Paired-type Homeodomain Research Articles

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Regulating transcriptional activity by phosphorylation: A new mechanism for the ARX homeodomain transcription factor.

Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in development. Here we identify that ARX protein is phosphorylated. Using mass spectrometry and in vitro kinase assays we identify phosphorylation at serines 37, 67 and 174. Through yeast-2-hybrid and CoIP we identified PICK1 (Protein interacting with C kinase 1) binding with the C-terminal region of ARX. PICK1 is a scaffold protein known to facilitate phosphorylation of protein partners by protein kinase C alpha (PRKCA). We confirm that ARX is phosphorylated by PRKCA and demonstrate phosphorylation at serine 174. We demonstrate that phosphorylation is required for correct transcriptional activity of the ARX protein using transcriptome-wide analysis of gene expression of phospho-null mutants (alanines replacing serines) compared to ARX wild-type (ARX-WT) overexpressed in pancreatic alpha TC cells. Compared to untransfected cells, ARX-WT overexpression significantly altered expression of 70 genes (Log2FC >+/-1.0, P-value <0.05). There were fewer genes with significantly altered expression compared to untransfected cells with the double phospho-null mutant Ser37Ala+Ser67Ala (26%) and Ser174Ala (39%), respectively. We demonstrate that the c-terminal region of ARX required to bind PICK1 causes a shift in PICK1 subcellular localisation to the nucleus to co-locate with the ARX protein, and truncation of this C-terminal region leads to the same loss of transcriptional activation as S174A mutant. In conclusion, we show that ARX is phosphorylated at several sites and that this modification affects its transcriptional activity.

MUTATIONS IN THE ARX GENE: CLINICAL, ELECTROENCEPHALOGRAPHIC AND NEUROIMAGING FEATURES IN 3 PATIENTS

The Aristaless-related homeobox (ARX) gene is a member of the paired-type homeodomain transcription factor family with critical roles in embryonic development, particularly in the developing brain. Mutations in ARX gene demonstrate striking intra- and interfamilial pleiotropy together with genetic heterogeneity and lead to a broad spectrum of diseases. They give rise to 4 key phenotypic features: a different types of brain malformation, abnormal genitalia, epilepsy and intellectual disability. Authors present 3 clinical cases: a girl with duplication on the short arm of X-chromosome (Xp11.22-p22.33), which include genes ARX and CDKL5; a girl and a boy with a missense mutation in ARX gene that have not been previously described (chrX:25031522C>A), causes the substitution of an amino acid in the 197 protein position (p.Gly197Val, NM_139058.2). All patients suffer from severe epilepsy, that is refractory to antiepileptic drugs, and all of them have different degrees of psychomotor delay. The patients with missense mutation also have movement disorders: stereotypic movements in the girl and choreo athetosis and dystonia in the boy. Electroencephalographic abnormalities have been identified in all patients, and there were not significant abnormalities on magnetic resonance imaging in all cases. The described cases broaden the clinical spectrum of mutations in ARX gene.

Embryonic forebrain transcriptome of mice with polyalanine expansion mutations in theARXhomeobox gene

The Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in embryonic development. Mutations in ARX give rise to intellectual disability (ID), epilepsy and brain malformation syndromes. To capture the genetics and molecular disruptions that underpin the ARX-associated clinical phenotypes, we undertook a transcriptome wide RNASeq approach to analyse developing (12.5 dpc) telencephalon of mice modelling two recurrent polyalanine expansion mutations with different phenotypic severities in the ARX gene. Here we report 238 genes significantly deregulated (Log2FC > +/-1.1, P-value <0.05) when both mutations are compared to wild-type (WT) animals. When each mutation is considered separately, a greater number of genes were deregulated in the severe PA1 mice (825) than in the PA2 animals (78). Analysing genes deregulated in either or both mutant strains, we identified 12% as implicated in ID, epilepsy and autism (99/858), with ∼5% of them as putative or known direct targets of ARX transcriptional regulation. We propose a core pathway of transcription regulators, including Hdac4, involved in chromatin condensation and transcriptional repression, and one of its targets, the transcription factor Twist1, as potential drivers of the ID and infantile spasms in patients with ARX polyalanine expansion mutations. We predict that the subsequent disturbance to this pathway is a consequence of ARX protein reduction with a broader and more significant level of disruption in the PA1 in comparison to the PA2 mice. Identifying early triggers of ARX-associated phenotypes contributes to our understanding of particular clusters/pathways underpinning comorbid phenotypes that are shared by many neurodevelopmental disorders.

