nubbin/POU domain protein 1
pdm-1 and pdm-2 belong to the class II type of POU genes.
As a POU domain transcription factor, VVL is related to an evolutionary complex group of genes consisting of at least 5 classes. Pit-1 is a class one POU domain transcription factor. Pit-1 is involved in the development of the anterior pituitary gland in mammals. Class II POU domain transcription factors include mammalian Oct1, Oct2, Oct11 and Drosophila PDM-1 and PDM-2. Mammalian Brn1, Brn2, Brn4, SCIP/Oct6 and Xenopus XLPOU1 and XLPOU2 and well as Drosophila Ventral veins lacking (Drifter/Cf1a) and C. elegans ceh6 are Class III proteins. Acj-6 is in POU domain group IV, along with C. elegans unc86 and vertebrate Brn3. Oct-3/4 is a class V POU domain protein. There is no known Drosophila class 1 or class V homolog (Verrijzer, 1993).
The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and
the University of Geneva provides extensive documentation for 'POU' domain signatures.
nubbin expression patterns were studied in three hemimetabolous insect groups: zygentomans (Thermobia domestica, firebrat), dyctiopterans (Periplaneta americana, cockroach), and hemipterans (Oncopeltus fasciatus, milkweed bug). Three major findings are reported: (1) nub patterns were observed in the ventral central nervous system ectoderm represent a synapomorphy (shared derived feature) that is not present in other arthropods. Furthermore, each of the analyzed insects exhibits a species-specific nub expression in the central nervous system. (2) Recruitment of nub for a role in leg segmentation occurred early during insect evolution. Subsequently, in some insect lineages (cockroaches and flies), this original role was expanded to include joints between all the leg segments. (3) The nub expression in the head region shows a coordinated change in association with particular mouthpart morphology. This suggests that nub has also gained an important role in the morphological diversification of insect mouthparts. Overall, the obtained data reveal an extraordinary dynamic and diverse pattern of nub evolution that has not been observed previously for other developmental genes (Li, 2004).
Overall, the cloned Nub/Pdm sequences showed a high degree of conservation with other arthropod nub/pdm orthologs and were quite distinct from sequences of a related vvl gene. Among insects, nub sequences showed perfect conservation in the POU domain except for a single amino acid replacement. In addition, insect Nub/Pdm sequences showed a high degree of sequence similarity in the homeodomain. Even a variable 'linker' region was conserved between firebrats, cockroaches, and milkweed bugs. Conservation was especially noticeable at the beginning of the 'linker' region, where all examined insect species (except Drosophila) contained an NSLN motif, compared with a KTIT/S motif in crustaceans (Pc nub and Ps nub) or to an ASMN motif in spiders (St nub). Intriguingly, the Drosophila nub sequence in this region differed from those of other insects and other arthropods. Although suggestive, the functional significance of these changes is not known (Li, 2004).
nub is one of the few developmental genes for which extensive comparative data are available. Among arthropods, nub expression has been examined in chelicerates, including spiders and horseshoe crabs, and in several crustacean species. Within insects, this gene was studied only in Drosophila. Because higher flies have a highly derived mode of development and a relatively recent phylogenetic origin, it is unclear whether the pattern observed in Drosophila is representative of all insects. With this analysis of an apterygote and two hemimetabolous insects, it is now possible to consider the evolution of nub expression in arthropods in general. nub expression is class specific and sometimes even species specific. Nonetheless, all examined arthropods share a common expression pattern in appendages, indicating that nub was originally an 'appendage' gene. In chelicerates, generally thought to be basal arthropods, nub is localized exclusively to the walking legs and other leg-derived structures. In the spider Steatoda triangulosa, a single band of nub expression was detected in the tarsus of all prosomal legs. This basic chelicerate pattern was substantially altered in the crustacean Porcelio scaber. (1) nub expression spread anteriorly into the head region. Note that this head expression is incomplete, encompassing some but not all the segments. (2) Although nub is still restricted to the distal leg segments, it is expressed in a set of rings (instead of in a single band as in spider embryos). This refinement of the nub expression continues even further in insect embryos. In firebrat and cockroach embryos, there was no expression in the antennal and mandibular appendages. However, milkweed bug embryos exhibit a strong antennal and mandibular staining Thus, the spread of nub expression in the anterior direction is complete in insects and now includes all the head segments. In addition, insect embryos also exhibit a further proximal expansion of nub expression in the legs. Whereas nub is localized only in the distal leg segments in spiders and crustaceans, its expression in insects encompasses proximal leg regions as well. This is particularly pronounced in cockroaches, in which nub is expressed in all leg segments (Li, 2004).
