Three Amino Acid Loop Extension Research Articles

Identification of Key Transcription Factors Related to Bacterial Spot Resistance in Pepper through Regulatory Network Analyses.

Bacterial spot (BS), caused by Xanthomonas campestris pv. Vesicatoria (Xcv), severely affects the quality and yield of pepper. Thus, breeding new pepper cultivars with enhanced resistance to BS can improve economic benefits for pepper production. Identification of BS resistance genes is an essential step to achieve this goal. However, very few BS resistance genes have been well characterized in pepper so far. In this study, we reanalyzed public multiple time points related to RNA-seq data sets from two pepper cultivars, the Xcv-susceptible cultivar ECW and the Xcv-resistant cultivar VI037601, post Xcv infection. We identified a total of 3568 differentially expressed genes (DEGs) between two cultivars post Xcv infection, which were mainly involved in some biological processes, such as Gene Ontology (GO) terms related to defense response to bacterium, immune system process, and regulation of defense response, etc. Through weighted gene co-expression network analysis (WGCNA), we identified 15 hub (Hub) transcription factor (TF) candidates in response to Xcv infection. We further selected 20 TFs from the gene regulatory network (GRN) potentially involved in Xcv resistance response. Finally, we predicted 4 TFs, C3H (p-coumarate 3-hydroxylase), ERF (ethylene-responsive element binding factor), TALE (three-amino-acid-loop-extension), and HSF (heat shock transcription factor), as key factors responsible for BS disease resistance in pepper. In conclusion, our study provides valuable resources for dissecting the underlying molecular mechanism responsible for Xcv resistance in pepper. Additionally, it also provides valuable references for mining transcriptomic data to identify key candidates for disease resistance in horticulture crops.

Genome-Wide Identification and Characterization of TALE Superfamily Genes in Soybean (Glycine max L.).

The three-amino-acid-loop-extension (TALE) superfamily genes broadly existed in plants, which played important roles in plant growth, development and abiotic stress responses. In this study, we identified 68 Glycine max TALE (GmTALE) superfamily members. Phylogenetic analysis divided the GmTALE superfamily into the BEL1-like (BLH/BELL homeodomain) and the KNOX (KNOTTED-like homeodomain) subfamilies. Moreover, the KNOX subfamily could be further categorized into three clades (KNOX Class I, KNOX Class II and KNOX Class III). The GmTALE genes showed similarities in the gene structures in the same subfamily or clade, whose coding proteins exhibited analogous motif and conserved domain compositions. Besides, synteny analyses and evolutionary constraint evaluations of the TALE members among soybean and different species provided more clues for GmTALE superfamily evolution. The cis-element analyses in gene promoter regions and relevant gene expression profiling revealed different regulating roles of GmTALE genes during soybean plant development, saline and dehydration stresses. Genome-wide characterization, evolution, and expression profile analyses of GmTALE genes can pave the way for future gene functional research and facilitate their roles for applications in genetic improvement on soybean in saline and dehydration stresses.

Возможная роль гомеодоменсодержащего транскрипционного фактора PREP1 в мезенхимальных клетках сердца

Homeodomain transcription factors play a significant role in mesenchymal stromal cells (MSCs). Previously, the role of Meis1, Pbx1 and Prep1 proteins from the TALE (Three Amino acid Loop Extension) family in adipocytic and osteogenic differentiation of mouse mesenchymal stromal cells was established. In this work, using ChIP-seq and bioinformatic analysis we investigated the binding pattern of PREP1 with the genomic DNA of human heart MSCs, identified nearby genes, and analyzed their ontology. The target genes of the PREP1 factor in cardiac MSCs have been established. Based on the results, the possible involvement of transcription factor PREP1 in the direct reprogramming of fibroblasts into cardiomyocytes is discussed.

Identification of BELL Transcription Factors Involved in Nodule Initiation and Development in the Legumes Pisum sativum and Medicago truncatula.

Single three-amino acid loop extension (TALE) homeodomain proteins, including the KNOTTED-like (KNOX) and BEL-like (BELL) families in plants, usually work as heterodimeric transcription factor complexes to regulate different developmental processes, often via effects on phytohormonal pathways. Nitrogen-fixing nodule formation in legumes is regulated by different families of homeodomain transcription factors. Whereas the role of KNOX transcription factors in the control of symbiosis was studied early, BELL transcription factors have received less attention. Here, we report the identification and expression analysis of BELL genes in the legume plants Medicago truncatula and Pisum sativum, which are involved in regulating symbiosis initiation and development. A more precise analysis was performed for the most significantly upregulated PsBELL1-2 gene in pea. We found that the PsBELL1-2 transcription factor could be a potential partner of PsKNOX9. In addition, we showed that PsBELL1-2 can interact with the PsDELLA1 (LA) protein-regulator of the gibberellin pathway, which has a previously demonstrated important role in symbiosis development.

