nubbin/POU domain protein 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - nubbin

Synonyms - dOct1, dPOU-19, twain, POU domain protein 1

Cytological map position - 33-F1

Function - transcription factor

Keyword(s) - CNS, PNS and endoderm

Symbol - nub

FlyBase ID:FBgn0085424

Genetic map position - 2 -[46]

Classification - homeodomain and POU domain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | HomoloGene | UniGene

Recent literature
Corty, M.M., Tam, J. and Grueber, W.B. (2016). Dendritic diversification through transcription factor-mediated suppression of alternative morphologies. Development 143: 1351-1362. PubMed ID: 27095495
Summary:
Neurons display a striking degree of functional and morphological diversity, and the developmental mechanisms that underlie diversification are of significant interest for understanding neural circuit assembly and function. This study finds that the morphology of Drosophila sensory neurons is diversified through a series of suppressive transcriptional interactions involving the POU domain transcription factors Pdm1 (Nubbin) and Pdm2, the homeodomain transcription factor Cut, and the transcriptional regulators Scalloped and Vestigial. Pdm1 and Pdm2 are expressed in a subset of proprioceptive sensory neurons and function to inhibit dendrite growth and branching. A subset of touch receptors show a capacity to express Pdm1/2, but Cut represses this expression and promotes more complex dendritic arbors. Levels of Cut expression are diversified in distinct sensory neurons by selective expression of Scalloped and Vestigial. Different levels of Cut impact dendritic complexity and, consistent with this, it was found that Scalloped and Vestigial suppress terminal dendritic branching. This transcriptional hierarchy therefore acts to suppress alternative morphologies to diversify three distinct types of somatosensory neurons. 

Dantoft, W., Lundin, D., Esfahani, S.S. and Engström, Y. (2016). The POU/Oct transcription factor Pdm1/nub is necessary for a beneficial gut microbiota and normal lifespan of Drosophila. J Innate Immun 8: 412-426. PubMed ID: 27231014
Summary:
Maintenance of a stable gut microbial community relies on a delicate balance between immune defense and immune tolerance. This study used Drosophila to study how the microbial gut flora is affected by changes in host genetic factors and immunity. Flies with a constitutively active gut immune system, due to a mutation in the POU transcriptional regulator Pdm1/nubbin (nub) gene, have higher loads of bacteria and a more diverse taxonomic composition than controls. In addition, the microbial composition shifts considerably during the short lifespan of the nub1 mutants. This shift is characterized by a loss of relatively few OTUs (operational taxonomic units) and a remarkable increase in a large number of Acetobacter spp. and Leuconostoc spp. Treating nub1 mutant flies with antibiotics prolongs their lifetime survival by more than 100%. Immune gene expression is also persistently high in the presence of antibiotics, indicating that the early death is not a direct consequence of an overactive immune defense but rather an indirect consequence of the microbial load and composition. Thus, changes in host genotype and an inability to regulate the normal growth and composition of the gut microbiota lead to a shift in the microbial community, dysbiosis and early death. 

Lindberg, B. G., Tang, X., Dantoft, W., Gohel, P., Seyedoleslami Esfahani, S., Lindvall, J. M. and Engstrom, Y. (2018). Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis. PLoS Pathog 14(3): e1006936. PubMed ID: 29499056
Summary:
Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. This study shows that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-kappaB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.
Tang, X., Zhao, Y., Buchon, N. and Engstrom, Y. (2018). The POU/Oct transcription factor Nubbin controls the balance of intestinal stem cell maintenance and differentiation by isoform-specific regulation. Stem Cell Reports 10(5):1565-1578. PubMed ID: 29681543
Summary:
Drosophila POU/Oct transcription factors are required for many developmental processes, but their putative regulation of adult stem cell activity has not been investigated. This stuy shows that Nubbin (Nub)/Pdm1, homologous to mammalian OCT1/POU2F1 and related to OCT4/POU5F1, is expressed in gut epithelium progenitor cells. The nub-encoded protein isoforms, Nub-PB and Nub-PD, play opposite roles in the regulation of intestinal stem cell (ISC) maintenance and differentiation. Depletion of Nub-PB in progenitor cells increased ISC proliferation by derepression of escargot expression. Conversely, loss of Nub-PD reduced ISC proliferation, suggesting that this isoform is necessary for ISC maintenance, analogous to mammalian OCT4/POU5F1 functions. Furthermore, Nub-PB is required in enteroblasts to promote differentiation, and it acts as a tumor suppressor of Notch RNAi-driven hyperplasia. It is suggested that a dynamic and well-tuned expression of Nub isoforms in progenitor cells is required for maintaining gut epithelium homeostasis.
BIOLOGICAL OVERVIEW

pdm-1 and pdm-2 are closely linked genes. They are coordinately regulated, with overlapping functions. Their expression is mostly ectodermal and both are essential for proper neuron development. The effects of mutation show that pdm-2 is quantitatively more important in neuroblast differentiation than pdm-1 (Yang, 1995). There is significant independently regulated endodermal expression for pdm-1 as well as expression of pdm-1in the wing.

