Uri Gat

Novel Assembly Properties of Recombinant Spider Dragline Silk Proteins

Spider dragline silk, which exhibits extraordinary strength and toughness, is primarily composed of two related proteins that largely consist of repetitive sequences. In most spiders, the repetitive region of one of these proteins is rich in prolines, which are not present in the repetitive region of the other. The absence of prolines in one component was previously speculated to be essential for the thread structure. Here, we analyzed dragline proteins of the garden spider Araneus diadematus, ADF-3 and ADF-4, which are both proline rich, by employing the baculovirus expression system. Whereas ADF-3 represented an intrinsically soluble protein, ADF-4 was insoluble in vitro and self-assembled into filaments in the cytosol of the host insect cells. These ADF-4 filaments displayed the exceptional chemical stability of authentic silk threads. We provide evidence that the observed properties of ADF-3 and ADF-4 strongly depend on intrinsic characteristics such as hydropathicity, which differs dramatically between the two proteins, as in most other pairs of dragline silk proteins from other Araneoidea species, but not on their proline content. Our findings shed new light on the structural components of spider dragline silk, allowing further elucidation of their assembly properties, which may open the door for commercial applications.

Identification, basic characterization and evolutionary analysis of differentially spliced mRNA isoforms of human YAP1 gene.

The YAP1 gene encodes a potent new oncogene and stem cell factor. However, in some cancers, the YAP1 gene plays a role of tumor suppressor. At present, the gene and its products are intensely studied and its cDNAs are used as transgenes in cellular and animal models. Here, we report 4 new potential mRNA splicing isoforms of the YAP1 gene, bringing the total number of isoforms to 8. We detected all 8 YAP1 isoforms in a panel of human tissues and evaluated the expression of the longest isoform of YAP1 (YAP1-2δ) using Real Time PCR. All YAP1 isoforms are barely detectable in human leukocytes compared to fair levels of expression found in other human tissues. We analyzed the structure of the genomic region that gave rise to alternatively spliced YAP1 transcripts in different metazoans. We found that YAP1 isoforms, which utilize exon 6 emerged in evolution with the appearance of amniotes. Interestingly, 6 YAP1 isoforms, which contain the exon 5 extension, exon 6 or both would have their leucine zipper region disrupted in the predicted protein product, compared to the intact leucine zipper found in two YAP1 (α) isoforms. This observation has direct functional ramifications for YAP1 signaling. We also propose a normalized nomenclature for the mRNA splice variants of the YAP1 gene, which should aid in the characterization of signaling differences among the potential protein products of the YAP1 gene.

The evolutionary origin of the Runx/CBFbeta transcription factors – Studies of the most basal metazoans

BackgroundMembers of the Runx family of transcriptional regulators, which bind DNA as heterodimers with CBFβ, are known to play critical roles in embryonic development in many triploblastic animals such as mammals and insects. They are known to regulate basic developmental processes such as cell fate determination and cellular potency in multiple stem-cell types, including the sensory nerve cell progenitors of ganglia in mammals.ResultsIn this study, we detect and characterize the hitherto unexplored Runx/CBFβ genes of cnidarians and sponges, two basal animal lineages that are well known for their extensive regenerative capacity. Comparative structural modeling indicates that the Runx-CBFβ-DNA complex from most cnidarians and sponges is highly similar to that found in humans, with changes in the residues involved in Runx-CBFβ dimerization in either of the proteins mirrored by compensatory changes in the binding partner. In situ hybridization studies reveal that Nematostella Runx and CBFβ are expressed predominantly in small isolated foci at the base of the ectoderm of the tentacles in adult animals, possibly representing neurons or their progenitors.ConclusionThese results reveal that Runx and CBFβ likely functioned together to regulate transcription in the common ancestor of all metazoans, and the structure of the Runx-CBFβ-DNA complex has remained extremely conserved since the human-sponge divergence. The expression data suggest a hypothesis that these genes may have played a role in nerve cell differentiation or maintenance in the common ancestor of cnidarians and bilaterians.

Dynamic expression of Runx1 in skin affects hair structure.

