Homeotic Proteins Research Articles

Reconstitution of ‘floral quartets’ in vitro involving class B and class E floral homeotic proteins

Homeotic MADS box genes encoding transcription factors specify the identity of floral organs by interacting in a combinatorial way. The ‘floral quartet model’, published several years ago, pulled together several lines of evidence suggesting that floral homeotic proteins bind as tetramers to two separated DNA sequence elements termed ‘CArG boxes’ by looping the intervening DNA. However, experimental support for ‘floral quartet’ formation remains scarce. Recently, we have shown that the class E floral homeotic protein SEPALLATA3 (SEP3) is sufficient to loop DNA in floral-quartet-like complexes in vitro. Here, we demonstrate that the class B floral homeotic proteins APETALA3 (AP3) and PISTILLATA (PI) do only weakly, at best, form floral-quartet-like structures on their own. However, they can be incorporated into such complexes together with SEP3. The subdomain K3 of SEP3 is of critical importance for the DNA-bound heterotetramers to be formed and is capable to mediate floral quartet formation even in the sequence context of AP3 and PI. Evidence is presented suggesting that complexes composed of SEP3, AP3 and PI form preferentially over other possible complexes. Based on these findings we propose a mechanism of how target gene specificity might be achieved at the level of floral quartet stability.

The homeotic protein HOXC13 is a member of human DNA replication complexes

The homeotic (and oncogenic) HOXC13 protein was shown to have an affinity for a DNA fragment corresponding to the sequence covered by the pre-replicative complex of the human lamin B2 replication origin. We show here that HOXC13 is a member of human replicative complexes. Our fluorescent fusion-protein data demonstrate that it co-localizes with replication foci of early-S cells and that this peculiar behaviour is driven by the homeodomain. By ChIP analysis we also show that HOXC13 binds the lamin B2 replication origin and the origins located near the TOP1 and MCM4 genes in asynchronously growing cells, whereas it does not bind these origins in G0 resting cells, consistently with its involvement in origin function.

Developmental Robustness by Obligate Interaction of Class B Floral Homeotic Genes and Proteins

DEF-like and GLO-like class B floral homeotic genes encode closely related MADS-domain transcription factors that act as developmental switches involved in specifying the identity of petals and stamens during flower development. Class B gene function requires transcriptional upregulation by an autoregulatory loop that depends on obligate heterodimerization of DEF-like and GLO-like proteins. Because switch-like behavior of gene expression can be displayed by single genes already, the functional relevance of this complex circuitry has remained enigmatic. On the basis of a stochastic in silico model of class B gene and protein interactions, we suggest that obligate heterodimerization of class B floral homeotic proteins is not simply the result of neutral drift but enhanced the robustness of cell-fate organ identity decisions in the presence of stochastic noise. This finding strongly corroborates the view that the appearance of this regulatory mechanism during angiosperm phylogeny led to a canalization of flower development and evolution.

Blocking internalization of pBP1, a secreted homeoprotein, kills breast cancer cells but not normal breast epithelial cells.

Abstract Abstract #3068 Background: BP1 is a member of the homeobox gene family, a highly conserved family of transcription factor genes. pBP1 is a homeotic protein that is upregulated in 80% of invasive ductal breast tumors. We have previously shown that the percentage of pBP1 positive breast cancer cases increases with the extent of cellular proliferation and carcinogenesis. BP1 expression is associated with aggressive tumors: 100% of ER negative tumors were BP1 positive, compared with 73% of ER positive tumors. pBP1 is secreted by MCF-7 cells, T47D cells, and MDA-MB-231 breast cancer cells, but not by MCF10A or H16N2 normal breast epithelial cells. In all cell lines tested, secreted pBP1 is internalized, resulting in stimulation of growth. Moreover, incubation of MCF-7 or MCF10A cells in recombinant pBP1 results in stimulation of several oncogenes, including bcl-2, Twist, and Met. These results led us to ask whether we could abrogate mitogenic stimulation by addition of anti-pBP1 antibody.
 Materials and Methods. Cells were incubated in 10 µg/ml anti-BP1 Ab (Novus) or 10 µg/ml IgG for three days. Growth was measured using MTT assays and apoptosis was assessed by flow cytometry after staining with Annexin V (Trevigen).
 Results. Addition of anti-BP1 Ab to the growth medium of MCF-7 cells, T47D cells, and MDA-MB-231 cells caused significant growth inhibition and apoptosis. In contrast, addition of anti-BP1 Ab to the growth medium of MCF10A and H16N2 cells did not lead to significant growth inhibition or apoptosis.
 Discussion. Our novel finding that pBP1 is secreted by breast cancer cells but not normal breast epithelial cells was the impetus to test the effect of adding anti-BP1 Ab to the growth media of both types of cells. Remarkably, anti-BP1 Ab kills only the breast cancer cells, suggesting they may be BP1-dependent. Our data indicate it may be possible to specifically target BP1 positive breast cancer cells in the 80% of women with BP1 positive tumors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3068.