pdm-1, more properly termed nubbin is expressed in both the anterior and posterior midgut primordia and in the developing endoderm. However, pdm-1 is not expressed in the central domain of the gut. This lack of central domain expression is thought to be due to both Ubx and dpp expression in the visceral mesoderm. Two gaps in pdm-1 expression are found: one in the anterior domain, underlying a region of dpp expression, and the second in parasegment 7, underlying a region of Ubx induced dpp expression. No developmental defects are known to result from pdm-1 expression in endoderm (Affolter, 1995).

Expression of pdm-1 in the wing disc is ubiquitous. In spite of this, it appears to be required locally in the wing hinge. Removal of pdm-1 activity from the hinge region results in a severe wing phenotype, while removal from more distal regions results in a less severe disruption. It is hypothesized that the effects in the hinge region are due to a disruption in the proximal-distal axis (Ng, 1995). pdm-1 expression in the wing disc is regulated by wingless which has a primary role in specifying the proximal-distal axis of the wing (Ng, 1996).

Cell interactions mediated by Notch-family receptors have been implicated in the specification of tissue boundaries in vertebrate and insect development. Although Notch ligands are often widely expressed, tightly localized activation of Notch is critical for the formation of sharp boundaries. Evidence is presented that the POU domain protein Nubbin contributes to the formation of a sharp dorsoventral (DV) boundary in the Drosophila wing. Nubbin represses Notch-dependent target genes and sets a threshold for Notch activity that defines the spatial domain of boundary-specific gene expression (Neumann, 1998).

Certain features of the abnormal wings in flies mutant for nubbin suggest a possible role for Nubbin protein in spatially limiting Notch activity at the DV boundary of the wing. The row of sensory bristles that makes up the wing margin is disorganized in nubbin wing mutants, suggesting a defect in Wingless or Notch activity. In preparations where the wing margin is viewed edge on, this disorganization reflects a broadening of the region where bristles form. Margin bristles are normally specified in cells very close to the DV boundary, reflecting a requirement for high levels of Wingless signaling activity. The broadening of the margin suggests that Wingless might be ectopically expressed in nubbin mutant wing discs. Wingless is normally expressed in a stripe of two to three cells straddling the DV boundary. In nubbin mutant discs, this stripe is widened considerably. Expression of the Notch targets vestigial and cut are similarly expanded at the DV boundary in nubbin mutants (Neumann, 1998).

To determine whether the effect on bristle specification is a direct consequence of removing nubbin activity, clones of nubbin mutant cells were generated in a wild-type background. Ectopic wing margin bristles are found in nubbin mutant clones located near the endogenous wing margins. The nubbin mutant clones show ectopic expression of neuralized, a molecular marker for precursors of the sensory neurons that innervate the bristles. The nubbin mutant clones misexpress wingless and vestigial. The largely autonomous effect of nubbin mutant clones on bristle specification may be due to the relatively low levels of Wg protein expressed in nubbin mutant clones. Together with the results on cut expressioon, these observations suggest that Notch target genes are transcriptionally up-regulated in nubbin mutant cells near the DV boundary (Neumann, 1998).

To test whether ectopic activation of these genes in nubbin mutant clones directly depends on Notch signaling activity, clones of cells were generated that were simultaneously mutant for nubbin and Suppressor of Hairless [Su(H)]. Su(H) encodes a DNA binding protein that mediates transcriptional activity of Notch target genes. Su(H) is autonomously required for the expression of wingless, vestigial, and cut at the DV boundary and binds directly to the vestigial DV boundary enhancer. Clones of cells mutant for both nubbin and Su(H) do not ectopically activate wingless, demonstrating that ectopic expression of wingless in nubbin mutant cells depends on activity of the Notch pathway. To confirm that Nubbin acts downstream of Notch, a test was performed to see whether overexpression of Nubbin could suppress the effects of a ligand-independent form of Notch. When Nubbin is coexpressed with such a constitutively active Notch, ectopic Wingless expression is strongly reduced. Together, these observations suggest that Nubbin may act as a direct repressor of Notch-dependent target gene expression. These findings argue that the effects of Nubbin are unlikely to be mediated by indirect effects on the expression of Notch ligands (Neumann, 1998).