The three mammalian Runx transcription factors, some of which are known to be involved in human genetic diseases and cancer, are pivotal players in embryo development and function as key regulators of cell fate determination and organogenesis. Here, we report the expression of Runx1 during the development of hair and other skin appendages in the mouse and describe the effect of Runx1 on the structural hair output. In hair follicles, where the three Runx proteins are expressed, Runx1 expression is most prominent in both mesenchymal and epithelial compartments. The epithelial expression includes the hair keratin forming layers of the hair shaft and the bulge, where interestingly, Runx1 is co-expressed with keratin 15, a putative hair follicle stem cell marker. In the hair mesenchyme, during early stages of hair morphogenesis, Runx1 is expressed in a discrete dermal sub-epithelial layer, while at later stages it is found in a hair cycle dependent pattern in the dermal papilla. To elucidate the function of Runx1 in the hair follicle we have generated a Runx1 epidermal conditional knockout and found that the mutant mice display a remarkable structural deformation of the zigzag hair type. The data delineate Runx1 as a novel specific marker of several hair follicle cell types and sheds light on its role in hair morphogenesis and differentiation.

Runx3 is involved in hair shape determination

Transcriptional regulators of the Runx family play critical roles in normal organ development and, when mutated, lead to genetic diseases and cancer. Runx3 functions during cell lineage decisions in thymopoiesis and neurogenesis and mediates transforming growth factor‐β signaling in dendritic cells. Here, we study the function of Runx3 in the skin and its appendages, primarily the hair follicle, during mouse development. Runx3 is expressed predominantly in the dermal compartment of the hair follicles as they form and during the hair cycle, as well as in the nail and sweat gland skin appendages. Distinct expression is also detected periodically in isolated cells of the epidermis and in melanocytes, populating the hair bulb. Runx3‐deficient mice display a perturbation of the normal hair coat, which we show to be due to hair type and hair shape changes. Thus, one of the functions of Runx3 in skin may be to regulate the formation of the epithelial derived structural hair by affecting dermal to epidermal interactions. Developmental Dynamics 233:1478–1487, 2005.

A chromatin immunoprecipitation screen in mouse keratinocytes reveals Runx1 as a direct transcriptional target of ΔNp63

Development of the skin epidermis and appendages such as hair follicles involves coordinated processes of keratinocyte proliferation and differentiation. The transcription factor p63 plays a critical role in these steps as evident by a complete lack of these structures in p63 null mice. The p63 gene encodes for two proteins TAp63 and ΔNp63, the latter being the more prevalent and dominant isoform expressed in keratinocytes. Although numerous p63 target genes have been identified, these studies have been limited to transformed human keratinocyte cell lines. Here, we have employed a genomic screening approach of chromatin immunoprecipitation (ChIP) coupled with an enrichment strategy to identify ΔNp63 response elements in primary mouse keratinocytes. Analysis of p63‐ChIP‐derived DNA segments has revealed more than 100 potential target genes including novel as well as mouse counterparts of established human p63 targets. Among these is Runx1, a transcription factor important for hair follicle development. We demonstrate that ΔNp63 binds to a p63‐response element located within a well‐conserved enhancer of the Runx1 gene. Furthermore, siRNA mediated reduction of ΔNp63 in mouse keratinocytes reduces Runx1 expression. Consistent with this, endogenous Runx1 levels are lower in the skin of p63+/− animals as compared to wild type animals. Lastly, we demonstrate that ΔNp63 and Runx1 are co‐expressed in specific compartments of the hair follicle in a dynamic fashion. Taken together our data demonstrate that p63 directly regulates Runx1 gene expression through a novel enhancer element and suggests a role for these two transcription factors in dictating skin keratinocyte and appendage development. J. Cell. Biochem. 104: 1204–1219, 2008.

A proposed model for dragline spider silk self‐assembly: Insights from the effect of the repetitive domain size on fiber properties

Dragline spider silk has been intensively studied for its superior qualities as a biomaterial. In previous studies, we made use of the baculovirus mediated expression system for the production of a recombinant Araneus diadematus spider silk dragline ADF4 protein and its self-assembly into intricate fibers in host insect cells. In this study, our aim was to explore the function of the major repetitive domain of the dragline spider silk. Thus, we generated an array of synthetic proteins, each containing a different number of identical repeats up to the largest recombinantly expressed spider silk to date. Study of the self-assembly properties of these proteins showed that depending on the increasing number of repeats they give rise to different assembly phenotypes, from a fully soluble protein to bona fide fibers with superior qualities. The different assembly forms, the corresponding chemical resistance properties obtained as well as ultrastructural studies, revealed novel insights concerning the structure and intermolecular interactions of the repetitive and nonrepetitive domains. Based on these observations and current knowledge in the field, we hereby present a comprehensive hypothetical model for the mechanism of dragline silk self-assembly and fiber formation.