Genome-wide Identification and Expression Analysis of TALE Gene Family in Pomegranate (Punica granatum L.)

The three-amino-acid-loop-extension (TALE) gene family is a pivotal transcription factor that regulates the development of flower organs, flower meristem formation, organ morphogenesis and fruit development. A total of 17 genes of pomegranate TALE family were identified and analyzed in pomegranate via bioinformatics methods, which provided a theoretical basis for the functional research and utilization of pomegranate TALE family genes. The results showed that the PgTALE family genes were divided into eight subfamilies (KNOX-Ⅰ, KNOX-Ⅱ, KNOX-Ⅲ, BELL-Ⅰ, BELL-Ⅱ, BELL-Ⅲ, BELL-Ⅳ, and BELL-Ⅴ). All PgTALEs had a KNOX domain or a BELL domain, and their structures were conservative. The 1500 bp promoter sequence had multiple cis-elements in response to hormones (auxin, gibberellin) and abiotic stress, indicating that most of PgTALE were involved in the growth and development of pomegranates and stress. Function prediction and protein-protein network analysis showed that PgTALE may participate in regulating the development of apical meristems, flowers, carpels, and ovules. Analysis of gene expression patterns showed that the pomegranate TALE gene family had a particular tissue expression specificity. In conclusion, the knowledge of the TALE gene gained in pomegranate may be applied to other fruit as well.

Overexpression of Populus transcription factor PtrTALE12 increases axillary shoot development by regulating WUSCHEL expression

The TALE (Three Amino acid Loop Extension) transcription factor family has been shown to control meristem formation and organogenesis in plants. To understand the functional roles of the TALE family in woody perennials, each of the TALE members of Populus trichocarpa was overexpressed in Arabidopsis as a proxy. Among them, the overexpression of PtrTALE12 (i.e., 35S::PtrTALE12) resulted in a dramatic increase of axillary shoot development with early flowering. Interestingly, expression of WUSCHEL (WUS), a central regulator of both apical and axillary meristem formation, was significantly increased in the 35S::PtrTALE12 Arabidopsis plants. Conversely, WUS expression was downregulated in 35S::PtrTALE12-SRDX (short transcriptional repressor domain) plants. Further analysis found that PtrTALE12, expressed preferentially in meristem tissues, directly regulates WUS expression in transient activation assays using Arabidopsis leaf protoplast. Yeast two-hybrid assays showed that PtrTALE12 interacts with SHOOT MERISTEMLESS (STM); however, the interaction does not affect the WUS expression. In addition, expression of both CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) genes was suppressed accordingly for early flowering 35S::PtrTALE12 Arabidopsis. Indeed, transgenic poplars overexpressing PtrTALE12 as well as Arabidopsis plants overexpressing AtBLH11, a close homolog of PtrTALE12, phenocopied the 35S::PtrTALE12 Arabidopsis (i.e., increased axillary shoot development). Taken together, our results suggest that PtrTALE12 functions as a positive regulator of axillary shoot formation in both Arabidopsis and poplar.

HOXA10 co-factor MEIS1 is required for the decidualization in human endometrial stromal cell.

Decidualization is a critical process for embryo implantation and pregnancy maintenance in humans. The homeobox gene HOXA10 has been widely studied in endometrial receptivity establishment and decidualization. MEIS1, a three-amino-acid loop extension (TALE) family homeobox gene, has been proven to be a co-factor for HOXA10 in mouse uterus. However, the interaction between MEIS1 and HOXA10 in the human decidual cells remains to be elucidated. siRNA and CRISPR-Cas9 were employed to knockdown and knockout MEIS1 in the cultured human endometrial stromal cells, and it was found that MEIS1 deficiency leads to impaired decidualization. The physical interaction between the MEIS1 and HOXA10 in human endometrial stromal cell was confirmed by immunoprecipitation. Moreover, KAT2B and ETA were proved to be downregulated in the absence of MEIS1, and luciferase reporter and ChIP assays demonstrated that MEIS1-HOXA10 complex binds to the promoters of KAT2B and ETA and regulates their activity. Overexpression of KAT2B and ETA can partially rescue the decidualization defects in MEIS1-knockout HESCs. Taken together, these data suggest that MEIS1 plays an indispensable role in decidualization in human endometrial stromal cells, and MEIS1 interacts with HOXA10 to regulate the downstream genes, such as KAT2B and ETA. These findings will contribute to our understanding about the regulatory network in the process of decidualization in humans.