cis-regulatory analysis of the Drosophila pdm locus reveals a diversity of neural enhancers

One of the major challenges in developmental biology is to understand the regulatory events that generate neuronal diversity. During Drosophila embryonic neural lineage development, cellular temporal identity is established in part by a transcription factor (TF) regulatory network that mediates a cascade of cellular identity decisions. Two of the regulators essential to this network are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. The focus of this study is the discovery and characterization of cis-regulatory DNA that governs their expression. Phylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. To determine which of these regions function as cis-regulatory enhancers that regulate the dynamics of pdm gene expression, this study tested each for in vivo enhancer activity during embryonic development and postembryonic neurogenesis. The screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. To facilitate data accessibility, the results of this analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers. These studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, clusters of functionally related enhancers were discovered throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, only partial evidence of sequence duplication between their enhancers was found, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences (Ross, 2015).

This study found 41 enhancers that directed embryonic expression, an overlapping set of 46 activated larval expression, and another overlapping set of 46 activated expression in the adult CNS. While many of these enhancers were activated only in the nervous system, a subset activated reporter gene expression outside of the nervous system, including in larval appendages and in the trachea. Roughly a third of the tested CSCs did not exhibit any detectable cis-regulatory activity in the nervous system. Since this study focused on identifying neural enhancers, the possibility exists that some or all of these CSCs that lack neural system activity may regulated gene expression in the larval and adult tissues that were not examined (Ross, 2015).

There are other online resources of documented enhancers in the Drosophila genome, namely, FlyLight and Vienna Tiles. While these cis-regulatory libraries provide useful information, the coverage of the pdm locus in these databases is not complete. For example, FlyLight analysis did not detect 14 enhancers that flank the nub transcribed sequence. These include those located upstream to the nub long transcript (nub-12 and nub-13), its first intron (nub-28), second exon (nub-32a), second intron (nub-32b, nub-32c, nub-33, nub-36, nub-40b, nub-41, nub-42, nub-44, and nub-45a), and third intron (nub-49b). The FlyLight library also does not include seven pdm-2 enhancers: located in the upstream region (pdm2-21); within the second intron (pdm2-27 and pdm2-28) and lacks information regarding its downstream region (pdm2-45, pdm2-46, pdm2-47 and pdm2-48). Vienna Tiles also provides only partial coverage of the pdm locus, omitting the following 11 pdm locus enhancers: nub-58a, nub-58b, pdm2-13, pdm2-17, pdm2-21, pdm2-22, pdm2-23a, pdm2-31b, pdm2-32, pdm-33, and pdm2-48 . While the Vienna Tiles database provides information on embryonic and adult enhancers, it does not supply information on cis-regulatory activity during larval development. In addition, based on the current analysis, most of the reporter transgenes in these two libraries contain multiple enhancers. For example, he Vienna Tiles enhancer denoted as VT6436 enhancer is made up of two embryonic enhancers (nub-28 and nub-29) (Ross, 2015).

Analysis of the pdm locus enhancers identified four functionally related enhancers (nub-46, nub-49b, pdm2-34, and pdm2-37a) that activated expression during NB lineage development. The nub-46 and pdm2-34 enhancers are both located in the third intron of the nub and pdm-2 long transcript, respectively, whereas nub-49b and pdm2-37a are positioned immediately 5' to the transcriptional start site of their respective short isoform. While the nub-46 and pdm2-34 enhancers drove overlapping but nonidentical expression during embryonic and larval NB lineage development, nub-49b and pdm2-37a regulated similar expression patterns during postembryonic NB lineage development. Analysis of nub-46 and pdm2-34 revealed that these enhancers share multiple conserved DNA elements, albeit in largely unique configurations. Although these observations suggest these enhancers are related, additional studies are needed to further resolve subtle differences between their regulatory activities (Ross, 2015).

Comparative analysis of the nub and pdm-2 coding sequences revealed that their sequence relationship was mostly limited to the exons that encode their POU domains and homeodomains. In contrast, no evidence of collinearity was detected within their noncoding regions, suggesting that they have diverged at a faster rate than the coding sequences. Only one pdm ortholog was found in the mosquito, whereas the medfly and housefly carry both genes. Given this observation and accounting for the divergence of Drosophila from these distant Diptera, the pdm duplication event may have occurred in the Dipteran line between 100 and 260 million (Ross, 2015).