The other key trend in the evolution of nub expression in arthropods is in its expansion from the periphery of the embryo (appendages) to the mid-ventral region. In both chelicerates and crustaceans, there is complete absence of nub in the center of the embryo. In insects, however, mid-ventral expression in neuroectoderm is one of the most noticeable aspects of nub expression. Intriguingly, each insect species exhibits a unique species-specific CNS pattern. Functional studies in Drosophila show that nub is part of a gene regulation cascade specifying the identity of developing neuroblasts in the CNS. These findings, combined with the current observations, suggest that nub expression in the CNS is unique to the insects and has continued to evolve toward more elaborate spatial and temporal patterns that coincide with diversification of insect morphology (Li, 2004).
nub expression patterns exhibit substantial differences between and within the major arthropod classes. However, in groups such as spiders and crustaceans, these differences are rather static. More specifically, once the pattern of expression is established, it does not change during development. In contrast, the data clearly indicate that variation is a key feature of nub expression within insect embryos. The best illustration of this variation is provided by dynamic temporal and spatial changes of nub expression in the CNS and antennae (Li, 2004).
If one compares the timing of its expression in the CNS versus appendages, it is evident that the onset of nub expression within the CNS is highly variable. In firebrat embryos, nub is first expressed in the CNS and is subsequently detected in the appendages. In cockroach embryos, the earliest expression of nub is detected in the appendages followed by the CNS. And in milkweed bug embryos, expression of nub in appendages and CNS occurs simultaneously at a very early stage. In addition to this temporal variation, nub also exhibits spatial variation in its pattern. nub expression in the CNS was found to have a unique species-specific pattern for each insect species studied. In firebrats, nub is expressed in groups of cells aligned along the midline of each segment in a uniform pattern that persists throughout development. In the cockroach, nub expression appears in an irregular triangular pattern encompassing a wider region in the thoracic than in the abdominal segments. Once established, this pattern does not change from early to late development. In contrast, CNS expression of nub in milkweed bugs is very dynamic. In early developmental stages, nub is expressed in cells aligned along either side of the midline, with expression excluded from any of those cells within the midline. As development proceeds, cells within the midline form an 'x' pattern of expression in each segment, beginning in T1 and ending at the posterior abdominal segment (Li, 2004).
Antennal expression represents another example of a highly variable nub pattern. In embryos of basal insect groups such as firebrats and cockroaches, nub is absent in antennae. This observation is also consistent with those on spiders and crustaceans. No nub expression is found in the first two prosomal segments (cheliceral and pedipalpal) in spiders. Similarly, nub is absent from the antennal 1 segment of crustaceans (Li, 2004).
These observations indicate that lack of nub expression in the anterior-most appendages is an ancestral arthropod feature. However, in more derived hemimetabolous insects such as milkweed bugs, nub is observed in this new domain. Moreover, its expression undergoes dramatic temporal and spatial change during development. Early on, nub is strongly expressed throughout the antennae. Then, as the appendages start to elongate, its expression is lost. As development of appendages continues, nub accumulation reappears again in a cluster of cells at the posterior-distal portion of antennae and then shifts to their most distal part and continues to be strongly expressed during dorsal closure. Toward the end of development, its expression disappears again. nub accumulation has also been detected in the eye/antennal disc of Drosophila larvae, further suggesting this novel pattern to be of relatively recent origin and to be shared among phylogenetically more derived insect groups (Li, 2004).
This study revealed that nub function may be evolving more rapidly than that of homeotic or other leg patterning genes such as Dll and dac. Also, the most dynamic temporal and spatial changes in expression occurred in milkweed bugs, a relatively derived hemimetabolous insect. Additional dynamic changes in nub pattern were observed in firebrat and cockroach embryos, especially in legs and mouthparts. These observations pose an intriguing question as to what level of developmental variation actually exists in nature. Whereas traditional views support relatively high levels of conservation in developmental pathways, the present analysis of nub suggests that variation may be more prevalent than previously thought (Li, 2004).
Mainly based on insights from leg development in Drosophila, it is now known that the Notch (N) signaling pathway plays a fundamental role in the process of segmental boundary formation in legs. In flies, the actual joint formation is mediated by establishing distinct rings of N expression in these regions of the leg. nubbin is also expressed in a series of concentric rings in third instar Drosophila leg discs, and its mutant expression results in shortened and gnarled legs. Mutant clones of Notch also cause loss of nub expression in Drosophila legs. Conversely, ectopic expression of nub is induced within clones of cells expressing activated Notch. Thus, in Drosophila legs, nub is positively regulated downstream of Notch (Li, 2004).