The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in ‘floral quartet’-like complexes in vitro

The organs of a eudicot flower are specified by four functional classes, termed class A, B, C and E, of MADS domain transcription factors. The combinatorial formation of tetrameric complexes, so called ‘floral quartets’, between these classes is widely believed to represent the molecular basis of floral organ identity specification. As constituents of all complexes, the class E floral homeotic proteins are thought to be of critical relevance for the formation of floral quartets. However, experimental support for tetrameric complex formation remains scarce. Here we provide physico-chemical evidence that in vitro homotetramers of the class E floral homeotic protein SEPALLATA3 from Arabidopsis thaliana bind cooperatively to two sequence elements termed ‘CArG boxes’ in a phase-dependent manner involving DNA looping. We further show that the N-terminal part of SEPALLATA3 lacking K3, a subdomain of the protein–protein interactions mediating K domain, and the C-terminal domain, is sufficient for protein dimerization, but not for tetramer formation and cooperative DNA binding. We hypothesize that the capacity of class E MADS domain proteins to form tetrameric complexes contributes significantly to the formation of floral quartets. Our findings further suggest that the spacing and phasing of CArG boxes are important parameters in the molecular mechanism by which floral homeotic proteins achieve target gene specificity.

The serum levels of the EGF‐like homeotic protein dlk1 correlate with different metabolic parameters in two hormonally different children populations in Spain*

The Dlk1 gene encodes for dlk1, a transmembrane protein belonging to the EGF-like repeat-containing family. Dlk1 has been shown to act as a regulator of adipogenesis. Fc-dlk1 transgenic mice show a decrease in adipose tissue and glucose tolerance, hypertriglyceridaemia and lower insulin sensitivity. Dlk1-deficient mice show growth retardation, increased serum lipid metabolites and develop obesity. These data advocate for a role of dlk1 in the maintenance of lipid homeostasis, and suggest that dlk1 levels may influence the development of cardiovascular disease. In this study, we analysed whether dlk1 serum levels could be indicative of the different hormonal or metabolic status shown by two Spanish children populations (6-8 years-old), Orense and Murcia. We determined dlk1 serum levels by ELISA assay, using an antibody raised against the recombinant protein, and performed a correlation analysis against measurements of several hormonal and biochemical parameters in samples from 494 subjects. We found a statistically significant positive correlation between serum levels of dlk1 and those of glucose (P < 0.05), total cholesterol (P < 0.01) and high-density lipoprotein-cholesterol (HDL-C) (P < 0.01) in children from Murcia, but not from Orense's population, where dehydroepiandrosterone-sulphate (DHEA-S) levels were significantly higher (P < 0.01) and dlk1 correlated positively with insulin (P < 0.01), homeostasis model assessment (HOMA) (P < 0.01) and free fatty acids (FFA) (P < 0.05). dlk1 serum levels appear related to the anabolic status of the children in association with changes in the levels of DHEA-S, which have been associated with hyperinsulinaemia and diabetes. Monitoring dlk1 levels may be important to evaluate the metabolic and hormonal stage of child development.

Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid

In our previous studies, we identified four DEFICIENS (DEF)-like genes and one GLOBOSA (GLO)-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA binding properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial for the elucidation of the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by the yeast two-hybrid system (Y2H) and glutathione S-transferase (GST) pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 in Y2H assay, yet with different strengths of interaction. Generally, the PeMADS3/PeMADS4 lineage interacted more strongly with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) was detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using a GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein-binding motif CArG. The heterodimeric complexes PeMADS2-PeMADS6, PeMADS4-PeMADS6 and PeMADS5-PeMADS6 showed CArG binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.

Dlk1 Specifically Interacts with Insulin-Like Growth Factor Binding Protein 1 to Modulate Adipogenesis of 3T3-L1 Cells

dlk1 is an epidermal growth factor (EGF)-like homeotic protein containing an intracellular region, a single transmembrane domain, and an extracellular region possessing six EGF-like repeats and a protease-target sequence. dlk1 functions as a modulator of adipogenesis, and other differentiation processes. The molecular mechanisms by which dlk1 regulates these processes are unclear. It has been reported that different Dlk1 mRNA spliced variants, encoding for isoforms possessing the protease-target sequence or not, determine the production of membrane-associated or soluble, secreted extracellular dlk1 proteins that appear to affect adipogenesis of 3T3-L1 cells differently. In particular, only soluble variants inhibit this process. Some recent evidence suggest that dlk1 may modulate extracellular stimuli inducing differentiation. Thus, an enforced decrease of Dlk1 expression in BALB/c 3T3 cells, which results in an increase of their adipogenic potential in response to insulin-like growth factor 1 (IGF-1), modifies the kinetics and levels of activation of ERK1/2 triggered by it. In this work, we identified a strong and specific interaction between the protease-target dlk1 region and the non-IGF binding region of IGF binding protein 1 (IGFBP1), a protein that binds to IGFs and modulates their action. We also observed that the increased adipogenic potential of 3T3-L1 cells caused by diminishing Dlk1 expression through transfection with an antisense Dlk1 expression construct was inhibited by the presence of IGFBP1 in the differentiation medium. On the other hand, the presence of IGFBP1 in the culture medium slightly increased the adipogenic potential of control 3T3-L1 cells, expressing regular levels of Dlk1. These data suggest that membrane dlk1 variants bind to extracellular IGFBP1/IGF-1 complexes, which may favor the release of IGF-1 and increase the local concentration of free IGF-1 that can enhance IGF receptor signaling, leading to adipogenesis.

The MIK region rather than the C‐terminal domain of AP3‐like class B floral homeotic proteins determines functional specificity in the development and evolution of petals

In core eudicots, euAP3-type MADS-box genes encode a PISTILLATA (PI)-derived motif, as well as a C-terminal euAP3 motif that originated from a paleoAP3 motif of an ancestral APETALA3 (AP3)-like protein through a translational frameshift mutation. To determine the functional and evolutionary relevance of these motifs, a series of point mutation and domain-swap constructs were generated, involving CsAP3, a paleoAP3-type gene from the basal angiosperm Chloranthus spicatus encoding a truncated paleoAP3 motif, and AtAP3, a euAP3-type gene from the core eudicot Arabidopsis thaliana. The chimeric constructs were expressed in A. thaliana under the control of the AP3 promoter or the CaMV 35S promoter in an ap3 mutant or wild-type background, respectively. Significant recovery of AP3 function was obtained in both complementation and ectopic expression experiments whenever the region upstream of the C-terminal motifs (MIK region) from A. thaliana was taken, even when the PI-derived motif and the truncated paleoAP3 motif of CsAP3 substituted for the corresponding sequences from AtAP3. However, no or very weak complementation or gain-of-function was seen when the MIK region was from CsAP3. Our data suggest that changes in the MIK region rather than mutations in the C-terminal domain were of crucial importance for the evolution of the functional specificity of euAP3-type proteins in stamen and petal development.

