InteractiveFly: GeneBrief

nab: Biological Overview | References

Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA-binding domains and act by associating with other transcription factors, but their precise roles in development are not known. This study analyzed the role of nab in Drosophila development. By employing genetic approaches it was found that nab is required for proximodistal patterning of the wing imaginal disc and also for determining specific neuronal fates in the embryonic CNS. Two partners of Nab were identified: the zinc-finger transcription factors Rotund and Squeeze. Nab is co-expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. These results indicate that Nab is a co-activator of Squeeze and is required to limit the number of neurons that express the LIM-homeodomain gene apterous and to specify Tv neuronal fate. Conversely, Nab is a co-repressor of Rotund in wing development and is required to limit the expression of wingless (wg) in the wing hinge, where wg plays a mitogenic role. Pull-down assays show that Nab binds directly to Rotund and Squeeze via its conserved C-terminal domain. Two mechanisms are described by which the activation of wg expression by Rotund in the wing hinge is repressed in the distal wing (Félix, 2007).

Precise temporal and spatial control of gene transcription is crucial for development. Sequence-specific DNA-binding factors and their association with a variety of modulator proteins, the co-factors, achieve this control. Co-factors do not bind DNA but act as adaptors between DNA-binding factors and other proteins. A number of transcription factors have been characterized, many of which act by recruiting multiprotein complexes with chromatin-modifying activities. By recruiting co-factors, a DNA-binding protein can act as co-activator or as co-repressor depending on the context. An example of a co-repressor is the retinoblastoma protein that converts the E2F transcription factor into a repressor of cell-cycle genes. The identification of co-factors and the determination of their precise roles are crucial for understanding the mechanisms that govern development (Félix, 2007).

Nab (NGFI-A-binding protein) proteins form an evolutionarily conserved family of transcriptional regulators. Nab was originally identified in mouse as a strong co-repressor by virtue of its capacity to interact directly with the Cys2-His2 zinc-finger transcription factor Egr1 (Krox24; NGFI-A) and inhibit its activity. Two Nab genes, Nab1 and Nab2, have been identified in vertebrates. Nab proteins do not bind DNA but they can repress (Svaren, 1998) or activate (Sevetson, 2000) gene expression by interacting with Egr transcription factors. Nab proteins have two regions of strong homology: NCD1 and NCD2. The NCD1 domain interacts with the R1 domain of Egr1 (Svaren, 1998). The NCD2 domain is required for transcriptional regulation (Swirnoff, 1998). Mice harboring targeted deletions of Nab1 and Nab2 have phenotypes very similar to Egr2 (Krox20)-deficient mice, suggesting that they act as co-activators of this gene (Le, 2005). In zebrafish, egr2 controls expression of the Nab gene homologs in the r3 and r5 rhombomeres of the developing hindbrain (Mechta-Grigoriou, 2000). Egr2 has been implicated in determining the segmental identities of r3 and r5 by controlling the expression of several target genes as well as cell proliferation. Misexpression experiments suggest that Nab1/Nab2 antagonize Egr2 transcriptional activity by a negative-feedback regulatory loop. Nevertheless, Nab proteins might have additional functions as these experiments also led to alterations of the neural tube not found in Egr2-deficient embryos (Mechta-Grigoriou, 2000). Conversely, Egr2-deficient mice have a severe hindbrain segmentation defect that is not found in mice deficient in Nab1 and Nab2. Nab might also have Egr-independent functions in mice because, although epidermal hyperplasia has been observed in Nab1 Nab2 double mutant mice, this phenotype has not been observed in mice lacking any of the Egr proteins (Le, 2005; Félix, 2007 and references therein).