Whereas this information indicates that nub is also involved in leg patterning in Drosophila, in addition to its previously described roles in CNS and wing development, the question remains as to when this acquisition of leg function occurred. In spider embryos nub is localized to a single distal band on all walking legs. In crustacean embryos with joint-less trunk appendages such as Artemia franciscana, no expression of nub has been detected. However, in crustaceans with segmented endopods (main leg branches), nub is localized to a series of rings that correspond to joints in the distal leg region. These findings indicate that nub may have a partial role in leg segmentation in chelicerates and crustaceans, mainly during the establishment of distal leg segments. However, formation of most leg joints in those groups has to be under the control of other genes (Li, 2004).
There are two dominant bands of nub expression in firebrat legs, a proximal band and a mid-distal band. The locations of these bands roughly correspond to the positions of the future coxa-trochanter and femur-tibia boundaries, respectively. But leg segmentation in firebrats occurs very late in development when it is technically very difficult to perform the in situ experiments (due to the deposition of embryonic cuticle). This prevents inference of the complete pattern of nub in this species and relation of this expression to the formation of joints. Leg development in Periplaneta embryos is quite different, and clearly identifiable leg segments can be recognized by mid-stages of embryogenesis. In this species, the appearance of five nub stripes precedes any visible demarcation of distinct leg regions. Only afterward does continuing nub expression begin to coincide with the formation of segmental boundaries in the legs. This coordination between the precise patterning of nub expression and leg segmentation in the cockroach embryos is highly indicative of a role in formation of joints. The observed pattern in Periplaneta is also reminiscent of Notch signaling-regulated nub expression in Drosophila leg development. These similarities suggest that regulation of leg segmentation in two widely divergent insect species may be homologous (Li, 2004).
The timing of leg segmentation in milkweed bug embryos is intermediate compared with firebrats and cockroaches. Proximal leg segments in Oncopeltus are established relatively early during germ-band extension, but segmentation of distal leg regions does not occur until late development. As in Periplaneta, the leg patterns of nub expression in Oncopeltus roughly correspond to the position of future joints and its appearance precedes formation of segments. There are also two main differences between observed nub patterns between milkweed bugs and cockroaches: (1) nub expression in Oncopeltus legs is composed of only three bands versus five bands in Periplaneta; (2) two of these bands in Oncopeltus begin to fade much earlier (compared with cockroach). The possible explanation for the first discrepancy is that milkweed bug nub may be involved in formation of some, but not all, leg segments. The likely explanation for the second discrepancy is that nub may play a role in initiating the formation of some leg joints in Oncopeltus but is not required for its maintenance (Li, 2004).
Overall, this analysis suggests that recruitment of nub for a role in leg segmentation may have occurred early in insect evolution. In both insects and crustaceans, nub expression is associated with establishment of some but not all leg segments. In insects, the primary association is with proximal and mid-leg segments. However, only the two most distal segments may be involved in crustaceans. These findings indicate that nub cannot be a necessary element in the regulation of overall leg segmentation in arthropods. Rather, the observed expression patterns suggest that this gene was independently recruited in insects and crustaceans for a possible role in development of specific leg joints. Subsequently, in some insect lineages (cockroaches and flies) this original role was expanded to include joints between all leg segments (Li, 2004).
The insect head has a modular organization and is composed of the three pregnathal (ocular, antennal, and intercalary) and three gnathal segments (mandibular, maxillary, and labial). A pair of appendages grows from each gnathal segment and forms the future mouthparts. Diversity in organization of the insect head is best represented by the variation in structure and morphology of the mouthparts. There are two basic types of mouthparts: mandibulate (specialized for chewing and biting), and haustellate (specialized for piercing and sucking). The mandibular type represents the ancestral form and is characteristic of members of most basal hexapod lineages (Zygentoma, Orthoptera, Blattoidea, etc.). Its main feature is that the mandibles function as 'jaws,' whereas the maxillary and labial limbs form branched appendages that are structurally identical until the midline fusion of the latter. In the haustellate type, it is the mandibular and maxillary appendages that are similar, whereas the labial appendages exhibit a completely different structure (Li, 2004).
Studies in firebrats, crickets, milkweed bugs, and flies have shown that the basic programming and identity of the gnathal segments are determined by three Hox genes: proboscipedia (pb), Deformed (Dfd), and Sex combs reduced (Scr). Changes in pb expression have been directly associated with changes in mouthpart structure. In insects with biting and chewing mouthparts, pb is expressed in the labium and maxillae but not in the mandibles. However, in insects with piercing and sucking mouthparts, such as milkweed bugs, pb expression is mainly detected in the labial appendages Thus, it has been proposed that loss of pb expression in the maxillae was responsible for the transformation of the mandibulate to the haustellate mouth type. More specifically, the loss of pb expression in milkweed bug embryos would 'free' the maxillary appendages to diverge from their original labium-like phenotype. However, such change in pb regulation cannot account for the development of stylets in both maxillary and mandibular segments. This suggests that other nonhomeotic genes are involved in the latter process (Li, 2004).