Epigenetic Inhibition of Nuclear Receptor Small Heterodimer Partner Is Associated With and Regulates Hepatocellular Carcinoma Growth

Aberrant hypermethylation of promoter regions in cytosine-guanine dinucleotides (CpG) islands has been shown to be associated with transcriptional silencing of tumor-suppressor genes in many cancers. This study evaluated the methylation profile and the tumor-suppressive function of the small heterodimer partner (SHP, NR0B2) in the development of human hepatocellular carcinoma (HCC). Human HCC pathologic specimens and cell lines were used as model systems in this study. The expression of SHP is diminished in HCC pathologic specimens and cell lines by epigenetic silencing owing to SHP promoter hypermethylation. In vitro methylation decreased SHP promoter transactivation and nuclear receptor LRH-1 binding, an event that was reversed by demethylation. Overexpression of SHP inhibited HCC foci formation, arrested HCC tumor growth in xenografted nude mice, and increased the sensitivity of HCC cells to apoptotic stimuli. Further analysis of a total of 19 normal liver and 57 HCC specimens showed that down-regulation of SHP gene expression may be a common denominator of HCC. We propose that SHP functions as a novel tumor suppressor in the development of HCC. These findings provide new insight into the molecular mechanisms leading to this common cancer and may have both diagnostic and therapeutic applications.

Chimeric AGAMOUS repressor induces serrated petal phenotype in Torenia fournieri similar to that induced by cytokinin application

The homeotic protein AGAMOUS (AG) terminates the floral meristem and promotes the development of stamens and carpels in Arabidopsis. Disruption of its function or expression of the chimeric AG repressor (AGSRDX) results in redundant petals, known as a double flower phenotype. To investigate whether this morphological change in Arabidopsis is applicable to ornamental flowers to increase their horticultural value, we introduced AGSRDX into torenia (Torenia fournieri Lind.) plants. Transgenic torenia plants expressing AGSRDX showed no redundancy in petal number, although they exhibited serration in petal margins, anthocyanin accumulation and morphological change in the stigma surface, and formation of extra vascular bundles in petals and styles. Anatomical observation of petals and styles revealed that these phenotypes are highly similar to those of forchlorfenuron (CPPU)-treated torenia plants especially in the derangement of vascular bundles. Phenotypes similar to AGSRDX transgenic torenia plants were also observed when the chimeric repressors for torenia C-function genes TfFAR or TfPLE1, homologs of Antirrhinum FARINELLI and PLENA respectively, were expressed. These results suggest that the morphological changes in AGSRDX transgenic torenia plants are induced by the disruption of C-function. These novel phenotypes might be caused by the modification of cytokinin-dependent regulation in vascular bundle formation and ectopic expression of the chimeric repressors in all whorls by the cauliflower mosaic virus (CaMV) 35S promoter.

Molecular Mechanisms Underlying Origin and Diversification of the Angiosperm Flower

Understanding the mode and mechanisms of the evolution of the angiosperm flower is a long-standing and central problem of evolutionary biology and botany. It has essentially remained unsolved, however. In contrast, considerable progress has recently been made in our understanding of the genetic basis of flower development in some extant model species. The knowledge that accumulated this way has been pulled together in two major hypotheses, termed the 'ABC model' and the 'floral quartet model'. These models explain how the identity of the different types of floral organs is specified during flower development by homeotic selector genes encoding transcription factors. We intend to explain how the 'ABC model' and the 'floral quartet model' are now guiding investigations that help to understand the origin and diversification of the angiosperm flower. Investigation of orthologues of class B and class C floral homeotic genes in gymnosperms suggest that bisexuality was one of the first innovations during the origin of the flower. The transition from dimer to tetramer formation of floral homeotic proteins after establishment of class E proteins may have increased cooperativity of DNA binding of the transcription factors controlling reproductive growth. That way, we hypothesize, better 'developmental switches' originated that facilitated the early evolution of the flower. Expression studies of ABC genes in basally diverging angiosperm lineages, monocots and basal eudicots suggest that the 'classical' ABC system known from core eudicots originated from a more fuzzy system with fading borders of gene expression and gradual transitions in organ identity, by sharpening of ABC gene expression domains and organ borders. Shifting boundaries of ABC gene expression may have contributed to the diversification of the angiosperm flower many times independently, as may have changes in interactions between ABC genes and their target genes.

Dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

dlk1/FA1 (delta-like 1/fetal antigen-1) is a member of the epidermal growth factor-like homeotic protein family whose expression is known to modulate the differentiation signals of mesenchymal and hematopoietic stem cells in bone marrow. We have demonstrated previously that Dlk1 can maintain the human bone marrow mesenchymal stem cells (hMSC) in an undifferentiated state. To identify the molecular mechanisms underlying these effects, we compared the basal gene expression pattern in Dlk1-overexpressing hMSC cells (hMSC-dlk1) versus control hMSC (negative for Dlk1 expression) by using Affymetrix HG-U133A microarrays. In response to Dlk1 expression, 128 genes were significantly up-regulated (with >2-fold; p < 0.001), and 24% of these genes were annotated as immune response-related factors, including pro-inflammatory cytokines, in addition to factors involved in the complement system, apoptosis, and cell adhesion. Also, addition of purified FA1 to hMSC up-regulated the same factors in a dose-dependent manner. As biological consequences of up-regulating these immune response-related factors, we showed that the inhibitory effects of dlk1 on osteoblast and adipocyte differentiation of hMSC are associated with Dlk1-induced cytokine expression. Furthermore, Dlk1 promoted B cell proliferation, synergized the immune response effects of the bacterial endotoxin lipopolysaccharide on hMSC, and led to marked transactivation of the NF-kappaB. Our data suggest a new role for Dlk1 in regulating the multiple biological functions of hMSC by influencing the composition of their microenvironment "niche." Our findings also demonstrate a role for Dlk1 in mediating the immune response.

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana

Floral patterning and morphogenesis are controlled by many transcription factors including floral homeotic proteins, by which floral organ identity is determined. Recent studies have uncovered widespread regulation of transcription factors by microRNAs (miRNAs), approximately 21-nucleotide non-coding RNAs that regulate protein-coding RNAs through transcript cleavage and/or translational inhibition. The regulation of the floral homeotic gene APETALA2 (AP2) by miR172 is crucial for normal Arabidopsis flower development and is likely to be conserved across plant species. Here we probe the activity of the AP2/miR172 regulatory circuit in a heterologous Solanaceae species, Nicotiana benthamiana. We generated transgenic N. benthamiana lines expressing Arabidopsis wild type AP2 (35S::AP2), miR172-resistant AP2 mutant (35S::AP2m3) and MIR172a-1 (35S::MIR172) under the control of the cauliflower mosaic virus 35S promoter. 35S::AP2m3 plants accumulated high levels of AP2 mRNA and protein and exhibited floral patterning defects that included proliferation of numerous petals, stamens and carpels indicating loss of floral determinacy. On the other hand, nearly all 35S::AP2 plants accumulated barely detectable levels of AP2 mRNA or protein and were essentially non-phenotypic. Overall, the data indicated that expression of the wild type Arabidopsis AP2 transgene was repressed at the mRNA level by an endogenous N. benthamiana miR172 homologue that could be detected using Arabidopsis miR172 probe. Interestingly, 35S::MIR172 plants had sepal-to-petal transformations and/or more sepals and petals, suggesting interference with N. benthamiana normal floral homeotic gene function in perianth organs. Our studies uncover the potential utility of the Arabidopsis AP2/miR172 system as a tool for manipulation of floral architecture and flowering time in non-model plants.