In Drosophila, only one Nab gene has been identified; it is highly homologous to vertebrate Nab genes in the NCD1 and NCD2 domains. Drosophila nab mutants are early larval lethal. Detection of nab transcripts by in situ hybridization indicates expression in a subset of neuroblasts of the embryonic and larval CNS and weak expression in imaginal discs. Targeted deletion mutations in the nab gene have been created, and the identification of additional, EMS-induced dnab mutations by genetic complementation analysis has been described. Null alleles in nab cause larval locomotion defects and early larval lethality (L1-L2). A putative hypomorphic allele in nab instead causes early adult lethality due to severe locomotion defects. In the nab -/- CNS, axon outgrowth/guidance and glial development appear normal; however, a subset of eve+ neurons forms in reduced numbers. In addition, mosaic analysis in the eye reveals that nab -/- clones are either very small or absent. Similarly, Nab overexpression in the eye causes eyes to be very small with few ommatidia. These dramatic eye-specific phenotypes should prove useful for enhancer/suppressor screens to identify nab-interacting genes (Clements, 2003; full text of article).

During imaginal disc development, nab is strongly expressed in the wing presumptive domain under the control of vg, and nab is required in proximodistal axis development to control the expression of wg in the wing hinge. In early larval development, the wing fate is established in the distal-most region of the wing disc by a combination of two factors: activation of the gene vestigial (vg) and repression of the gene teashirt (tsh). Later, in early third instar larvae, wingless (wg) is activated in a ring of cells (the inner ring, IR) that borders the vg expression domain in the presumptive wing region. It has been suggested that activation of the IR involves a signal from the vg-expressing cells to the adjacent cells. Interpretation of this signal by the adjacent cells requires the transcription factors encoded by rotund (rn) and nubbin (nub). Expression of wg in the IR plays a mitogenic role; hence, as a consequence of wg expression, cells proliferate and the IR moves away from the vg border. At a distance from the source of the signal that drives the initial activation, wg IR expression is maintained by an autoregulatory loop that involves homothorax (hth). It is thought that an additional mechanism distally represses wg IR expression and, in so doing, controls cell proliferation in the wing hinge. This study shows nab is required in proximodistal axis development to control the expression of wg (Félix, 2007).

nab is also a component of the combinatorial code that determines the number of neurons that express the gene apterous (ap) in embryonic neural development, and nab specifies the Tv neuronal fate in the ap thoracic cluster of neurons. Two putative partners of Nab have been identified: Rn and Squeeze (Sqz). These proteins are members of the Krüppel family of zinc-finger proteins. Pull-down assays show that that Nab interacts with both proteins via a conserved C-terminal domain, and evidence is presented that Nab acts as co-activator of Sqz in embryo development and as co-repressor of Rn in wing development. Finally, it is proposed that there are two mechanisms to repress the activation of wg expression by Rn in the wing pouch: the first involves Nab as a co-repressor of Rn; the second involves Sqz as a competitor of Rn for binding to specific DNA target sites (Félix, 2007).

Expression of Nab

Antibody against Nab revealed a low level of expression in all imaginal discs. In late third instar wing discs, Nab was strongly expressed in a circular domain that delimits the expression of wg in the inner ring. Nab expression was first detected in early third instar larvae, in a group of cells of the distal-most wing, and was maintained throughout the remainder of the larval and pupal stages. There was a low level of expression in the rest of the wing disc, except in the hinge where there was no detectable expression. In the eye disc, Nab was detected in a stripe corresponding to the morphogenetic furrow (Félix, 2007).

Vestigial controls nab expression in the wing

It was asked whether, as with other genes involved in proximodistal patterning, nab expression in the wing is dependent upon vg. No expression of nab was detected in the distal wing of vg^83b27r wing discs. However, nab is ectopically expressed in clones of vg-expressing cells. Together, these results indicate that the expression of nab in the wing depends on vg. In wild-type discs and vg ectopic-expressing clones, the domain of nab expression is broader than that of vg, pointing to the nonautonomous control of nab expression. A similar mechanism has been proposed for other genes, such as rn and nub, whose expression depends on vg. Expression of vg in the wing starts in second instar larvae, whereas nab expression is first detected at early third instar. This suggests that some other mechanism controls the initiation of nab expression (Félix, 2007).

nab delimits the expression of wg in the wing inner ring

The nab^SH143 allele is a P(lacW) insertion in the first exon. Most larvae homozygous for this allele die in first instar. Thus, to analyze the role of nab in development of the wing nab^SH143 homozygous mutant clones were generated by mitotic recombination using the FLP/FRT mitotic recombination system. In the wing, these clones activated wg ectopically. However, it was noted that not all the clones activated wg. It is therefore possible that there is functional redundancy between Nab and other proteins (Félix, 2007).