A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Genome-wide identification and characterization of TALE superfamily genes in cotton reveals their functions in regulating secondary cell wall biosynthesis

BackgroundCotton fiber length and strength are both key traits of fiber quality, and fiber strength (FS) is tightly correlated with secondary cell wall (SCW) biosynthesis. The three-amino-acid-loop-extension (TALE) superclass homeoproteins are involved in regulating diverse biological processes in plants, and some TALE members has been identified to play a key role in regulating SCW formation. However, little is known about the functions of TALE members in cotton (Gossypium spp.).ResultsIn the present study, based on gene homology, 46, 47, 88 and 94 TALE superfamily genes were identified in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. Phylogenetic and evolutionary analysis showed the evolutionary conservation of two cotton TALE families (including BEL1-like and KNOX families). Gene structure analysis also indicated the conservation of GhTALE members under selection. The analysis of promoter cis-elements and expression patterns suggested potential transcriptional regulation functions in fiber SCW biosynthesis and responses to some phytohormones for GhTALE proteins. Genome-wide analysis of colocalization of TALE transcription factors with SCW-related QTLs revealed that some BEL1-like genes and KNAT7 homologs may participate in the regulation of cotton fiber strength formation. Overexpression of GhKNAT7-A03 and GhBLH6-A13 significantly inhibited the synthesis of lignocellulose in interfascicular fibers of Arabidopsis. Yeast two-hybrid (Y2H) experiments showed extensive heteromeric interactions between GhKNAT7 homologs and some GhBEL1-like proteins. Yeast one-hybrid (Y1H) experiments identified the upstream GhMYB46 binding sites in the promoter region of GhTALE members and defined the downstream genes that can be directly bound and regulated by GhTALE heterodimers.ConclusionWe comprehensively identified TALE superfamily genes in cotton. Some GhTALE members are predominantly expressed during the cotton fiber SCW thicking stage, and may genetically correlated with the formation of FS. Class II KNOX member GhKNAT7 can interact with some GhBEL1-like members to form the heterodimers to regulate the downstream targets, and this regulatory relationship is partially conserved with Arabidopsis. In summary, this study provides important clues for further elucidating the functions of TALE genes in regulating cotton growth and development, especially in the fiber SCW biosynthesis network, and it also contributes genetic resources to the improvement of cotton fiber quality.

Genome Wide Identification and Expression Profiles of TALE Genes in Pineapple (Ananas comosus L)

TALE (Three Amino-acid Loop Extension) transcription factors belong to a large gene family in plants and are also widespread in other Eukaryotes, including animals. We have in detail studied TALE genes in the economically important crop plant pineapple (Ananas comosus) but also compared these gene sequences with other plant TALE-genes. Using the webtool HMMER we found 18 TALE gene sequences in the pineapple genome, 33 genes in rice genome and 27 genes in Arabidopsis thaliana genome, 25 genes in Brachypodium distachyon genome, 22 genes in Vitis vinifera genome and 36 genes in Populus trichocarpa genome. The found TALE genes in pineapple genome were assigned to four groups (A, B, C, and D) based on similarity. The 18 TALE genes in pineapple were located on 11 of the 25 chromosomes. Structural heterogeneity was observed for the pineapple TALE genes and number of introns varied from 1 to 3 while the number of exons was 2–4. The pineapple TALE genes showed tissue specific expression. AcoTALE1 and AcoTALE15 were highly expressed in roots while AcoTALE16, AcoTALE2, and AcoTALE18 were highly expressed in fruit tissues. AcoTALE4 and AcoTALE7 were highly expressed at two different stages of stamen development. Abiotic shock treatment revealed that AcoTALE17 and AcoTALE8 were moderately upregulated by cold shock while heat shock firmly upregulated AcoTALE16 and AcoTALE5. AcoTALE2, AcoTALE4, AcoTALE12 and AcoTALE16 was highly expressed in response to salt shock while AcoTALE5 and AcoTALE12 was upregulated in response to osmotic (mannitol) shock. None of the TALE gene was differentially regulated in response to sudden darkness. Our study, identifying these TALE transcription factors and their involvement in different stress responses lays a foundation for the possible use of genetic modifications of TALE genes for pineapple crop improvements.

