Three-amino Acid Loop Extension Homeodomain Research Articles

Three-amino acid loop extension homeodomain proteins regulate stress responses and encystation in Entamoeba.

It is interesting to identify factors involved in the regulation of the encystation of Entamoeba histolytica that differentiate trophozoites into cysts. Evolutionarily conserved three amino acid loop extension (TALE) homeodomain proteins act as transcription factors and execute a variety of functions that are essential for life. A TALE homeodomain (EhHbox) protein-encoding gene has been identified in E. histolytica (Eh) that is highly upregulated during heat shock, glucose, and serum starvation. Its ortholog, EiHbox1, a putative homeobox protein in E. invadens (Ei), is also highly upregulated during the early hours of encystation, glucose starvation, and heat shock. They belong to the PBX family of TALE homeobox proteins and have conserved residues in the homeodomain that are essential for DNA binding. Both are localized in the nucleus during encystation and under different stress conditions. The electrophoretic mobility shift assay confirmed that the recombinant GST-EhHbox binds to the reported TGACAG and TGATTGAT motifs. Down-regulation of EiHbox1 by gene silencing reduced Chitin synthase, Jacob, and increased Jessie gene expression, resulting in defective cysts and decreased encystation efficiency and viability. Overall, our results suggest that the TALE homeobox family has been conserved during evolution and acts as a transcription factor to control the differentiation of Entamoeba by regulating the key encystation-induced genes.

Floral organ abscission in Arabidopsis requires the combined activities of three TALE homeodomain transcription factors.

Floral organ abscission is a separation process in which sepals, petals, and stamens detach from the plant at abscission zones. Here, we investigated the collective role of three amino-acid-loop-extension (TALE) homeobox genes ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1), KNAT6 (for KNOTTED LIKE from Arabidopsis thaliana) and KNAT2, which form a module that patterns boundaries under the regulation of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) co-activators. These TALE homeodomain transcription factors were shown to maintain boundaries in the flower, functioning as a unit to coordinate the growth, patterning, and activity of abscission zones. Together with BOP1 and BOP2, ATH1 and its partners KNAT6 and KNAT2 collectively contribute to the differentiation of lignified and separation layers of the abscission zone. The genetic interactions of BOP1/2 and ATH1 with INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) were also explored. We showed that BOP1/2 co-activators and ATH1 converge with the IDA signalling pathway to promote KNAT6 and KNAT2 expression in the abscission zone and cell separation. ATH1 acts as a central regulator in floral organ abscission as it controls the expression of other TALE genes in abscission zone cells.

Pbx loss in cranial neural crest, unlike in epithelium, results in cleft palate only and a broader midface.

Orofacial clefting represents the most common craniofacial birth defect. Cleft lip with or without cleft palate (CL/P) is genetically distinct from cleft palate only (CPO). Numerous transcription factors (TFs) regulate normal development of the midface, comprising the premaxilla, maxilla and palatine bones, through control of basic cellular behaviors. Within the Pbx family of genes encoding Three Amino‐acid Loop Extension (TALE) homeodomain‐containing TFs, we previously established that in the mouse, Pbx1 plays a preeminent role in midfacial morphogenesis, and Pbx2 and Pbx3 execute collaborative functions in domains of coexpression. We also reported that Pbx1 loss from cephalic epithelial domains, on a Pbx2‐ or Pbx3‐deficient background, results in CL/P via disruption of a regulatory network that controls apoptosis at the seam of frontonasal and maxillary process fusion. Conversely, Pbx1 loss in cranial neural crest cell (CNCC)‐derived mesenchyme on a Pbx2‐deficient background results in CPO, a phenotype not yet characterized. In this study, we provide in‐depth analysis of PBX1 and PBX2 protein localization from early stages of midfacial morphogenesis throughout development of the secondary palate. We further establish CNCC‐specific roles of PBX TFs and describe the developmental abnormalities resulting from their loss in the murine embryonic secondary palate. Additionally, we compare and contrast the phenotypes arising from PBX1 loss in CNCC with those caused by its loss in the epithelium and show that CNCC‐specific Pbx1 deletion affects only later secondary palate morphogenesis. Moreover, CNCC mutants exhibit perturbed rostro‐caudal organization and broadening of the midfacial complex. Proliferation defects are pronounced in CNCC mutants at gestational day (E)12.5, suggesting altered proliferation of mutant palatal progenitor cells, consistent with roles of PBX factors in maintaining progenitor cell state. Although the craniofacial skeletal abnormalities in CNCC mutants do not result from overt patterning defects, osteogenesis is delayed, underscoring a critical role of PBX factors in CNCC morphogenesis and differentiation. Overall, the characterization of tissue‐specific Pbx loss‐of‐function mouse models with orofacial clefting establishes these strains as unique tools to further dissect the complexities of this congenital craniofacial malformation. This study closely links PBX TALE homeodomain proteins to the variation in maxillary shape and size that occurs in pathological settings and during evolution of midfacial morphology.