Two enhancers drive the expression of wg in the wing: the wing margin enhancer, which is activated by the Notch signaling pathway, and the spade (spd) enhancer, which drives wg expression in the inner ring. Previous results suggest that activation by the latter depends on a nonautonomous signal coming from the vg-expressing cells. nab co-expresses with wg in the wing margin and abuts on wg expression in the inner ring. It was therefore assumed that Nab should repress activation of the inner ring enhancer derepressed in nab clones. To obtain independent evidence that the inner ring enhancer is being activated, tests were performed to see whether other genes activated in the wing margin were activated in the nab clones. To this end, cut (ct) was analyzed, and no ectopic expression was detected. It has been reported that wg expression can be detected in the wing after induction of cell death. To detect cell death in the nab clones, use was maed of an antibody that recognizes the activated form of Caspase 3, but no cell death was detected. These results, together with the pattern of expression, strongly suggest that the inner ring enhancer is being activated in the nab clones and, therefore, that in normal development Nab acts as a repressor of the wg inner ring enhancer in the distal wing. To confirm this hypothesis, nab was expressed ectopically in the inner ring domain using the nubGAL4 driver, which is expressed in a circular domain that includes the inner ring. In nubGAL4>UASnab larvae, expression of wg in the inner ring was lost, whereas its expression in the wing margin was not affected. Clones of nab-expressing cells were generated, and it was found that wg expression was cell-autonomously lost in these clones, whereas wg expression in the wing margin was not affected. In the light of these results, it is proposed that the function of nab in wing development is to delimit, distally, the domain of wg expression in the inner ring by inhibiting the mechanism of inner ring activation (Félix, 2007).

The Rn zinc-finger transcription factor is a potential partner of Nab in wing development

The mammalian Nab partner Egr1 contains an inhibitory domain called R1. When this domain is deleted the transcriptional activity of Egr1 increases 15-fold. It has been shown that the R1 domain mediates a functional interaction between Nab and Egr1. Since no R1 domain has been identified in the fly genome and all the previously identified partners of Nab are Krüppel-type zinc-finger transcription factors, transcription factors of the Krüppel family expressed in the wing were examined as potential Nab partners in the fly. The gene rn encodes a Krüppel-like zinc-finger protein that in the wing is expressed in a circular domain slightly broader than the nab domain. The wg inner ring enhancer is active only in the cells that express rn and that do not express nab. Previous studies have shown that Rn is required for activation of the wingless spd enhancer. The results so far suggest that Rn could be a partner of Nab in the wing: first, nab is expressed in the rn-expressing cells that do not express wg; second, nab loss-of-function clones contain ectopic Wg; and third, nab misexpression represses the wg inner ring enhancer (Félix, 2007).

rn was also expressed in leg discs in a broad ring that corresponded to three tarsal segments (T2-4). In rn mutant legs, the T2-4 tarsal segments were deleted. It would therefore be expected that if Rn were a partner of Nab, ectopic expression of nab in the leg would generate the same phenotype as the lack of Rn. This proved to be the case when nab was misexpressed in the rn expression domain under the control of the rnGal4 driver. The phenotype of these flies was indistinguishable from the rn mutant phenotype in both legs and wings. The specificity of this interaction was examined by rescuing the phenotype caused by nab misexpression by co-expressing rn (rnGal4>UASrn+UASnab), as well as by misexpressing nab in a broader domain using Distal-less Gal4 (DllGal4), which is expressed from mid-tibia to distal leg (DllGal4>UASrn). In the first experiment, the phenotype was markedly reduced in both wing and leg, indicating that adding more rn antagonizes the inhibitory effect of nab misexpression. In the second experiment, although nab was misexpressed in a broader domain of the leg, the phenotype was unaltered and was restricted to the area where rn was expressed. Taken together, these results support a role for Rn as a potential partner of Nab and that Nab acts as co-repressor of Rn function in the cells where both are expressed. The rn mutant phenotype in the wing is caused by the loss of wg expression in the inner ring. Whether wg expression was affected in rnGal4 UASnab and rnGal4 UASnab UASrn wings was examined. In the first case, the inner ring was found to be absent, whereas in the second it was partially restored. In summary, these results indicate that Nab functions in wing development by antagonizing the transcriptional activation function of Rn (Félix, 2007).