Consistent with the morphological diversification of head appendages, the sharpest difference of nub expression is observed in this region. Patterns of expression show a coordinated change in association with particular mouthpart morphology. In firebrat and cockroach embryos, which have typical mandibulate mouthparts, nub is localized in the maxillary and labial appendages in a similar pattern. This is particularly pronounced in Periplaneta embryos, which exhibit highly coordinated patterns of expression in the labium and maxillae from early to late development. Such an observation is consistent with the fact that these segments are structurally very similar and differ from the mandibular segment. nub is generally not expressed in the mandibles except locally and transiently in cockroaches. This suggests that in insects with biting and chewing mouthparts, mandibles were originally devoid of nub expression (Li, 2004).
In milkweed bug embryos, which have the stylate-haustellate (sucking) mouthparts, nub is expressed in a very different pattern: (1) there is novel expression along the mid-ventral region of the mandibular appendages; (2) this mandibular expression is similar to the maxillary pattern. Expression in the labium, however, is localized only in the distal portion of the appendage and exhibits a completely different timing and pattern. This differential expression correlates with a change in the morphology of the maxillary and labial segments that is characteristic for piercing and sucking mouthparts: development and morphology of the maxillary segment parallels that of the mandibular segment. These observations provide the first nonhomeotic gene example of a linkage between a change in expression pattern and a corresponding change in the morphology of insect mouthparts. They also provide a strong indication that nub may function in the development of haustellate mouthparts (Li, 2004).
The emerging insight from this and other recent studies suggests that the evolution of the maxillae and mandibles in Oncopeltus and other hemipterans was governed by regulatory changes at both higher levels (Hox genes such as pb and Dfd) and lower levels (genes such as nub) in the developmental hierarchy. This underlines the critical importance of understanding the functional roles and relationships between these two classes of genes. For example, Dfd is expressed in the mandibular segments in both Tribolium (which has mandibulate mouthparts) and in Oncopeltus (which has haustellate mouthparts). Functional studies have shown that in the absence of Dfd, mandibles are transformed into antenna-like appendages in these two insects. These findings show that Dfd orthologs control mandibular identity in both species. Yet the actual morphology of mandibles in Tribolium and Oncopeltus is very different, a result of distinct mandibulate and haustellate modifications. Additional factors, either acting in parallel with Dfd, or downstream targets, or independently of Dfd, must be involved in the establishment of these distinct mandibular morphologies. Based on this analysis of its expression patterns, nub is a good candidate for being such a target gene in the mandibular segment. Further studies of other genes at lower levels in the developmental hierarchy will be necessary to fully understand the mechanisms underlying the morphological diversification of insect mouthparts (Li, 2004).
The homeodomain defines a family of transcription factors broadly involved in the regulation of gene expression. DNA recognition, as observed in three representative complexes (Engrailed, Antennapedia, and MAT alpha 2), is mediated in the major groove by a helix-turn-helix (HTH) element and in the minor groove by an N-terminal arm. The three complexes share similar overall features, but they also exhibit significant differences in DNA interactions. Because these differences may distinguish the biological activities of different classes of homeodomains, the contribution of the Oct-2 POU-specific homeodomain (POUHD) to the specificity of the bipartite POU motif has been investigated. Comparative studies of variant protein-DNA complexes demonstrate the following: The bipartite DNA binding domain of the POU family of transcription factors contains a 'POU-specific' domain unique to this class of factors and a 'POU homeodomain' homologous to other homeodomains. DNA binding of the Oct-2 factor POU domain and the Antennapedia (ANTP) homeodomain were compared with a chimeric Oct-2/ANTP protein in which the distantly related Antp homeodomain was substituted for the Oct-2 POU homeodomain. The Oct-2/Antp chimeric protein binds both the octamer and the Antp sites efficiently, indicating that DNA binding specificity is contributed by both components of the POU domain (Brugnera, 1992).
Two crystal structures of Oct-1 POU domain bound to DNA provide a rationale for differential, conformation-dependent recruitment of transcription cofactors. The POU-homeo and POU-specific subdomains of Oct-1 contain two different nonoverlapping pairs of surface patches that are capable of forming unrelated protein-protein interfaces. Members of the POU factor family contain one or two conserved sequence motifs in the interface that are known to be phosphorylated, as noted for Oct-1 and Pit-1. Modeling of Oct-4 reveals the unique case where the same conserved sequence is located in both interfaces. These studies provide the basis for two distinct dimeric POU factor arrangements that are dictated by the architecture of each DNA response element. It is suggested that interface swapping in dimers could be a general mechanism of modulating the activity of transcription factors (Remenyi, 2001).