RBM28, a protein deficient in ANE syndrome, regulates hair follicle growth via miR-203 and p63

Alopecia–neurological defects–endocrinopathy (ANE) syndrome is a rare inherited hair disorder, which was shown to result from decreased expression of the RNA‐binding motif protein 28 (RBM28). In this study, we attempted to delineate the role of RBM28 in hair biology. First, we sought to obtain evidence for the direct involvement of RBM28 in hair growth. When RBM28 was downregulated in human hair follicle (HF) organ cultures, we observed catagen induction and HF growth arrest, indicating that RBM28 is necessary for normal hair growth. We also aimed at identifying molecular targets of RBM28. Given that an RBM28 homologue was recently found to regulate miRNA biogenesis in C. elegans and given the known pivotal importance of miRNAs for proper hair follicle development, we studied global miRNA expression profile in cells knocked down for RBM28. This analysis revealed that RBM28 controls the expression of miR‐203. miR‐203 was found to regulate in turn TP63, encoding the transcription factor p63, which is critical for hair morphogenesis. In conclusion, RBM28 contributes to HF growth regulation through modulation of miR‐203 and p63 activity.

A transcriptional time-course analysis of oral vs. aboral whole-body regeneration in the Sea anemone Nematostella vectensis

BackgroundThe ability of regeneration is essential for the homeostasis of all animals as it allows the repair and renewal of tissues and body parts upon normal turnover or injury. The extent of this ability varies greatly in different animals with the sea anemone Nematostella vectensis, a basal cnidarian model animal, displaying remarkable whole-body regeneration competence.ResultsIn order to study this process in Nematostella we performed an RNA-Seq screen wherein we analyzed and compared the transcriptional response to bisection in the wound-proximal body parts undergoing oral (head) or aboral (tail) regeneration at several time points up to the initial restoration of the basic body shape. The transcriptional profiles of regeneration responsive genes were analyzed so as to define the temporal pattern of differential gene expression associated with the tissue-specific oral and aboral regeneration. The identified genes were characterized according to their GO (gene ontology) assignations revealing groups that were enriched in the regeneration process with particular attention to their affiliation to the major developmental signaling pathways. While some of the genes and gene groups thus analyzed were previously known to be active in regeneration, we have also revealed novel and surprising candidate genes such as cilia-associated genes that likely participate in this important developmental program.ConclusionsThis work highlighted the main groups of genes which showed polarization upon regeneration, notably the proteinases, multiple transcription factors and the Wnt pathway genes that were highly represented, all displaying an intricate temporal balance between the two sides. In addition, the evolutionary comparison performed between regeneration in different animal model systems may reveal the basic mechanisms playing a role in this fascinating process.

The novel Cthrc gene family in the development and regeneration of the sea-anemone Nematostella vectensis

Dictyostelium discoideum is a free living soil amoeba that feeds on bacteria and proliferates in large numbers as long as food is abundant. On starvation the cells come together by chemotaxis, aggregate and further differentiate to a slug comprising of two distinct cell types-the prestalk and the prespore cells. The prestalk cells occupy the slug anterior and terminally differentiate to a dead stalk. The prespore cells occupy the slug rear, and finally form spores of a fruiting body. The choice to become either a prestalk or prespore cell depends on several factors such as, cell cycle phases, cellular Ca2+ levels, nutritional status, exposure to signaling molecules etc. For instance, cells at S/G1 phase of cell cycle display high cellular Ca2+ levels and preferentially become pre-stalk cells. Whereas, cells at G2/M boundary that demonstrates relatively low cellular Ca2+ levels tends to become prespore cells. Though the social amoebae isolated so far are known to be haploid, diploid strains can be constructed by parasexual genetics. Here, we wanted to ascertain if ploidy levels play a role in cell-fate choices and for this, we generated an isogenic diploid strain using the haploid parents (Ax2 wild-type and Ax2 thymidine auxotroph). The haploid and diploid cells were mixed in different proportions after one population was selectively labeled with the stable, fluorescent, live-cell marker Carboxyfluorescein succinimidyl ester (CFSE) and thereafter, the cell fate was tracked. Independently, the haploid cell populations were transfected with plasmids that express fluorescent reporters (GFP/RFP). Reconstitution of cells with different ploidy levels suggest that irrespective of the cell proportion mixes, the haploid cells always occupy the prespore region, while the diploid cells occupy the prestalk region of the slugs, suggesting that ploidy levels are also crucial in cell-fate decisions during Dictyostelium differentiation and development.