'Building a perfect body': control of vertebrate organogenesis by PBX-dependent regulatory networks.

Pbx genes encode transcription factors that belong to the TALE (three-amino-acid loop extension) superclass of homeodomain proteins. We have witnessed a surge in information about the roles of this gene family as leading actors in the transcriptional control of development. PBX proteins represent a clear example of how transcription factors can regulate developmental processes by combinatorial properties, acting within multimeric complexes to implement activation or repression of transcription depending on their interaction partners. Here, we revisit long-emphasized functions of PBX transcription factors as cofactors for HOX proteins, major architects of the body plan. We further discuss new knowledge on roles of PBX proteins in different developmental contexts as upstream regulators of Hox genes-as factors that interact with non-HOX proteins and can work independently of HOX-as well as potential pioneer factors. Committed to building a perfect body, PBX proteins govern regulatory networks that direct essential morphogenetic processes and organogenesis in vertebrate development. Perturbations of PBX-dependent networks can cause human congenital disease and cancer.

Comprehensive analysis of the three-amino-acid-loop-extension gene family and its tissue-differential expression in response to salt stress in poplar

The three-amino-acid-loop-extension (TALE) transcription factor gene family is widely present in plants and plays an important role in its growth and development. However, studies on the gene family are limited in poplar. In this study, we investigated 35 TALE gene family members in terms of their evolutionary relationship, classification, physicochemical properties, gene structures, and protein motifs. We divided the genes into four classes, based on their protein sequences similarity. The members from each class share similar gene structures and motif compositions. Evidence from transcript profiling indicated that the majority of the TALE genes exhibited distinct expression patterns over leaf, stem, and root tissues. Out of the 35 genes, 17 genes are highly expressed in stems, suggesting that the TALE gene family may play an important role in secondary growth and wood formation. Furthermore, out of the 35 genes, 11 genes are responsive to salt stress, and the spatio-temporal expression patterns of these 11 genes under salt stress were analysed using RT-qPCR. Yeast two-hybridization analysis indicated that poplar TALE proteins from different classes can form heterodimers. These results lay the foundation for future studies on biological functions of poplar TALE genes.

Functional testing of a human PBX3 variant in zebrafish reveals a potential modifier role in congenital heart defects.

ABSTRACTWhole-genome and exome sequencing efforts are increasingly identifying candidate genetic variants associated with human disease. However, predicting and testing the pathogenicity of a genetic variant remains challenging. Genome editing allows for the rigorous functional testing of human genetic variants in animal models. Congenital heart defects (CHDs) are a prominent example of a human disorder with complex genetics. An inherited sequence variant in the human PBX3 gene (PBX3 p.A136V) has previously been shown to be enriched in a CHD patient cohort, indicating that the PBX3 p.A136V variant could be a modifier allele for CHDs. Pbx genes encode three-amino-acid loop extension (TALE)-class homeodomain-containing DNA-binding proteins with diverse roles in development and disease, and are required for heart development in mouse and zebrafish. Here, we used CRISPR-Cas9 genome editing to directly test whether this Pbx gene variant acts as a genetic modifier in zebrafish heart development. We used a single-stranded oligodeoxynucleotide to precisely introduce the human PBX3 p.A136V variant in the homologous zebrafish pbx4 gene (pbx4 p.A131V). We observed that zebrafish that are homozygous for pbx4 p.A131V are viable as adults. However, the pbx4 p.A131V variant enhances the embryonic cardiac morphogenesis phenotype caused by loss of the known cardiac specification factor, Hand2. Our study is the first example of using precision genome editing in zebrafish to demonstrate a function for a human disease-associated single nucleotide variant of unknown significance. Our work underscores the importance of testing the roles of inherited variants, not just de novo variants, as genetic modifiers of CHDs. Our study provides a novel approach toward advancing our understanding of the complex genetics of CHDs.