Members of the POU transcription factor family are involved in a broad range of biological processes ranging from housekeeping gene functions (Oct-1) to programming of embryonic stem cells (Oct-4) and the development of immune responses (Oct-1, Oct-2). However, according to the latest global sequencing reports, human, fly, and worm genomes encode only fifteen, five, and four POU factors, respectively. Therefore, members of this transcription factor family need to rely on multilevel control mechanisms such as posttranslational modification, interaction with heterologous transcription regulators, and flexible DNA binding to perform these multiple tasks. A linker joining the POU-specific (POUS) and the POU-homeo domain (POUH) confers the flexibility inherent to members of the POU factor family. This linker is variable both in sequence and length (15-56 residues). Since both domains are structurally and functionally autonomous in DNA binding, various arrangements on DNA are possible (Remenyi, 2001 and references therein).
POU factors were originally identified to function as monomeric transcription regulators, for instance, when they bind to the DNA octamer motif. However, more recently, their capability to homo- and hetero-dimerize on specific DNA response motifs has received substantial attention. The Palindromic Oct factor Recognition Element (PORE), ATTTGAAATGCAAAT, within the first intron of the osteopontin (OPN) gene, was initially identified as an Oct-4 DNA responsive element. It mediates strong transcriptional activation in preimplantation mouse embryos and cell lines derived thereof. Oct-4 binds to the PORE in a monomer/dimer equilibrium, in which single nucleotide replacements are sufficient to enhance or diminish dimerization. In vitro, the PORE behaves as a general Oct factor recognition element. To further investigate the general dimerization potential of Oct factors, sequences related to the Prl Pit-1 response element were characterized. These so-called MORE sequences (More palindromic Oct factor Recognition Elements: ATGCATATGCAT) are found in various promoters. All members of the Oct family tested bind cooperatively as homo and heterodimers to the consensus MORE (Remenyi, 2001 and references therein).
The transcriptional activity of Oct-1 on the octamer motif in B cells is regulated by the lymphoid-specific coactivator OBF-1 (OCA-B, BOB-1) that clamps the POUH and POUS subdomains together and thus enhances their DNA binding affinity. However, the Oct-1 dimer formed on MOREs within immunoglobulin heavy chain promoters (VH) fails to interact with OBF-1. In contrast, the Oct-1/PORE dimeric complex can interact and synergize in transcriptional activation with this cofactor. These findings established the paradigm of differential transcriptional regulation mediated by two distinct POU dimer configurations (Remenyi, 2001).
To elucidate the structural basis of this phenomenon, two crystal structures of the Oct-1 POU domain bound to the MORE and PORE have been solved. By direct comparison, these structures demonstrate how the same polypeptide chain can form two different dimer arrangements with two distinct protein-protein interfaces. These data introduce the concept of distinct transcription factor dimerization that depends on the sequence and the spacing of the protein domain binding motifs of the DNA response element. Thus, it extends previous models of protein-DNA complex formation mediated by ligand-induced allosteric effects. The members of the nonsteroid nuclear receptor family, for example, provide another mode of transcription factor dimerization dictated by the binding site, the hormone response element (HRE). HREs consist of two core hexad sequences, AGGTCA. These could form direct, inverted, or everted repeats. On inverted and everted repeats, the receptors dimerize only via a specific surface patch within the carboxy-terminal ligand binding domain. On direct repeats, however, the receptors form an additional interface between the conserved zinc finger DNA binding domains. The DNA sequence-mediated dimerization of POU proteins is different since the distinct surface patches involved in dimer interface formation are both located on the DNA binding POU domain. The results of this study show how swapping protein-protein interfaces between different quaternary arrangements of POU factor/DNA complexes can regulate transcriptional activity (Remenyi, 2001).
The Oct-1 POU factor binds to DNA in a bipartite fashion. The binding topography is the same in both complexes in the sense that the POUS domains are situated proximal to the center of each DNA element, while the POUH domains bind at distal positions. In the MORE complex, the DNA binding sites for POUH and POUS overlap by 2 base pairs within each MORE half-site, whereas in the PORE complex, the two POUS domains share 1 base pair in the center of the PORE motif. Due to the nonpalindromic nature of the PORE, the two POUS domains bind to nonidentical sites, AGGC and TTTC (Remenyi, 2001).
Each POUH domain is bound to a distal AT-motif. On the MORE, this motif is located at positions 1-2 and 11-12, while on the PORE it is located at positions 1-2 and 14-15. In both complexes, the C-terminal helix 3 of each POUH domain is situated in the major groove, with base-specific interactions of the side chains of Asn151 and Gln154. In the PORE complex, the arginine residues of the N-terminal part of POUH (Arg102 and Arg105) form DNA sequence-specific hydrogen bonds (H bonds) with the base pairs in positions 3-4 and 12-13. These interactions are formed via the minor groove within each half-site of the PORE motif. They are not visible in the Oct-1/MORE complex because this part is disordered. The POUS domains interact with bases in the major groove via their third alpha helix. In the two complexes, the side chains of residues Gln44, Thr45, and Arg49 almost identically contribute with base-specific interactions to each POUS binding site (Remenyi, 2001).