Xenopus Teashirt1 regulates posterior identity in brain and cranial neural crest

We have isolated two related Xenopus homologues of the homeotic zinc finger protein Teashirt1 (Tsh1), XTsh1a and XTsh1b. While Drosophila teashirt specifies trunk identity in the fly, the developmental relevance of vertebrate Tsh homologues is unknown. XTsh1a/b are expressed in prospective trunk CNS throughout early neurula stages and later in the migrating cranial neural crest (CNC) of the third arch. In postmigratory CNC, XTsh1a/b is uniformly activated in the posterior arches. Gain- and loss-of-function experiments reveal that reduction or increase of XTsh1 levels selectively inhibits specification of the hindbrain and mid/hindbrain boundary in Xenopus embryos. In addition, both overexpression and depletion of XTsh1 interfere with the determination of CNC segment identity. In transplantation assays, ectopic XTsh1a inhibits the routing of posterior, but not of mandibular CNC streams. The loss of function phenotype could be rescued with low amounts either of XTsh1a or murine Tsh3. Our results demonstrate that proper expression of XTsh1 is essential for segmentally restricted gene expression in the posterior brain and CNC and suggest for the first time that teashirt genes act as positional factors also in vertebrate development.

Hyped‐up for Hox in Hyderabad

The EMBO Workshop on Upstream and Downstream of Hox Genes took place in Hyderabad, India, between 14 and 17 December 2005, and was organized by R. Mishra and F. Karch. ![][1] For many years, EMBO workshops dealing with new insights into the regulation and function of Hox genes have been held in Switzerland. However, last year, the EMBO World Programme, together with the Centre for Cellular and Molecular Biology (CCMB), held a workshop entitled Upstream and Downstream of Hox Genes in the CCMB's hometown of Hyderabad, India. Imagine beautiful gardens full of butterflies and palm trees, a delightful guest house neighbouring a state‐of‐the‐art research centre, all in a peaceful oasis surrounded by a pulsating and crowded Indian city. The question at the beginning of the meeting was whether the talks and discussions would match the stunning environment; it was answered rapidly and affirmatively. Hox genes have had an illustrious and curious history. Perhaps one of the most famous images in biology from the past century is the four‐winged mutant of the fruit fly Drosophila melanogaster caused by mutations in the Hox gene Ultrabithorax ( Ubx ). This fascinating mutant fly, discovered and characterized by Edward B. Lewis, has motivated many investigators to join the field to understand how these genes are responsible for such complex changes in morphology. Another highlight of Hox history was Matthew Scott's, William McGinnis's and Walter Gehring's discovery of the homeodomain—a 60‐amino‐acid DNA‐binding motif now known to be a key domain in hundreds of genes throughout the animal, fungal and plant kingdoms. Although a lot has been learned through experiments stemming from these important discoveries, there are still many open questions. Among the questions being debated in the field and at the meeting are: How is the regulation of the Hox genes initiated and maintained? How do Hox proteins … [1]: /embed/graphic-1.gif

Transcriptional activation by extradenticle in the Drosophila visceral mesoderm

decapentaplegic ( dpp) is a direct target of Ultrabithorax ( Ubx) in parasegment 7 (PS7) of the embryonic visceral mesoderm. We demonstrate that extradenticle ( exd) and homothorax ( hth) are also required for dpp expression in this location, as well as in PS3, at the site of the developing gastric caecae. A 420 bp element from dpp contains EXD binding sites necessary for expressing a reporter gene in both these locations. Using a specificity swap, we demonstrate that EXD directly activates this element in vivo. Activation does not require Ubx, demonstrating that EXD can activate transcription independently of homeotic proteins. Restoration is restricted to the domains of endogenous dpp expression, despite ubiquitous expression of altered specificity EXD. We demonstrate that nuclear EXD is more extensively phosphorylated than the cytoplasmic form, suggesting that EXD is a target of signal transduction by protein kinases.

Mutant analysis, protein–protein interactions and subcellular localization of the Arabidopsis Bsister (ABS) protein