Three-amino-acid-loop-extension homeodomain factor Meis3 regulates cell survival via PDK1

Three-amino-acid-loop-extension (TALE) homeodomain proteins including Meis and Pbx families are generally recognized for their roles in growth and differentiation during vertebrate embryogenesis and tumorigenesis. Whereas genetic studies indicate that Pbx1 regulates the development and function of insulin-producing pancreatic β-cells, the role of Meis family members in β-cells is still unknown. Here we show that Meis3 is abundantly expressed in pancreatic islets and β-cells and that it regulates β-cell survival. We further identify the 3-phosphoinositide-dependent protein kinase 1 (PDK1), a well-known kinase involved in the PI3K-Akt signaling pathway, as a direct Meis3 target, which mediates its role in β-cell survival. This regulatory module appears to function broadly as we also identify Meis3 regulation of cell survival and PDK1 expression in ovarian carcinoma cells, suggesting a unique function for Meis3 beyond the traditional roles for TALE homeodomain factors during embryogenesis.

Pbx1 is essential for growth of zebrafish swim bladder

pbx1, a TALE (three-amino acid loop extension) homeodomain transcription factor, is involved in a diverse range of developmental processes. We examined the expression of pbx1 during zebrafish development by in situ hybridization. pbx1 transcripts could be detected in the central nervous system and pharyngeal arches from 24 hpf onwards. In the swim bladder anlage, pbx1 was detected as early as 28 hpf, making it the earliest known marker for this organ. Morpholino-mediated gene knockdown of pbx1 revealed that the swim bladder failed to inflate, with eventual lethality occurring by 8 dpf. The knockdown of pbx1 did not perturb the expression of prdc and foxA3, with both early swim bladder markers appearing normally at 36 and 48 hpf, respectively. However, the expression of anxa5 was completely abolished by pbx1 knockdown at 60 hpf suggesting that pbx1 may be required during the late stage of swim bladder development.

Essential Role for Pbx1 in Corneal Morphogenesis

The Pbx TALE (three-amino-acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in the corneal epithelium of mice. Pbx1(f/f) mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently, the eyes of these mice were dissected and prepared for histologic or molecular analysis. Tissue-specific deletion of Pbx1 in the corneal epithelium of mice resulted in corneal dystrophy and clouding that was apparent in newborns and progressively worsened with age. Thickening of the cornea epithelium was accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover was associated with perturbed expression of developmental regulators and aberrant differentiation, suggesting an important function for Pbx1 in determining corneal identity. These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.

TALE Homeodomain Proteins Regulate Gonadotropin-releasing Hormone Gene Expression Independently and via Interactions with Oct-1

Gonadotropin-releasing hormone (GnRH) is the central regulator of reproductive function. Expression of the GnRH gene is confined to a rare population of neurons scattered throughout the hypothalamus. Restricted expression of the rat GnRH gene is driven by a multicomponent enhancer and an evolutionarily conserved promoter. Oct-1, a ubiquitous POU homeodomain transcription factor, was identified as an essential factor regulating GnRH transcription in the GT1-7 hypothalamic neuronal cell line. In this study, we conducted a two-hybrid interaction screen in yeast using a GT1-7 cDNA library to search for specific Oct-1 cofactors. Using this approach, we isolated Pbx1b, a TALE homeodomain transcription factor that specifically associates with Oct-1. We show that heterodimers containing Pbx/Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the GnRH regulatory region, each in close proximity to an Oct-1-binding site. Cotransfection experiments indicate that TALE proteins are essential for GnRH promoter activity in the GT1-7 cells. Moreover, Pbx1 and Oct-1, as well as Prep1 and Oct-1, form functional complexes that enhance GnRH gene expression. Finally, Pbx1 is expressed in GnRH neurons in embryonic as well as mature mice, suggesting that the associations between TALE homeodomain proteins and Oct-1 regulate neuron-specific expression of the GnRH gene in vivo.