In the Oct-1/MORE structure, there are two identical protein-protein contacts forming between a POUS and a POUH domain within each half-site of the palindromic MORE motif. The buried surface per domain is about 550 Å2. A central feature of this interface is the docking of the C-terminal residues 157-160 of the POUH domain onto the loop region between helix 3 and 4 of the POUS domain. The side chain of Ile159 fits into a hydrophobic cavity of the POUS subdomain, which forms a 'knob-in-the-hole' structure. Furthermore, additional interactions are mediated by several H bonds, mostly from main chain atoms except for one side chain contribution by Asn160. This interface is reminiscent of those found in the Pit-1/DNA complexes (Remenyi, 2001).
The two main interfaces formed in the Oct-1/PORE complex are located across the center of the PORE motif, as opposed to the interface observed in the Oct-1/MORE structure. These interfaces are not identical, reflected in the different amounts of buried surface areas of 220 Å2 and 500 Å2, hence termed IF1 and IF2, respectively. Central to this interface is the N terminus of the POUH domain, which interacts with the minor groove of the PORE-DNA as well as with residues of a POUS domain. In the larger interface (IF2), one phosphate group within the minor groove (position 10 of the PORE) forms H bonds with Arg20 from the POUS and Ser107 from the POUH domain. This DNA-mediated POUS-POUH contact is surrounded by two POUS-POUH salt bridges, Asp29-Lys104 and Lys22-Glu109. Like the knob-in-the-hole interaction by Ile159 in the Oct-1/MORE complex, the exposed Ile21 from the POUS domain penetrates into a hydrophobic surface patch of the POUH domain (Remenyi, 2001).
Within the smaller interface (IF1), the only specific interaction is the POUS-POUH salt bridge between Asp29 and Lys104. Ser107 is more exposed to the solvent than in the IF2 interface. Thus, the phosphate group from the minor groove forms only one H bond with Arg20. The observed asymmetry in the PORE interface (IF1 and IF2) can be explained by the different minor groove parameters at the two half-sites of the semipalindromic PORE. Only the minor groove at the nonoctamer half-site, which is about 10% narrower and deeper than that of the octamer half-site, allows a tight fit of the two interacting POU domains within the IF2 interface (Remenyi, 2001).
It is concluded that the two structures of the Oct-1 POU factor in complex with the MORE and PORE DNA motifs reveal two alternative and unrelated dimer arrangements of the same polypeptide chain. Direct evidence is provided for the unique property of POU transcription factors to regulate their functional properties by forming different dimer assemblies. The members of the POU transcription factor family could serve as a paradigm to regulate protein function by protein-protein interface swapping (Remenyi, 2001).
Nerve growth factor (NGF) has been found to have differential effects on the levels of three POU protein transcription factors that are expressed in adult rat sensory neurons. A sensory neuron octamer-binding protein with the properties of the transcription factor Oct-2 is up-regulated 3-4-fold by NGF, as measured using nuclear extracts from adult rat dorsal root ganglion neurons grown in the presence or absence of NGF. There is a parallel increase in Oct-2 mRNA levels. In contrast, the levels of mRNA encoding the ubiquitous POU protein Oct-1 or the neuron-specific POU protein Brn-3, also present in sensory neurons, are unaffected by NGF. These observations suggest a role for Oct-2 in mediating transcriptional effects induced by NGF. In particular, as Oct-2 is known to inhibit herpes simplex virus immediate-early gene expression in neuronal cells, these findings provide a mechanism for the known action of NGF in the maintenance of latent herpes virus infections in sensory neurons (Wood, 1992).
During S phase, transcription of mammalian histone H2B genes requires a specific promoter element and its cognate transcription factor Oct1 (OTF1). A possible mechanism for regulating histone H2B transcription during the cell cycle is direct modulation of Oct1 activity by phase-specific posttranslational modifications. Analysis of Oct1 during progression through the cell cycle reveal a complex temporal program of phosphorylation. A p34cdc2-related protein kinase that is active during mitosis may be responsible for one mitotic phosphorylation of Oct1. However, the temporally controlled appearance of Oct1 phosphopeptides suggests the involvement of multiple kinases and phosphatases. These results support the idea that cell cycle-regulated transcription factors may be
direct substrates for phase-specific regulatory enzymes (Roberts, 1991).