Given the presence of the pdm genes in the medfly and housefly genomes, it was asked whether some or all of the Drosophila CSCs could also be identified in these distant species. Submitting the D. melanogaster genomic sequences surrounding nub and pdm-2 to BLAST searches using the medfly and housefly genomes revealed sequences conserved in the three Dipteran species within several pdm locus CSCs (see Three-way alignment of ultraconserved sequences in conserved sequence clusters identified in Drosophila, housefly, and medfly) that were typically found within their longest conserved sequence blocks (CSBs). For example, a 48 bp sequence within the pdm2-26 CSC that is conserved in all drosophilids, in addition to the medfly and housefly (see The pdm2-26 enhancer contains ultraconserved sequences detected in multiple Diptera)(Ross, 2015).

These studies revealed that two-thirds of the CSCs function as cis-regulatory enhancers that regulate gene expression in a diverse array of spatiotemporal aspects, which taken together reflect pdm expression domains. These observations suggest that the pdm genes are dynamically regulated by multiple cis-regulatory modules, and that these enhancers are more amenable to evolutionary restructuring than their protein encoding exons. This is in agreement with recent reviews on the evolution of Dipteran enhancers highlighting the flexibility of enhancers to maintain their function after loss and/or gain of TF DNA binding sites. Also consistent with these observations, functionally related enhancers were found within the pdm locus that share conserved sequences, albeit in different arrangements and orientations (Ross, 2015).

From a mechanistic perspective, these observations suggest that enhancer behavior can be predicted based on the combination of the conserved elements shared among functionally related enhancers. Similar observations have been made by others. Hierarchical clustering analysis of shared conserved sequences revealed that pdm SOG enhancers may be grouped based on shared elements that are for the most part not present within other pdm locus CSCs. A similar analysis of adult median neurosecretory cell (mNSC) enhancers revealed that they grouped together, as evidenced by sharing of conserved sequence elements, which were largely absent in non-mNSC CSCs with the pdm locus. While further work is required to determine whether these shared elements are important for enhancer activity, these findings suggest a level of structural complexity in the presence and clustering of enhancers that requires further analysis. To construct a better representation of enhancer structure and thus cis-regulatory prediction, one would ideally prefer to use a larger training set of enhancers to improve the accuracy of prediction. These approaches will be addressed in future studies (Ross, 2015).

One of the principal findings of this study is the discovery of 77 enhancers that exhibit a remarkably diverse range of cis-regulatory activities during embryonic and postembryonic development. The biological significance of this enhancer diversity most likely reflects the diversity of the developmental programs in which these transcription factors participate. Functionally related enhancers that share multiple conserved DNA sequences were also identified, and these enhancers could be classified using hierarchical clustering techniques. In addition, this analysis has revealed that the collinearity between the pdm genes is predominantly confined to their POU domain and homeodomain exons, suggesting that their noncoding sequences are diverging at a faster rate than their coding sequences. These results should provide further insight into the regulatory logic that controls cis-regulatory function and thus gene regulation (Ross, 2015).


GENE STRUCTURE

pdm-1 and pdm-2 are both transcribed in the same direction with respect to the centromere: distal to proximal. They are separated by 60 kb (Yeo, 1995).

Bases in 5' UTR - 192

Exons - four

Bases in 3' UTR - 485


PROTEIN STRUCTURE

Characterization of cDNA clones reveal two alternative forms of PDM-1 transcripts, which differ in a translated first exon, but have identical second and third exons (Ng, 1995).

Amino Acids - 498

Structural Domains

PDM-1 has a homeodomain (the POU homeodomain) and a POU domain (Lloyd, 1991).

The 75 amino acid POU-specific (POUs) domain and a 60 amino acid carboxy-terminal homeo (POUh) domain are joined by a hypervariable linker segment that can vary from 15 to 56 amino acids in length in different POU domain proteins. Thus the POU domain is not a single structural domain; indeed, the POUs and POUh segments form separate structurally independent domains. The POUs and POUh domains are, however, always found together and have therefore coevolved. Both POUs and POUh domains contain helix-turn-helix motifs. The POUs-domain structure is very similar to that of lambda and 434 bacteriophage proteins, but there are significant differences in the length of the first alpha helix, and the "turn" connecting the two HTH alpha helices is also longer. Both POUs and POUh bind DNA, and the length of the linker regulates the efficacy of binding various DNA sequence motifs, especially because POUs and POUh DNA binding sites have different spacings in different promoter elements (Herr, 1995).


nubbin/POU domain protein 1: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 3 MAR 97 

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