Characterization of the interaction interface and conformational dynamics of human TGIF1 homeodomain upon the binding of consensus DNA

The TG interacting factor-1 homeodomain (TGIF1-HD) binds with the consensus DNA motif 5′-TGTCA-3′ in gene promoters through its three-amino acid loop extension (TALE) type homeodomain, and then recruits co-regulators to regulate gene expression. Although the solution NMR structure of human TGIF1-HD has been reported previously, little is known about its DNA binding mechanism. NMR titrations have been extensively used to study mechanisms of ligand binding to target proteins; however, an intermediate exchange occurred predominantly between TGIF1-HD in the free and bound states when titrated with the consensus DNA, which resulted in poor-quality NMR spectra and precluded further exploration of its interaction interface and conformational dynamics. Here, the helix α3 of TGIF1-HD was speculated as the specific DNA binding interface by hydrogen–deuterium exchange mass spectrometry (HDX-MS) experiments, and subsequently confirmed by chemical exchange saturation transfer (CEST) spectroscopy. In addition, simultaneous conformational changes in other regions, including α1 and α2, were induced by DNA binding, explaining the observation of chemical shift perturbations from extensive residues besides those located in α3. Further, low-populated DNA-bound TGIF1-HD undergoing a slow exchange at a rate of 130.2 ± 3.6 s−1 was derived from the analysis of the CEST data, and two residues, R220 and R221, located in the middle of α3 were identified to be crucial for DNA binding. Our study provides structural and dynamic insights into the mechanisms of TGIF1-HD recognition of extensive promoter DNA.

Prep1, A Homeodomain Transcription Factor Involved in Glucose and Lipid Metabolism.

The three-amino acid loop extension (TALE) homeodomain proteins are a family of transcription factor including the mammalian Pbx, MEIS and Prep proteins. TALE proteins can bind other transcription factors such as Pdx-1 and play an important role in the regulation of glucose metabolism. Experiments performed in mutant mice have shown that while the single Pbx1 or Pdx-1 knockout mice feature pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia, the trans-heterozygous Pbx1+/− Pdx1+/− mice develop age-dependent overt diabetes mellitus. In contrast, Prep1 plays a different role with respect to these proteins. Indeed, Prep1 hypomorphic mice, expressing low levels of protein, feature pancreatic islet hypoplasia accompanied by hypoinsulinemia similar to Pbx1 or Pdx1. Nevertheless, these animals show increased insulin sensitivity in skeletal muscle, liver and adipose tissue accompanied by protection from streptozotocin-induced diabetes. In addition, Prep1 hypomorphic mice feature reduced triglyceride synthesis and do not develop steatohepatitis after a methionine and coline deficient diet. In this review we have underlined how important metabolic functions are controlled by TALE proteins, in particular by Prep1, leading to hypothesis that its suppression might represent beneficial effect in the care of metabolic diseases.

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.

Identification and functional characterization of a novel BEL1-LIKE homeobox transcription factor GmBLH4 in soybean

The interaction between BEL1- and KNOTTED1-type homeobox transcription factors from TALE (Three-Amino-acid-Loop Extension) class plays key roles in plant growth and development. In our previous study, a soybean KNOTTED1-like gene GmSBH1, which played important roles in soybean growth and development as well as in response to high temperature and humidity (HTH) stress was identified. In the present study, 15 GmSBH1-interacting candidate proteins were identified by yeast two-hybrid system. From the 15 candidate proteins, a novel BEL1-LIKE homeodomain 4 protein, GmBLH4, was selected to isolate and characterize through sequence alignment, phylogenetic analysis and subcellular localization. GmBLH4 showed tissues specific expression and was involved in response to HTH stress in developing seeds. Overexpression of GmBLH4 in Arabidopsis altered leaf phenotype and silique length, and enhanced seed tolerance to HTH stress. Glutathione-S-transferase pull-down (GST) and bimolecular fluorescence complementation (BiFC) assays confirmed that GmBLH4 specifically interacted with GmSBH1 in vivo and in vitro. The conserved domains of GmSBH1 and GmBLH4 were sufficient for their interaction in yeast expression system. Our results indicated that GmBLH4 might heterodimerize with GmSBH1 to form functional complexes and function in modulating plant growth and development as well as in response to HTH stress in soybean.