The duplicated paired box protein 7 (pax7) genes differentially transcribed during Japanese flounder (Paralichthys olivaceus) embryogenesis.

PAX are important regulators of developmental processes. PAX7 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest (NC), and skeletal muscle. Here, we identified six spliced isoforms of pax7a and one pax7b and characterized their expression patterns. All of flounder Pax7a-1, Pax7a-2, Pax7a-3, and Pax7b contain a conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). However, the PD of Pax7a-4 and the HD of Pax7a-5 are not intact, and there is no HD in Pax7a-4 and Pax7a-6. pax7a and pax7b show distinct spatiotemporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that the expression patterns of pax7a and pax7b are overlapping but distinguishable in the dorsal central nervous system. pax7a is expressed in most part of the brain and the neural tube, while pax7b is expressed exclusively in the diencephalon and the midbrain. In addition, pax7a is also expressed in the cranial NC and the trunk NC. RT-PCR results show that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated pax7 genes. The duplicated pax7 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis.

The homeodomain transcription factor Phox2 in the stellate ganglion of the squidLoligo pealei

ABSTRACTHomeodomain transcription factors regulate development of embryos and cellular physiology in adult systems. Paired-type homeodomain genes constitute a subclass that has been particularly implicated in establishment of neuronal identity in the mammalian nervous system. We isolated fragments of eight homeodomain genes of this subclass expressed in the stellate ganglion of the North Atlantic long finned squid Loligo pealei (lp) [Note: Loligo pealei has been officially renamed Doryteuthis pealei. For reasons of uniformity and clarity Loligo pealei (lp) is used here]. Of the most abundant ones, we cloned a full length cDNA which encoded the squid ortholog of the paired-type homeodomain proteins Phox2a/b. The homology of lpPhox2 to invertebrate and mammalian Phox2 was limited to the homeodomain. In contrast to mouse Phox2b, lpPhox2 was unable to transactivate the dopamine beta-hydroxylase (DBH) promoter in a heterologous mammalian transfection system. In vivo, lpPhox2 was expressed in the developing stellate ganglion of stage 27 squid embryos and continued to be expressed in the adult stellate neurons where expression was confined to the giant fiber lobe containing the neurons that form the giant axons. The expression of lpPhox was similarly timed and distributed as the Fmrf gene. Furthermore, the Fmrf upstream region contained putative Phox2a/b binding sites. These results suggest a role of lpPhox2 in the developmental specification of neuronal identity and regulation of neurons of the squid giant axon.

The olive flounder (Paralichthys olivaceus) Pax3 homologues are highly conserved, encode multiple isoforms and show unique expression patterns.

Pax genes encode a highly conserved family of transcription factors that play crucial roles in the formation of tissues and organs during development. Pax3 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest and skeletal muscle. Here, we identified two spliced isoforms of Pax3a and three spliced isoforms of Pax3b and characterized their expression patterns. Both of flounder Pax3a-1 and Pax3b-1 contain the conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). But the PD domain in Pax3a-2 and Pax3b-3 is not intact and there is no HD in Pax3b-2 and Pax3b-3. Pax3a and Pax3b show distinct temporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that Pax3a and Pax3b are expressed in overlapping patterns in the dorsal central nervous system, with some subtle regional differences between the two genes. In addition, Pax3a is scattered in the somites while Pax3b is specifically expressed in the newly forming somites. RT-PCR results have shown that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated Pax3 genes. The duplicated Pax3 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis.