The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

Identification and Characterization of Human PKNOX2, a Novel Homeobox-Containing Gene

The three-amino-acid loop extension (TALE) homeodomain proteins are highly conserved transcription regulators. Since cooperative function among members of this growing family is critical for regulating transcription, we have tried to explore novel members to understand their regulatory mechanisms in cellular proliferation and differentiation. Here we report identification of PKNOX2, a novel TALE homeodomain protein that shows distinct homology with PKNOX1, a stable partner of PBX proteins. PKNOX2 is composed of 460 amino acids and contains HR1, HR2, and homeodomain, which are highly similar to PKNOX1, suggesting that PKNOX2 may also interact with PBX proteins as well as the same DNA sequence as PKNOX1. Genomic organization of PKNOX2 also showed high similarity to PKNOX1, though PKNOX2 lies on a different chromosomal region, 11q24. Unlike PKNOX1, which was broadly expressed in many tissues, PKNOX2 showed a more restricted pattern of mRNA expression. Nuclear localization of PKNOX2 was confirmed by transfection of epitope-tagged cDNA. Taken together, these data indicate that PKNOX2 is a novel PKNOX-related protein and may interact with PBX proteins and play a tissue-specific regulation of transcription.

In vitro interactions between barley TALE homeodomain proteins suggest a role for protein-protein associations in the regulation of Knox gene function.

This paper describes two-hybrid interactions amongst barley homeodomain proteins encoded by the Three Amino acid Loop Extension (TALE) superfamily. The class I KNOX protein BKN3 is shown to homodimerise and to associate with proteins encoded by the class I and II Knox genes BKn-1 and BKn-7. Furthermore, JUBEL1 and JUBEL2, two BELL1 homologous proteins, are identified and characterised as interacting partners of BKN3. Differences in the requirements of BKN3 derivatives for interactions with KNOX and JUBEL proteins imply the involvement of overlapping but slightly different domains. This set of results is an example for interactions amongst different classes of plant TALE homeodomain proteins, as previously described for related animal proteins. Apparently identical spatial and temporal expression patterns of BKn-1, BKn-3, BKn-7, JuBel1 and JuBel2, as determined by in situ hybridisation, are compatible with possible interactions of their protein products in planta. Contradictory to the common model, that the transcriptional down-regulation of certain class 1 Knox-genes is the prerequisite for organ differentiation, transcripts of all five genes were, similar to Tkn1 and Tkn2/LeT6 of tomato, detected in incipient and immature leaves as well as in meristematic tissues. A characteristic phenotype is induced by the overexpression of JuBel2 in transgenic tobacco plants.

Functional and Cooperative Interactions between the Homeodomain PDX1, Pbx, and Prep1 Factors on the Somatostatin Promoter

Expression of the somatostatin gene in endocrine pancreatic cells is controlled by several regulatory cis-elements located in the promoter region. Among these, the adjacent UE-A and TSEI elements, located from -113 to -85 relative to the transcription initiation site, function in combination and act as a pancreas-specific mini-enhancer. The TSEI element is recognized by the pancreatic homeodomain factor PDX1. In the present study, we show that the UE-A element binds a heterodimeric complex composed of a Pbx factor and the Prep1 protein, both belonging to the atypical three-amino acid loop extension homeodomain family. Recombinant Pbx1 and Prep1 proteins bind cooperatively to the UE-A site, whereas neither protein can bind this site alone. Transient transfection experiments reveal that both Pbx1 and Prep1 are required to generate a strong transcriptional activation from the UE-A element when this element is inserted close to the TATA box. In contrast, in the context of the intact somatostatin promoter or mini-enhancer, Pbx1 and Prep1 alone have no effect, but they produce a drastic activation when the pancreatic homeodomain factor PDX1 is also coexpressed. Thus, the activity of the somatostatin mini-enhancer is mediated by a cooperative interaction between the Pbx-Prep1 heterodimeric complex and the pancreatic factor PDX1.