Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the ...intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, several affinity reagents against fluorescent proteins have also been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfil their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or in living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.
Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the ...intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, several affinity reagents against fluorescent proteins have also been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfil their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or in living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.
Summary:
Structural characterization of two novel DARPins (designed ankyrin repeat proteins) recognizing the monomeric teal fluorescent protein 1 (mTFP1) and their functionalization for protein manipulation strategies in cultured cells and potentially in living organisms.
Heterozygous mutations in the TP63 transcription factor underlie the molecular basis of several similar autosomal dominant ectodermal dysplasia (ED) syndromes. Here we provide a novel cellular model ...derived from embryonic stem (ES) cells that recapitulates
in vitro the main steps of embryonic skin development. We show that ES cells carrying AEC or EEC mutations are unable to differentiate into the epidermal fate. Comparative transcriptome analysis strongly reveals an embryonic epidermal signature and suggests that mutations in the SAM domain (AEC) provide activating properties while mutations in the DBD domain (EEC) induce strong inhibitory capabilities. Our model uncovers the effect of relevant ED mutations that otherwise are difficult to evaluate on the ectodermal embryonic stage, an embryonic event critical for proper skin formation.
The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An ...important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis‐regulatory sequences of optomotor‐blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence‐specific DNA binding protein. Mutations in the high‐affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.
The murine oct-3 gene encodes a transcription factor containing a POU-specific domain and a homeodomain. In marked contrast to other homeodomain-encoding genes, oct-3 is expressed in the totipotent ...and pluripotent stem cells of the pregastrulation embryo and is down-regulated during differentiation to endoderm and mesoderm, suggesting that it has a role in early development. The oct-3 gene is also expressed in primordial germ cells and in the female germ line.
A central theme during development and homeostasis is the generation of cell type-specific responses to the action of a limited number of extant signaling cascades triggered by extracellular ligands. ...The molecular mechanisms by which information from such signals are integrated in responding cells in a cell-type specific manner remain poorly understood. We have undertaken a detailed characterization of an enhancer that is regulated by DPP signaling and by the homeotic protein Labial and its partners, Extradenticle and Homothorax. The expression driven by this enhancer (lab550) and numerous deletions and point mutants thereof was studied in wild-type and mutant Drosophila embryos as well as in cultured cells. We find that the lab550 enhancer is composed of two elements, a Homeotic Response Element (HOMRE) and a DPP Response Element (DPPRE) that synergize. None of these two elements can reproduce the expression of lab550, either with regard to expression level or with regard to spatial restriction. The isolated DPPRE of lab550 responds extremely weakly to DPP. Interestingly, we found that the inducibility of this DPPRE is weak because it is tuned down by the action of a repressor element. This repressor element and an additional 50 bp element appear to be crucial for the cooperation of the HOMRE and the DPPRE, and might tightly link the DPP response to the homeotic input. The cooperation between the different elements of the enhancer leads to the segmentally restricted activity of lab550 in the endoderm and provides a mechanism to create specific responses to DPP signaling with the help of a HOX protein complex.
Signaling by Decapentaplegic (Dpp), a member of the TGFβ superfamily of signaling molecules similar to vertebrate BMP2 and BMP4, has been implicated in many developmental processes in Drosophila ...melanogaster. Notably, Dpp acts as a long‐range morphogen during imaginal disc growth and patterning. Genetic approaches led to the identification of a number of gene products that constitute the core signaling pathway. In addition to the ligand‐activated heteromeric receptor complex and the signal‐transducing intracellular Smad proteins, Dpp signaling requires two nuclear proteins, Schnurri (Shn) and Brinker (Brk), to prime cells for Dpp responsiveness. A complex interplay between the nuclear factors involved in Dpp signaling appears to control the transcriptional readout of the Dpp morphogen gradient. It remains to be seen whether similar molecular mechanisms operate in the nucleus in vertebrate systems.
Monocytes promote the early host response to infection releasing key pro-inflammatory cytokines, such as IL-1β. The biologically inactive IL-1β precursor is processed to active form by inflammasomes, ...multi-protein complexes activating caspase-1. Human monocytes exhibit an unconventional one-step pathway of inflammasome activation in response to lipopolysaccharide (LPS) alone. Although this lineage-restricted mechanism is likely to contribute to the pathology of endotoxin shock, signalling pathways regulating this mechanism are currently unknown. Here we report that caspase-4 and caspase-5 mediate IL-1α and IL-1β release from human monocytes after LPS stimulation. Although caspase-4 remains uncleaved, caspase-5 undergoes rapid processing upon LPS treatment. We also identify an additional caspase-5 cleavage product in LPS-stimulated monocytes, which correlates with IL-1 secretion. This one-step pathway requires Syk activity and Ca(2+) flux instigated by CD14/TLR4-mediated LPS internalization. Identification of caspase-4/5 as the key determinants of one-step inflammasome activation in human monocytes provides potential targets for therapeutic intervention in endotoxin shock.
p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The ...DeltaNp63alpha isoform is thought to play a 'master' role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing DeltaNp63alpha; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.
C/EBPs are a family of B-Zip transcription factors -TFs- involved in the regulation of differentiation in several tissues. The two most studied members -C/EBPalpha and C/EBPbeta- play important roles ...in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPdelta which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPdelta target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPdelta as well as overexpressions of two TF targets -MafB and SOX2- affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPdelta is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPdelta plays a role in late stages of keratinocyte differentiation.
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![C/EBP[delta] Gene Targets i...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/1932-6203/small "C/EBP[delta] Gene Targets in Human Keratinocytes.(Research Article)")