The Sqz zinc-finger transcription factor is a potential partner of Nab in neuronal fate specification

Although nab loss-of-function alleles are larval lethal, the rn-null condition is homozygous viable. This suggests that Nab may have at least one other partner in embryonic development. Rn belongs to a conserved subfamily of zinc-finger proteins that include Drosophila Sqz, C. elegans LIN-28 and rat Ciz. Sqz and Rn have two highly homologous domains: the zinc-finger domain (90% identity) and a 32 amino acid C-terminal domain (over 80% identity). sqz mutant alleles are larval lethal and have a motility defect. sqz is first required in embryonic CNS development to define the number of cells that express the LIM-homeodomain gene ap in the ap thoracic cluster of interneurons. Later on, it is also involved in the combinatorial code of transcription factors that specifies the fate of the Tv neuron in the ap cluster. The Tv neuron is distinguished from the rest of the neurons in the cluster by the fact that it contains the neuropeptide FMRFa [FMRFamide-related (Fmrf)]. In sqz mutant embryos, additional ap-expressing neurons are generated and the Tv neuron is not specified as no FMRFa expression is found (Allan, 2003). To determine whether Nab is a co-factor of Sqz, the expression of nab and sqz was examined in stage-17 embryos. It was found that a subset of the CNS neurons that express sqz also expressed nab, whereas other neurons express either sqz or nab. Two or three neurons in the ap cluster of stage-17 embryos express nab, one typically at a relatively high level of expression. By the first instar larval stage only one neuron in the ap cluster expressed nab. By double staining with anti-FMRFa and anti-Nab it was possible to identify this as the Tv neuron. At this stage, sqz was expressed at high levels in the Tv neuron and at low levels in two other neurons of the ap cluster. Next the expression of ap and FMRFa was analyzed in nab mutant larvae. In first instar nab^SH143 larvae, additional ap-expressing neurons were found in the ap cluster. In nab^SH143 embryos, additional cells expressed the bHLH gene dimmed (dimm), as shown for sqz mutants. The expression of FMRFa was analyzed in the ap clusters of first instar larvae, and FMRFa staining was lost or reduced in all the Tv neurons, mainly in the T1 cluster. It is concluded that lack-of-function alleles of nab and sqz generate the same embryonic phenotypes: the number of ap-expressing cells in the ap thoracic clusters is increased, additional dimm-expressing neurons are detected in the clusters, and Tv neuronal fate is absent. These results strongly suggest that, unlike the situation in imaginal disc development where Nab acts as a co-repressor of Rn, in CNS development Nab is required as a co-activator of Sqz (Félix, 2007).

