Transcriptional Network of p63 in Human Keratinocytes

p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin

Engineered kinases as a tool for phosphorylation of selected targets in vivo

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors

Nanobody-Based GFP Traps to Study Protein Localization and Function in Developmental Biology

Synthetic protein-binding tools based on anti-green fluorescent protein (GFP) nanobodies have recently emerged as useful resources to study developmental biology. By fusing GFP-targeting nanobodies to well-characterized protein domains residing in discrete sub-cellular locations, it is possible to directly and acutely manipulate the localization of GFP-tagged proteins-of-interest in a predictable manner. Here, we describe a detailed protocol for the application of nanobody-based GFP-binding tools, namely Morphotrap and GrabFP, to study the localization and function of extracellular and intracellular proteins in the Drosophila wing imaginal disc. Given the generality of these methods, they are easily applicable for use in other tissues and model organisms

Using Nanobodies to Study Protein Function in Developing Organisms

Polyclonal and monoclonal antibodies have been invaluable tools to study proteins over the past decades. While indispensable for most biological studies including developmental biology, antibodies have been used mostly in fixed tissues or as binding reagents in the extracellular milieu. For functional studies and for clinical applications, antibodies have been functionalized by covalently fusing them to heterologous partners (i.e., chemicals, proteins or other moieties). Such functionalized antibodies have been less widely used in developmental biology studies. In the past few years, the discovery and application of small functional binding fragments derived from single-chain antibodies, so-called nanobodies, has resulted in novel approaches to study proteins during the development of multicellular animals in vivo. Expression of functionalized nanobody fusions from integrated transgenes allows manipulating proteins of interest in the extracellular and the intracellular milieu in a tissue- and time-dependent manner in an unprecedented manner. Here, we describe how nanobodies have been used in the field of developmental biology and look into the future to imagine how else nanobody-based reagents could be further developed to study the proteome in living organisms

Reflections on the use of protein binders to study protein function in developmental biology

Studies in the field of developmental biology aim to unravel how a fertilized egg develops into an adult organism and how proteins and other macromolecules work together during this process. With regard to protein function, most of the developmental studies have used genetic and RNA interference approaches, combined with biochemical analyses, to reach this goal. However, there always remains much room for interpretation on how a given protein functions, because proteins work together with many other molecules in complex regulatory networks and it is not easy to reveal the function of one given protein without affecting the networks. Likewise, it has remained difficult to experimentally challenge and/or validate the proposed concepts derived from mutant analyses without tools that directly manipulate protein function in a predictable manner. Recently, synthetic tools based on protein binders such as scFvs, nanobodies, DARPins, and others have been applied in developmental biology to directly manipulate target proteins in a predicted manner. Although such tools would have a great impact in filling the gap of knowledge between mutant phenotypes and protein functions, careful investigations are required when applying functionalized protein binders to fundamental questions in developmental biology. In this review, we first summarize how protein binders have been used in the field, and then reflect on possible guidelines for applying such tools to study protein functions in developmental biology. This article is categorized under: Technologies > Analysis of Proteins Establishment of Spatial and Temporal Patterns > Gradients Invertebrate Organogenesis > Flies

Studying Protein Function Using Nanobodies and Other Protein Binders in Drosophila

The direct manipulation of proteins by nanobodies and other protein binders has become an additional and valuable approach to investigate development and homeostasis in Drosophila. In contrast to other techniques, that indirectly interfere with proteins via their nucleic acids (CRISPR, RNAi, etc.), protein binders permit direct and acute protein manipulation. Since the first use of a nanobody in Drosophila a decade ago, many different applications exploiting protein binders have been introduced. Most of these applications use nanobodies against GFP to regulate GFP fusion proteins. In order to exert specific protein manipulations, protein binders are linked to domains that confer them precise biochemical functions. Here, we reflect on the use of tools based on protein binders in Drosophila. We describe their key features and provide an overview of the available reagents. Finally, we briefly explore the future avenues that protein binders might open up and thus further contribute to better understand development and homeostasis of multicellular organisms

In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing

Precise genome engineering is essential for both basic and applied research, permitting the manipulation of genes and gene products in predictable ways. The irruption of the CRISPR/Cas technology accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. To this day, a number of strategies permit gene manipulation. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we propose an efficient method based on homology-directed repair (HDR) and single-strand annealing (SSA) repair pathways. In this method, HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. By engineering the Hedgehog (Hh) pathway components, we demonstrated fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. The use of homology arms as short as 30 base pairs further simplified and cheapened the process. In addition, SSA can be triggered in somatic cells, permitting conditional gene labeling in different tissues. Finally, to achieve conditional labeling and manipulation of proteins tagged with short protein tags, we have further developed a toolbox based on rational engineering and functionalization of the ALFA nanobody

An epigenetic profile of early T-cell development from multipotent progenitors to committed T-cell descendants

Cellular differentiation of the T-cell branch of the immune system begins with the hematopoietic stem cell, which undergoes a series of stages characterized by progressive restriction in multipotency and acquisition of specific lineage identity At the molecular level, the restriction of cell potential, commitment and differentiation to a specific lineage is achieved through the coordinated control of gene expression and epigenetic mechanisms. Here we analyzed and compared the gene expression profiles and the genome-wide histone modification marks H3K4me3 and H3K27me3 in i) in vitro propagated hematopoietic stem cells, ii) in vitro generated and propagated pro T dells derived from these stem cells and iii) Double positive thymocytes derived from these pro T cells after injection into Rag deficient mice. The combined analyses of the different datasets in this unique experimental system, highlighted the importance of both transcriptional and epigenetic repression in shaping the early phases of T-cell development. This article is protected by copyright. All rights reserved

DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

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

DARPins recognize mTFP1 as novel reagents for and protein manipulations

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