ARX homeodomain mutations abolish DNA binding and lead to a loss of transcriptional repression

Mutations in the Aristaless-related homeobox (ARX) gene are one of the most frequent causes of X-linked intellectual disability (ID). Several missense mutations, clustered in the paired-type homeodomain of ARX, have been identified. These mutations lead to a range of phenotypes from X-linked lissencephaly with abnormal genitalia to seizure disorders without brain malformations including X-linked infantile spasms with ID (ISSX-ID) and X-linked myoclonic epilepsy with spasticity and ID (XMESID). The effect of these mutations on the DNA-binding and transcriptional activity has been evaluated. Luciferase reporter assays showed altered repression activity of ARX by all mutations, causing brain malformations and ISSX-ID phenotypes, but not by the P353L mutation implicated in a milder phenotype of XMESID. Similarly, transient overexpression of wild-type ARX repressed endogenous expression of known ARX targets, LMO1 and SHOX2, when measured by real-time quantitative polymerase chain reaction. Overall, the molecular consequence of missense mutations correlated well with the severity of the clinical phenotype. In all mutations tested, except P353L, the DNA binding was abolished. Electrophoretic mobility shift assay results were validated using chromatin immunoprecipitation following overexpression of normal and selected missense mutations. Unlike wild-type ARX and clinically less severe mutations, the mutations leading to severe clinical phenotypes were not able to specifically bind to DNA upstream of known, endogenous ARX-regulated genes, LMO1 and SHOX2. In conclusion, the missense mutations in the ARX homeodomain represent loss-of-function mutations, which lead to a reduced or complete loss of DNA binding and as a consequence, a loss of transcriptional repression.

Postnatal expression of the homeobox gene Prrxl1 ( Drg11) is increased in inflammatory but not neuropathic pain

The paired-type homeodomain transcription factor Prrxl1 (also known as Drg11) is a key regulator of the differentiation and survival of dorsal root ganglia (DRG) and spinal nociceptive neurons in pre- and perinatal stages. Prrxl1 −/− mice exhibit abnormalities in DRG-spinal projections, defects in superficial dorsal horn structure and neurochemistry, and reduced nociceptive behaviour in several pain tests. Although a low expression of Prrxl1 persists in dorsal root ganglia beyond embryonic development, no data exist on its role in adult life. In this paper we evaluate whether DRG expression of Prrxl1 is affected both in inflammatory and neuropathic models of pain in adult mice. Ipsilateral versus contralateral relative expression of Prrxl1 in the DRG was compared in control and pain animals. The expression of Prrxl1 mRNA in mice with zymosan-induced peripheral inflammation presented a 3.06 ± 0.71-fold-increase in ipsilateral ganglia, which was significantly different from the value observed in control animals. In contrast, a slight, non-statistically significant decrease was detected in the SNI model of neuropathy. Interestingly, the expression of the mRNA splice variant Prrxl1b was unchanged in both pain conditions. Immunohistochemical studies showed an increase in the number of Prrxl1-positive neurons in the inflammatory pain model, which belonged both in the peptidergic and non-peptidergic categories. Our present results point to a role for Prrxl1 in sensitization of nociceptive neurons upon inflammatory pain.

MixL1 Expression Identifies Putative Hematopoietic Stem Cells In the Murine Allantois.