Nab binds directly to Rn and Sqz via a conserved C-terminal domain

In order to analyze the molecular role of Nab as a co-factor of Sqz and Rn GST pull-down assays were performed. The complete nab cDNA was cloned in a glutathione S-transferase (GST) vector and incubated with radioactively labeled Rn or Sqz. Nab-GST, but not GST alone, readily retained [³⁵S]methionine-labeled Rn or Sqz. Rn and Sqz share a C-terminal domain of 32 amino acids with a homology greater than 80%. To further test whether this domain mediates the interaction with Nab, the pull-down assays were repeated with an [³⁵S]Rn in which the C-terminal domain was deleted. This deletion removes the region from amino acid 894 to the C-terminus (943) of the protein (Rn^Δ894). The ability of Nab-GST to retain the [³⁵S]Rn^Δ894 was notably reduced. It is concluded that this conserved domain mediates the direct interaction of Nab with Rn and Sqz. To further test whether the C-terminal domain is sufficient to mediate this interaction, the Nab-GST was incubated with a 32 amino acid peptide containing just the sequence of the C-terminal domain. Nab-GST did not retain the peptide, indicating that the C-terminal domain is not sufficient to mediate Nab-Rn interaction. Since no other conserved domains have been identified between Rn and Sqz besides the zinc-finger and C-terminal domains, it is considered that either secondary structure or an additional modification of the protein is required for binding Nab. In order to provide an in vivo functional test of this hypothesis, the rn^Δ894 fragment was cloned into the pUAST vector and clones of cells misexpressing UASrn^Δ894 were generated (Act>Gal4>UASrn^Δ894). These clones activated the expression of wg throughout the wing pouch. As a control experiment, the wild-type version of rn (Act>Gal4>UASrn) was misexpressed. These clones only activated wg expression in the wing hinge, outside of the nab expression domain (Félix, 2007).

Sqz competes with Rn in wing disc development

sqz expression was examined in the wing disc. Because of the high degree of sequence homology between rn and sqz and to avoid interference with the rn mRNA present in the wing, in situ hybridization assay was performed in rn mutant discs. sqz expression was detected by in situ hybridization in rn²⁰ wing discs in a circular pattern that faded off laterally and whose proximal limit coincided with the limit of vg expression; this corresponded to the distal-most wing fold. To determine whether sqz plays a role in wing development the phenotype was analyzed of sqz mutant clones induced by mitotic recombination. These clones had no adult phenotype, nor did they alter the expression of wg. Since Sqz and Rn share zinc-finger and the C-terminal domains and differ in their N-terminal domains, the roles of Sqz and Nab might be functionally redundant, both repressing Rn activity but by different mechanisms: Nab would repress Rn activity by direct binding to Rn protein as a co-repressor, whereas Sqz would compete for binding to the same DNA targets. To test this hypothesis, the effect was analyzed of misexpressing sqz in the rn expression domain. rnGal4/UASsqz UASGFP flies had small deletions of the wing hinge and shortened legs, a phenotype that resembles the nab misexpression and rn mutant phenotypes. In agreement with these results, wg expression in the inner ring was downregulated in rnGal4/UASsqz wing discs. An alternative explanation for these results is that sqz activates nab expression, but no nab misexpression was seen in this experiment. It is suggested that there must be some functional redundancy, irrespective of whether Nab and Sqz play similar roles in the wing by repressing Rn activity, and this would account for the low penetrance of the nab mutant clones. Because nab and sqz map on different chromosome arms it was not possible to generate double-mutant clones. Therefore nab^SH143 homozygous clones were generated in a sqz^lacZ/+ background. In this situation, the frequency of clones misexpressing wg increased by 38%). It was also noted that the clones that showed wg misexpression were preferentially located in the lateral-most regions of the wing, which correspond to the regions with the lowest levels of sqz expression. Taken together, these observations support the hypothesis that Nab and Sqz play similar roles in wing development: Nab as a co-repressor of Rn via its conserved C-terminal domain, and Sqz by competing with Rn for binding to its DNA targets. This function of Sqz would differ from its above-proposed role as a transcriptional activator in CNS development, and would not require Nab (Félix, 2007).

This study presented evidence that Nab is a co-activator of Sqz. This protein has been implicated in two aspects of embryonic ventral nerve cord development: first, in a Notch-dependent lateral inhibition mechanism that specifies the number of cells that express ap in the ap thoracic neuronal cluster; and second, in the specification of the Tv neuronal fate. nab and sqz are co-expressed in a subset of neurons, including several of the ap cluster, as well as the Tv neuron. nab loss-of-function embryos reproduce all the phenotypes of sqz loss-of-function embryos: additional cells express ap in the cluster and the Tv neuronal fate is lost. In addition, in both nab and sqz mutants an increased number of cells in the clusters express dimm. These findings indicate that Nab is required for all identified Sqz functions in embryonic development. Although this analysis focused on the ap thoracic cluster of neurons, both sqz and nab are co-expressed in many cells in the ventral nerve cord and others expressed either sqz or nab. But no other functions have been identified for sqz and it is not known how the expression of sqz is controlled. It has been reported that the expression of nab in the ventral nerve cord depends on the gene castor (Clements, 2003). Thus, the results presented in this study reveal greater complexity in the mechanisms of neuronal fate specification. The combined expression of genes, whose expression is individually activated by different mechanisms, is required to determine specific neuronal fates (Félix, 2007).