Recently, close relatives of class B floral homeotic genes, termed B(sister) genes, have been identified in both angiosperms and gymnosperms. In contrast to the B genes themselves, B(sister) genes are exclusively expressed in female reproductive organs, especially in the envelopes or integuments surrounding the ovules. This suggests an important ancient function in ovule or seed development for B(sister) genes, which has been conserved for about 300 million years. However, investigation of the first loss-of-function mutant for a B(sister) gene (ABS/TT16 from Arabidopsis) revealed only a weak phenotype affecting endothelium formation. Here, we present an analysis of two additional mutant alleles, which corroborates this weak phenotype. Transgenic plants that ectopically express ABS show changes in the growth and identity of floral organs, suggesting that ABS can interact with floral homeotic proteins. Yeast-two-hybrid and three-hybrid analyses indicated that ABS can form dimers with SEPALLATA (SEP) floral homeotic proteins and multimeric complexes that also include the AGAMOUS-like proteins SEEDSTICK (STK) or SHATTERPROOF1/2 (SHP1, SHP2). These data suggest that the formation of multimeric transcription factor complexes might be a general phenomenon among MIKC-type MADS-domain proteins in angiosperms. Heterodimerization of ABS with SEP3 was confirmed by gel retardation assays. Fusion proteins tagged with CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein) in Arabidopsis protoplasts showed that ABS is localized in the nucleus. Phylogenetic analysis revealed the presence of a structurally deviant, but closely related, paralogue of ABS in the Arabidopsis genome. Thus the evolutionary developmental genetics of B(sister) genes can probably only be understood as part of a complex and redundant gene network that may govern ovule formation in a conserved manner, which has yet to be fully explored.

Differential expression of delta-like gene and protein in neuroblastoma, ganglioneuroblastoma and ganglioneuroma

Neuroblastoma is an extremely malignant solid tumor in children, characterized by spontaneous differentiation and regression. An epidermal growth factor-like homeotic protein, delta-like (dlk), has been involved in differentiation of neuroblastoma cell lines, but is unknown in in vivo expression of neuroblastoma. By using in situ hybridization and immunohistochemistry, dlk mRNA and protein expression were studied in formalin-fixed archival tissues from 10 patients with neuroblastoma, five with ganglioneuroblastoma, and five with ganglioneuroma. Three adrenal tissues from children died of diseases other than adrenal tumors and one from an adult with pheochromocytoma were severed as normal and disease controls. The results showed strong immunoreactive dlk staining in endothelial cells in neuroblastoma, ganglioneuroblastoma and ganglioneuroma. Dlk was detectable in mature neuromatous stroma and gangliocytes of ganglioneuroma, but not in neuroblasts of neuroblastoma and ganglioneuroblastoma, neither in gangliocytes of ganglioneuroblastoma. In contrast, dlk mRNA expression was mainly observed in the gangliocytes, but was less intense in the neuroblasts and neuromatous stroma cells. Endothelial cells were essentially devoid of dlk mRNA expression. The findings indicated that there is differential expression of dlk gene and protein among neuroblastoma, ganglioneuroblastoma and ganglioneuroma. The stronger expression of dlk in gangliocytes in ganglioneuroma, in contrast to weaker or no expression in gangliocytes in ganglioneuroblastoma and neuroblasts in neuroblastoma, suggests upregulation of dlk during differentiation of neuroblastoma into more benign form. Furthermore, higher dlk protein expression in the tumor endothelium than in the endothelium of normal adrenal gland implies that dlk may regulate the endothelial function in neuroblastic tumors.

Expression of DLK1 in hematopoietic cells results in inhibition of differentiation and proliferation

The Delta-like (DLK1) gene is overexpressed in CD34+ cells from myelodysplastic syndrome (MDS) patients. DLK1 encodes an EGF-like homeotic transmembrane protein homologous to the notch/delta/serrate family. Although exogenous DLK1 promotes maintenance of murine hematopoietic stem cells, the functional effects of DLK1 overexpression in hematopoietic cells are unknown. We show that ectopically expressed DLK1 significantly inhibits differentiation and proliferation of human promyelocytic HL-60 cells. Unlike preadipocytes, where proteolytic processing of membrane-bound protein and release of a soluble form mediates differentiation inhibition, proteolytic release of the extracellular domain was not required for inhibition of hematopoietic cell differentiation. However, intracellular domain interactions were critical to this DLK1 function. We conclude that DLK1 overexpression in hematopoietic cells has important functional consequences. Our studies identify novel molecular mechanisms and indicate that DLK1 has activity both as a soluble and a transmembrane expressed protein. Our results support further investigation of the role of DLK1 in abnormal hematopoiesis in MDS.