ueberschar, 2019).

Genes in the Drosophila genome are more compact than in vertebrates. There is a need to partition dense transcription units to ensure enhancer specificity. Thanks to many years of genetic studies in Drosophila, a list of individual genomic loci were identified that separate enhancers or promoters. Insulator proteins such as GAF, CTCF, CP190, and BEAF-32 were found to mediate these activities. It was shown that BEAF-32 binds to sequences in between closely apposed genes with a head-to-head configuration (divergent). This study showed that the neighboring genes that are differentially expressed in wild type become more equally expressed in ELBA mutant embryos. In this case, all three types of promoter configurations, divergent, tandem, and convergent, have similar requirement for ELBA-dependent insulation. Together with the evidence that genomic elements bound by ELBA and Insv are sufficient to block enhancer-promoter interactions in transgene assays, the results suggest ELBA is required to ensure the autonomous regulation of linked transcription units (Ueberschar, 2019).

New properties have been assigned to insulators recently, especially in chromatin organization and long-range cis-element interactions. This work focused on the functions of ELBA and Insv in active chromatin regions because of their enrichment in close proximity to active promoters. However, enrichment of ELBA and Insv was detected in several known elements that could mediate long-range interactions, such as the homie-nhomie and scs and scs' loci. Future studies will be needed to determine the roles of ELBA and Insv in chromatin organization (Ueberschar, 2019).


Functions of Insensitive orthologs in other species

Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins

The BEN domain (see Drosophila Insensitive) is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. This study characterized several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. Detailed functional, genomic, and structural studies of BEND3 were conducted. BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. A high-resolution structure was obtained of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins (Zheng, 2022).


REFERENCES

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

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Biological Overview

date revised: 15 April 2020

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