Abstract 500: Endothelial Specific Meis1 Knockout Protects Cells from Doxorubicin-Induced Apoptosis

Introduction: Meis1 belongs to the TALE (three amino-acid loop extension) subclass of the homeobox gene families. It is a highly conserved transcription factor in all eukaryotes and plays a crucial role in anatomical development during embryogenesis and maintaining homeostasis after birth. However, little is known about the role of Meis1 gene on regulating functions of endothelial cells (ECs). Our preliminary study found that EC-specific knockout (KO) of Meis1 showed protective benefits in response to hindlimb ischemia, such as increased blood flow and decreased toe apoptosis. We hypothesize that deletion of Meis1 may enhance EC viability or resistance to harsh environment. The present study aims to explore the potential underlying mechanisms using an in vitro apoptotic condition induced by doxorubicin (Dox). Methods: EC-specific deletion of Meis1 gene was generated by crossbreeding Meis1 flox/flox mice with Tie2-Cre mice. Following confirmation of genotyping, ECs were isolated from the lungs of 3-5 WT and KO newborn pups using CD31 microbeads. Cells were then plated and treated with 0, 0.5, and 1 μM Dox for 24 hours prior to various assays. Results: Our qPCR data indicated that KDR expression was ~100-times more in CD31 + fraction than in CD31 - fraction both in WT and KO groups (n=4); while in KO group, Meis1 expression was significantly decreased to 20% of WT control, suggesting the successful isolation of Meis1-KO ECs. Further analysis with immunocytochemistry indicated that 83.1±2.3% CD31 + cells were also KDR + , compared to 8.3±1.2% KDR + cells in CD31 - fraction (n=7-8). Using EC-specific dye uptake assay, we found that 84.6±2.4% CD31 + cells were positive for Dil-Ac-LDL, compared to 7.9±3.2% in CD31 - cells (n=6), which appears to correspond to more tube formation observed in KO than WT groups. As we expected, Meis1-KO ECs demonstrated greater cell viability (81.2±6.1%) than WT cells (58.0±1.6%) under 0.5 μM Dox treatment (n=5-6). The increased viability in KO ECs may be due to the decreased expression of Bax , a pro-apoptotic factor. Conclusions: We conclude that activation of Meis1 gene may facilitate EC death under ischemic condition while deletion of Mei1 gene promote protective effects on ECs.

Structural insights into the impact of two holoprosencephaly-related mutations on human TGIF1 homeodomain

Human protein TGIF1 is an essential regulator of cell fate with broad roles in different tissues, and has been implicated in holoprosencephaly (HPE) and many cancers. The function of TGIF1 in transcriptional regulation depends on its three-amino acid loop extension (TALE) type of homeodomain (HD). Two missense mutations that led to P192A and R219C substitutions in TGIF1-HD were previously found in HPE patients and suggested to be the causes for these cases. However, how these mutations affected TGIF1 function has not been investigated from a structural view. Here, we investigated the roles of P192 and R219 in TGIF1-HD structure packing through determining the NMR structure of TGIF1-HD. Surprisingly, P192 and R219 were found to play roles in packing α1 and α2 to α3 together with A190 and F215 through side-chain interactions. Circular dichroism (CD) showed that P192A and R219C mutants displayed structural change and less folding compared with wild-type TGIF1-HD, and 1H-15N HSQC spectrum of P192A mutant exhibited chemical shift perturbations in all three helices of TGIF1-HD. Thus, it is suggested that P192A and R219C mutations led to structure disturbances of TGIF1-HD, which subsequently reduced the DNA-binding affinity of TGIF1-HD by 23-fold and 10-fold respectively, as revealed by the isothermal titration calorimetry (ITC) experiments. Our study provides structural insights of the probable pathogenesis mechanism of two TGIF1-related HPE cases, and evidences for the roles of P192 and R219 in HD folding.

Emerging Roles of Meis1 in Cardiac Regeneration, Stem Cells and Cancer.

Meis1 is a member of three-amino-acid loop extension (TALE) homeodomain transcription factors. Studies in the last decade have shown that Meis1 has crucial roles in cardiac regeneration, stem cell function, and tumorigenesis. We have recently demonstrated that knocking out of Meis1 in adult cardiomyocytes resulted in the induction of cardiomyocyte proliferation. This suggests that targeting of Meis1 might be utilized in the manipulation of cardiomyocyte cell cycle post cardiac injuries. In addition, hematopoietic stem cell (HSC) specific deletion of Meis1 leads to in vivo expansion of HSCs pool. Thus, targeting Meis1 may lead to not only cell cycle entry but also ex vivo and in vivo expansion of HSCs. On the other hand, Meis1 transcriptionally regulates the expression of hypoxic tumor markers, namely Hif-1α and Hif-2α. Hif-1α and Hif-2α are involved in the induction of cytoplasmic glycolysis and scavenging of reactive oxygen species (ROS), respectively. Studies highlight emerging roles of Meis1 towards development of new therapeutic approaches in the treatment of myocardial injuries, bone failure, and cancer.