AbstractAbstract 1591MixL1, a paired-type homeodomain transcription factor, is implicated in pre-hematopoietic commitment of stem cell populations. In poorly-differentiated human lymphoma and leukemia lines, MixL1 is inappropriately over-expressed (Drakos et al., Human Pathol 2007:38:500). When constitutively expressed in mice, MixL1 is sufficient to induce Acute Myeloid Leukemia (Glaser et al., PNAS 2006:103:16460). On the basis of these observations, we hypothesize that MixL1 plays an important role in gating the cellular decision to remain in a poorly differentiated and proliferative phase rather than proceeding to definitive hematopoietic stem cell (HSC) identity.Several years ago, the placenta was shown to be a major site of hematopoiesis (Gekas et al., Dev Cell 2005:8:365; Ottersbach and Dzierzak, Dev Cell 2005:8:377). The placenta is composed of the chorionic disc and the allantois, the latter of which matures into the umbilical component of the placenta. The allantois exhibits definitive hematopoietic potential (Ziegler et al., Development 2006:133:4183; Corbel et al., Dev Biol 2007:301:478), and has recently been demonstrated to contain a core of stem cells referred to as the Allantoic Core Domain, or ACD, where potential placental hematopoietic activity may originate (Downs et al., Dev Dyn 2009:238:532).Our immediate goal is to evaluate whether MixL1 is expressed in the allantois, and to establish its precise spatiotemporal whereabouts with respect to early markers of hematopoietic cells, such as Runx1 (Chen et al., Nature 2009:457:887). Using immunohistochemistry in conjunction with the LacZ/Runx1 reporter mouse (North et al., Development 1999:126:2563), we have demonstrated that MixL1 is strongly expressed in the blood islands of the yolk sac, as well as in a broad, contiguous posterior domain of the embryo that extends to include the ACD stem cell core of the allantois. This domain does not include Runx1, and MixL1 expression temporally precedes that of Runx1 in the allantois.As development proceeds, the MixL1 signal becomes most prominent in putative nascent blood cells budding off from the poorly-described blood vessel common to the allantois, yolk sac and dorsal aortae, which we have called the “Vessel of Confluence” (VOC). Here, fetal blood is shuttled into the umbilical cord to the chorion for exchange with the mother. Shortly thereafter, Runx1 begins to appear within VOC, and is co-expressed with MixL1.These findings provide preliminary evidence that MixL1 is expressed within the allantois, and within nascent blood cells derived from a specific arterial site common to the allantois, yolk sac, and fetus. Moreover, MixL1 expression appears to precede that of Runx1. Thus, MixL1 may identify one of the earliest hematopoietic precursor cell populations thus far known in mammals. Further, these data provide additional evidence that the allantois is a promising model system for the study of definitive hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

The Roles of Multiple Importins for Nuclear Import of Murine Aristaless-related Homeobox Protein

Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin beta1, importin 9, or importin 13, with binding being strongest to importin beta1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impbeta1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin betas, importin beta1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin beta1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.

Importin 13 Mediates Nuclear Import of Histone Fold-containing Chromatin Accessibility Complex Heterodimers

The histone fold is a structural element that facilitates heterodimerization, and histone fold heterodimers play crucial roles in gene regulation. Here, we investigated the nuclear import of two human histone fold pairs, which belong to the H2A/H2B family: CHRAC-15/CHRAC-17 and p12/CHRAC-17. Our results from in vitro nuclear import assays with permeabilized cells and in vivo cotransfection experiments reveal that importin 13 facilitates nuclear import of both histone fold heterodimers. Using glutathione S-transferase pulldown experiments, we provide evidence that heterodimers are required for efficient binding of importin 13 because the monomers alone do not significantly interact. Mutational analysis shows that stepwise substitution of basic amino acid residues conserved among the histone fold subunits leads to a progressive loss of importin 13 binding and nuclear accumulation of CHRAC-15/CHRAC-17 and p12/CHRAC-17. The distribution of basic amino acid residues among the histone fold subunits essential for nuclear uptake suggests that heterodimerization of the histone fold motif-containing proteins forms an importin 13-specific binding platform.