Oct-1 and Oct-2 represent the prototypical example of related transcription factors with identical DNA
recognition properties. They bind functionally critical octamer elements found in diverse regulatory
sequences. It has not been possible to determine directly if these factors are functionally redundant or
selective when interacting with different regulatory sequences in the appropriate cell type. An
equivalent pair of altered DNA-binding specificity mutants of Oct-1 and Oct-2 were used to examine
their function from varied regulatory contexts in B cells. These factors function as redundant activators
of immunoglobulin (Ig) gene promoters (Vkappa and VH) and a histone H2B promoter. The structural
basis of redundancy resides in the highly conserved DNA-binding POU domain, because this domain
of either protein can activate transcription from both Ig and H2B promoters. The nature of
a distal enhancer dictates the relative potency of Oct-1 versus Oct-2 bound to a promoter. Oct-1
preferentially stimulates transcription from a VH or Vkappa promoter in combination with enhancers
from the IgH or Igkappa locus, respectively. In this context, the more potent action of Oct-1 is
dependent on a region external to the POU domain. These results suggest that Oct-1 may be the
critical regulator of Ig gene transcription during B cell development and provide an explanation for
selective Ig isotype expression defects in Oct-2 and OCA-B null mice (Shah, 1997).
The cellular aging-associated transcriptional repressor that has been previous termed Orpheus is identical to Oct-1, a member of the POU domain family. Oct-1 represses the collagenase gene, one of the cellular aging-associated genes, by interacting with an AT-rich cis-element upstream of the gene. This repression occurs in preimmortalized cells at earlier population-doubling levels and in immortalized cells. In cells at these stages, considerable fractions of the Oct-1 protein are prominently localized in the nuclear periphery and colocalize with lamin B. During the cellular aging process, however, this subspecies of Oct-1 disappears from the nuclear periphery. The cells lacking the nuclear peripheral Oct-1 protein exhibit strong collagenase expression and carry typical senescent morphologies. Concomitantly, the binding activity and the amount of nuclear Oct-1 protein is reduced during the aging process and resumes after immortalization. However, the amounts of Oct-1 protein in the cells are not significantly changed during either process. Thus, the cellular aging-associated genes, including the collagenase gene, seem to be derepressed by the dissociation of Oct-1 protein from the nuclear peripheral structure. Oct-1 may form a transcriptional repressive apparatus by anchoring nuclear matrix attachment regions onto the nuclear lamina in the nuclear periphery (Si, 1997).
An enhancer sequence found in the Protease Nexin-1 (PN-1) gene has been shown to drive lacZ expression specifically at the met-/mesencephalic junction in transgenic mouse embryos. A functional study of this enhancer has been performed to better understand the mechanisms regulating isthmic gene expression. An octamer-binding site for POU domain factors is crucial for the activity of the enhancer in vivo. Comparative expression studies of POU domain factors, electrophoretic mobility shift assays and transient transfection experiments, strongly suggest that Brn-1/-2 regulates the enhancer activity in vivo. In addition, in vitro experiments indicate that FGF-8 is required for the maintenance of the enhancer activity, but not for the synthesis of Brn-1/-2. These data represent the first functional evidence for a role of POU factors in the regulation of met-/mesencephalic gene expression. It also implies that at least two regulatory pathways, namely the FGF-8 signaling and the octamer-binding site pathway, synergistically interact to control the PN-1 enhancer activity in vivo (Mihailescu, 1999).
Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by
which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF promoter region, a single nucleotide polymorphism (SNP) has been identified that causes the
helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in
human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased
susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other
known TNF polymorphisms and linked HLA alleles (Knight, 1999).
Two hypotheses have been proposed to explain the origin of insect wings. One holds that wings evolved by modification of limb branches already present in multibranched ancestral appendages that probably first functioned as gills. The second hypothesis proposes that wings arose as novel outgrowths of the body wall, not directly related to any pre-existing limbs. If wings derive from dorsal structures of multibranched appendages, it is expected that some of their distinctive features have been built on genetic functions that were already present in the structural progenitors of insect wings, and in homologous structures of other arthropod limbs. Crustacean homologs have been isolated for two genes that have wing-specific functions in insects: pdm and apterous. Their expression patterns support the hypothesis that insect wings evolved from gill-like appendages that were already present in the aquatic ancestors of both crustaceans and insects. Artemia franciscana PDM and Apterous are specifically expressed in cells of the distal epipodite before these acquire their characteristic differentiated morphology (large nuclei, large intercellular spaces). These expression patterns contrast markedly with that of Distal-less which is expressed in all outgrowing appendages (including insect legs and wings, and all crustacean limb branches). Artemia pdm and apterous are associated specifically with a distal epipodite of crustacean limbs. Crustacean epipodites are dorsally located limb branches with respiratory and osmoregulatory functions, precisely the type of structures that would have given rise to insect wings, according to some hypotheses. An alternative interpretation might be that wings did not derive from epipodites but have nevertheless independently coopted a number of gene functions that already existed in epipodites (Averof, 1997).