Sqz and Rn share two regions of strong homology: the zinc finger and a stretch of 32 amino acids in the C-terminal domain. By contrast, only rn has a long N-terminal domain. The results indicate that the C-terminal domain mediates the interaction with Nab. By GST pull-down assays, it was shown that Nab binds to the full-length Rn protein but not to the Rn^Δ894 version, and clones of cells misexpressing rn^Δ894 activate wg expression in the nab expression domain. The similarity between sqz misexpression and rn loss-of-function phenotypes in leg and wing suggests that Sqz acts like a dominant-negative form of Rn in the rn domain: both proteins would bind to the same target sites but have opposite effects, and the results indicate that this role of Sqz would not require interaction with Nab. It is possible that the long N-terminal region of Rn is involved in interaction with other partners specifically required for Rn function (Félix, 2007).

Thus, these results indicate that Nab has a dual role as co-repressor of Rn and co-activator of Sqz. Previous studies in vertebrates also suggest that Nab is involved in both repression and activation of transcription. Co-repressors are proteins that bridge the interaction of the repressor with its target. Two main co-repressors have been identified in Drosophila: Groucho and CtBP. CtBP binds to a specific sequence motif (P-DLS-K) that has been found in the sequence of three repressors present in the early embryo: Snail, Knirps and Krüppel. All three are zinc-finger transcription factors, and genetic evidence suggests that they all require CtBP to repress their targets. Neither Rn nor Sqz have a CtBP-binding motif but one has been in Nab (P-DLS--K). Although the functional significance of this motif remains to be confirmed, itis suggested that Nab is acting as a bridge between Rn and CtBP (Félix, 2007).

Search PubMed for articles about Drosophila Nab

Clements, M., Duncan, D. and Milbrandt, J. (2003). Drosophila NAB (dNAB) is an orphan transcriptional co-repressor required for correct CNS and eye development. Dev. Dyn. 226: 67-81. Medline abstract: 12508226

Felix, J. T., Magarinos, M. and Diaz-Benjumea, F. J. (2007). Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. Development 134(10): 1845-52. Medline abstract: 17428824

Le, N., Nagarajan, R., Wang, J. Y., Svaren, J., LaPash, C., Araki, T., Schmidt, R. E. and Milbrandt, J. (2005). Nab proteins are essential for peripheral nervous system myelination. Nat. Neurosci. 8: 932-940. Medline abstract: 16136673

Mechta-Grigoriou, F., Garel, S. and Charnay, P. (2000). Nab proteins mediate a negative feedback loop controlling Krox-20 activity in the developing hindbrain. Development 127: 119-128. Medline abstract: 10654606

Sevetson, B. R., Svaren, J. and Milbrandt, J. (2000). A novel activation function for NAB proteins in EGR-dependent transcription of the luteinizing hormone beta gene. J. Biol. Chem. 275: 9749-9757. Medline abstract: 10734128

Svaren, J., Sevetson, B. R., Golda, T., Stanton, J. J., Swirnoff, A. H. and Milbrandt, J. (1998). Novel mutants of NAB corepressors enhance activation by Egr transactivators. EMBO J. 17: 6010-6019. Medline abstract: 9774344

Swirnoff, A. H., Apel, E. D., Svaren, J., Sevetson, B. R., Zimonjic, D. B., Popescu, N. C. and Milbrandt, J. (1998). Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain. Mol. Cell. Biol. 18: 512-524. Medline abstract: 9418898

date revised: 3 October 2007

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.