Increased expression of the transcription factor Prrxl1/Drg11 in dorsal root ganglia following peripheral inflammation in adult mice

The paired-type homeodomain transcription factor Prrxl1/Drg11 is a key regulator of the differentiation and survival of nociceptive neurons in pre and perinatal stages. Drg11-/- mice were shown to exhibit abnormalities in DRG spinal projections, defects in superficial dorsal horn structure and neurochemistry and reduced nociceptive behaviour in several pain tests. Here, we examined if the expression of Drg11 gene was affected by the induction of inflammatory pain. Peripheral inflammatory pain was induced by hindpaw plantar injection of zymosan in 21 days old male mice. After 4 hours the animals were sacrificed and L4-L5 DRGs ipsi and contralateral to the injection were removed for analysis of gene expression using Real Time PCR. Relative quantification of Drg11 gene expression was compared between ipsi- and contralateral DRGs. In addition, the amount of Drg11 expression was quantified by western blotting analysis and qualitative evaluation of Prrxl1-expressing neurons was also performed using immunohistochemistry analysis. Following zymosan injection Prrxl1 expression was 2.29 +/- 0.50 fold higher in the ipsilateral DRGs relative to the contralateral DRGs. Similar results were obtained by blotting analysis. Moreover, we observed an increase in immunolabeling of Drg11-positive DRG neurons of the side ipsilateral to the zymosan injection. The importance of Drg11 in the early development of nociceptive DRG and spinal cord neurons is well established. The present study shows for the first time that peripheral inflammation enhances the post-developmental level of Drg11 expression, and raises the possibility that this transcription factor may have a role in pain processing in adult mice. The paired-type homeodomain transcription factor Prrxl1/Drg11 is a key regulator of the differentiation and survival of nociceptive neurons in pre and perinatal stages. Drg11-/- mice were shown to exhibit abnormalities in DRG spinal projections, defects in superficial dorsal horn structure and neurochemistry and reduced nociceptive behaviour in several pain tests. Here, we examined if the expression of Drg11 gene was affected by the induction of inflammatory pain. Peripheral inflammatory pain was induced by hindpaw plantar injection of zymosan in 21 days old male mice. After 4 hours the animals were sacrificed and L4-L5 DRGs ipsi and contralateral to the injection were removed for analysis of gene expression using Real Time PCR. Relative quantification of Drg11 gene expression was compared between ipsi- and contralateral DRGs. In addition, the amount of Drg11 expression was quantified by western blotting analysis and qualitative evaluation of Prrxl1-expressing neurons was also performed using immunohistochemistry analysis. Following zymosan injection Prrxl1 expression was 2.29 +/- 0.50 fold higher in the ipsilateral DRGs relative to the contralateral DRGs. Similar results were obtained by blotting analysis. Moreover, we observed an increase in immunolabeling of Drg11-positive DRG neurons of the side ipsilateral to the zymosan injection. The importance of Drg11 in the early development of nociceptive DRG and spinal cord neurons is well established. The present study shows for the first time that peripheral inflammation enhances the post-developmental level of Drg11 expression, and raises the possibility that this transcription factor may have a role in pain processing in adult mice.

Eye-specific expression of an ancestral jellyfish PaxB gene interferes with Pax6 function despite its conserved Pax6/Pax2 characteristics

Pax transcription factors are evolutionarily conserved regulators of eye development and can be distinguished on the basis of three functional domains: two DNA-binding domains (the paired domain and the paired-type homeodomain), and the octapeptide motif. PaxB of the eyed cubozoan jellyfish, Tripedalia cystophora, is characterized by a Pax2-like paired domain and octapeptide, and a Pax6-like homeodomain. In mice, functionally distinct Pax6 and Pax2 proteins have unique as well as redundant roles in eye morphogenesis. Here, we show that expression of the jellyfish PaxB gene in mouse embryonic eye tissues impairs normal development of lens and retina. Our data show that PaxB misexpression leads to a downregulation of endogenous Pax6 protein in the prospective lens and in subsets of cells within the inner nuclear layer of transgenic retina. In addition to Pax6 downregulation, the expression of PaxB leads to an almost complete loss of amacrine cells in the adult transgenic retina, a phenotype that differs from a loss-of-function of the Pax6 gene. The present data suggest that PaxB, due to its Pax2-like paired domain and Pax-6 like homeodomain, disturbs the transcriptional network regulated by Pax6 in the developing lens and retina. Taken together, our data suggest that molecular properties of individual mouse Pax2 and Pax6 proteins are essential determinants of mouse eye development and cannot be substituted for by jellyfish PaxB which possesses elements of vertebrate Pax2 and Pax6.