The roles of the POU domain genes Skin-1a/i (Skn-1a/i/Epoc/Oct-11) and Testes-1 (Tst-1/Oct-6/SCIP), respectively related to Drosophila pdm-1 and drifter) have been examined in epidermis where proliferating basal keratinocytes withdraw from the cell cycle, migrate suprabasally, and terminally differentiate to form a multilayered, stratified epithelium. The expression of the Skn-1a/i and Tst-1 genes is linked to keratinocyte differentiation in vivo and in vitro, whereas the ubiquitous POU domain factor Oct-1 is expressed highly in both proliferating and post-mitotic keratinocytes. Analysis of Skn-1a/i gene-deleted mice reveals that the Skn-1a/i gene modulates the pattern of expression of the terminal differentiation marker loricrin and inhibits expression of genes encoding markers of the epidermal keratinocyte wounding response. Although epidermis from Tst-1 gene-deleted mice develops normally, epidermis from mice deleted for both Skn-1a/i and Tst-1 is hyperplastic and fails to suppress expression of K14 and Spr-1 in suprabasal cells when transplanted onto athymic mice. This suggests that Skn-1a/i and Tst-1 serve redundant functions in epidermis. Therefore, at least two POU domain genes, Skn-1a/i and Tst-1, serve both distinct and overlapping functions to regulate differentiation of epidermal keratinocytes during normal development and wound healing (Andersen, 1997).
To examine the role of the Oct-6 gene in Schwann cell differentiation, the chicken and zebrafish homologues of the mouse Oct-6 gene have been cloned and characterized. Oct-6 has no known close Drosophila homolog. While highly homologous in the Pit1-Oct1/2-Unc86 (POU) domain, sequence similarities are limited outside this domain. Both genes are intronless and both proteins lack the amino acid repeats that are a characteristic feature of the mammalian Oct-6 proteins. However as in mammals, the aminoterminal parts of the chicken and zebrafish Oct-6 proteins are essential for transactivation of octamer containing promoters. By immunohistochemistry it is found that the chicken Oct-6 protein is expressed in late embryonic ensheathing Schwann cells of the sciatic nerve and is rapidly downregulated when myelination proceeds. This expression profile in glial cells is identical to that in the mouse and rat. The zebrafish Oct-6 homolog is expressed in the posterior lateral nerve at a time when it contains actively myelinating Schwann cells. Thus, despite extensive primary sequence divergence among the vertebrate Oct-6 proteins, the expression of the chicken and zebrafish Oct-6 proteins is consistent with the notion that Oct-6 functions as a 'competence factor' in promyelin cells to execute the myelination program (Levavasseur, 1998).
The vertebrate midbrain-hindbrain boundary (MHB) organizes patterning and neuronal differentiation in the midbrain and anterior hindbrain. Formation of this organizing center involves multiple steps, including positioning of the MHB within the neural plate, establishment of the organizer and maintenance of its regional identity and signaling activities. Juxtaposition of the Otx2 and Gbx2 expression domains positions the MHB. How the positional information is translated into activation of Pax2, Wnt1 and Fgf8 expression during MHB establishment remains unclear. In zebrafish spiel ohne grenzen (spg) mutants, the MHB is not established, neither isthmus nor cerebellum form, the midbrain is reduced in size and patterning abnormalities develop within the hindbrain. In spg mutants, despite apparently normal expression of otx2, gbx1 and fgf8 during late gastrula stages, the initial expression of pax2.1, wnt1 and eng2, as well as later expression of fgf8 in the MHB primordium are reduced. spg mutants have lesions in pou2, which encodes a POU-domain transcription factor. Maternal pou2 transcripts are distributed evenly in the blastula, and zygotic expression domains include the midbrain and hindbrain primordia during late gastrulation. Microinjection of pou2 mRNA can rescue pax2.1 and wnt1 expression in the MHB of spg/pou2 mutants without inducing ectopic expression. This indicates an essential but permissive role for pou2 during MHB establishment. pou2 is expressed normally in noi/pax2.1 and ace/fgf8 zebrafish mutants, which also form no MHB. Thus, expression of pou2 does not depend on fgf8 and pax2.1. These data suggest that pou2 is required for the establishment of the normal expression domains of wnt1 and pax2.1 in the MHB primordium (Belting, 2001).
Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene (most closely related to Drosophila vvl). pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. It is proposed that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories (Hauptmann, 2002).
Continued: Evolutionary Homologs part 2/2
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
nubbin/POU domain protein 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Society for Developmental Biology's Web server.