Pias3-Dependent SUMOylation Directs Rod Photoreceptor Development

Specification of retinal rod photoreceptors is determined by several different transcription factors that activate expression of rod-specific genes and repress expression of cone photoreceptor-specific genes. The mechanism by which this dual regulation occurs is unclear. We have found that Pias3, a transcriptional coregulator and E3 SUMO ligase that is selectively expressed in developing photoreceptors, promotes the differentiation of rod photoreceptors while preventing rods from adopting cone photoreceptor-like characteristics. Pias3 binds the photoreceptor-specific transcription factors Crx and Nr2e3 and is specifically targeted to the promoters of photoreceptor-specific genes. Pias3 SUMOylates Nr2e3, converting it into a potent repressor of cone-specific gene expression. Rod- and cone-specific promoters are bound by hyperSUMOylated proteins in rod photoreceptors, and blocking SUMOylation in photoreceptors results in cells with morphological and molecular features of cones and an absence of rod-specific markers. Our data thus identify Pias3-mediated SUMOylation of photoreceptor-specific transcription factors as a key mechanism of rod specification.

A Core Paired-Type and POU Homeodomain-Containing Transcription Factor Program Drives Retinal Bipolar Cell Gene Expression

The diversity of cell types found within the vertebrate CNS arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200 bp Grm6 enhancer sequence, a 445 bp Cabp5 promoter sequence, and a 164 bp Chx10 enhancer sequence, were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and reverse transcription-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs Otx2 and Brn2 together appear to play a common role in regulating gene expression in retinal bipolar cells.

Cooperative Interactions between the Two DNA Binding Domains of Pax3: Helix 2 of the Paired Domain Is in the Proximity of the Amino Terminus of the Homeodomain

Pax3 is a transcription factor that plays an important role during neurogenesis and myogenesis, and Pax3 mutant animals display neural tube defects and lack limb muscles. Pax3 harbors two DNA binding domains, the paired domain (PD) and a paired-type homeodomain (HD). Genetic and biochemical data have (i) identified strong cooperative interactions between the PD and HD domains for DNA binding in the intact Pax3 protein and (ii) suggested an important role for the amino-terminal portions of both domains in such cooperativity. We have studied proximity relationships between the PD and HD of Pax3. For this, we have used a cross-linking strategy with the bifunctional thiol reagent bismaleimidoethane (BMOE) in 21 mutants bearing pairs of cysteine residues (DCM) inserted in strategic locations of a functional Pax3 protein otherwise devoid of endogenous cysteine residues. All 21 DCMs were characterized for protein stability, for DNA binding by the PD and HD, and for the effect of BMOE on protein binding to PD, HD, or PD-HD combined DNA targets. BMOE-induced cross-links in DCMs were detected as slower migrating species on immunoblots. Mutants bearing double cysteine insertions (I59C/S222C, S73C/Q219C, and V78C/K218C) showed the most robust cross-linking upon BMOE exposure. These cross-linking studies suggest that portions of helix 1 (I59), helix 2 (S73), and the loop between helices 2 and 3 (V78) of the PD are in the proximity of the N-terminal segment of the HD (K218, Q219, and S222) in the tertiary structure of Pax3. These results are compatible with a model in which the PD and HD are organized in an everted arrangement, with the N-terminal portion of the PD being in the proximity of the N-terminus of the HD. This arrangement may be important for the noted PD-HD cooperativity in DNA binding.

The bovine aristaless-like homeobox 4 (ALX4) as a candidate gene for syndactyly

The ALX4 (aristaless-like homeobox 4) gene encodes a paired-type homeodomain transcriptional activator and plays a major role in anterior-posterior pattern formation during limb development. Here, the cloning, genomic structure and expression of the bovine ortholog of the ALX4 gene are reported. The bovine ALX4 gene consists of four exons and is located on BTA15q28→q29 in a region syntenic to HSA11p11.2. The transcribed ALX4 mRNA encodes a 397-amino-acid protein showing a paired-type homeodomain and a C-terminal stretch of amino acids known as the OAR- or aristaless domain. The predicted protein shares 92.5% identity to human and mouse ALX4 proteins and all three species share almost complete identity in the conserved domains. ALX4 expression was detected by reverse transcriptase polymerase chain reaction in bovine fetal limb bones. The ALX4 gene was evaluated as a candidate gene for bovine syndactyly which has been mapped on the telomeric region of cattle chromosome 15. Sequencing of the four exons with flanking sequences of the bovine ALX4 gene from a panel of 14 affected animals belonging to German Holstein, German Fleckvieh and crossbreds, and 27 unaffected individuals from German Holstein revealed five silent SNPs within the coding region out of eleven SNPs in total. Four SNPs were polymorphic in the affected animals, but in comparison to the genotyped unaffected individuals the genotype distribution showed no evidence for an association to the phenotype. Therefore our data indicate that the ALX4 gene can probably be excluded as candidate gene for bovine syndactyly in the examined animals.

Identification of a new class of PAX3-FKHR target promoters: a role of the Pax3 paired box DNA binding domain.

Alveolar rhabdomyosarcoma (aRMS), an aggressive skeletal muscle cancer, carries a unique t(2;13) chromosomal translocation resulting in the formation of a chimeric transcription factor PAX3-FKHR. This fusion protein contains the intact DNA-binding domains (PD: paired box binding domain; HD: paired-type homeodomain) of Pax3 fused to the activation domain of FKHR. Cells expressing Pax3 and PAX3-FKHR show vastly different gene expression patterns, despite that they share the same DNA-binding domains. We present evidence of a gain of function mechanism that allows the fusion protein to recognize and transcriptionally activate response elements containing a PD-specific binding site. This DNA recognition specificity is in contrast to the requirement for Pax3-specific target sequences that must contain a composite of PD-and HD-binding sites. Domain swapping studies suggest that an increased structural flexibility could account for the relaxed DNA targeting specificity in PAX3-FKHR. Here, we identify myogenin gene as a direct target of PD-dependent PAX3-FKHR activation pathway in vitro and in vivo. We demonstrate that PAX3-FKHR could induce myogenin expression in undifferentiated myoblasts by a MyoD independent pathway, and that PAX3-FKHR is directly involved in myogenin expression in aRMS cells.

Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb

The vertebrate forelimb and hindlimb are serially homologous structures; however, their distinctive morphologies suggest that different mechanisms are associated with each limb type to give rise to limb-type identity. Three genes have been implicated in this process; T-box transcription factors Tbx5 and Tbx4, which are expressed in the forelimb and hindlimb, respectively, and a paired-type homeodomain transcription factor Pitx1, expressed in the hindlimb. To explore the roles of Pitx1 and Tbx4 in patterning the hindlimb, we have ectopically misexpressed these genes in the mouse forelimb using transgenic methods. We have developed a novel technique for visualising the structure and organisation of tissues in limbs in 3D using optical projection tomography (OPT). This approach provides unparalleled access to understanding the relationships between connective tissues during development of the limb. Misexpression of Pitx1 in the forelimb results in the transformation and translocation of specific muscles, tendons, and bones of the forelimb so that they acquire a hindlimb-like morphology. Pitx1 also upregulates hindlimb-specific factors in the forelimb, including Hoxc10 and Tbx4. In contrast, misexpression of Tbx4 in the forelimb does not result in a transformation of limb-type morphology. These results demonstrate that Pitx1, but not Tbx4, determines the morphological identity of hindlimb tissues.