Laurent Magnenat

RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation

Although the role of the TH1 and TH17 subsets of helper T cells as disease mediators in autoimmune neuroinflammation remains a subject of some debate, none of their signature cytokines are essential for disease development. Here we report that interleukin 23 (IL-23) and the transcription factor RORγt drove expression of the cytokine GM-CSF in helper T cells, whereas IL-12, interferon-γ (IFN-γ) and IL-27 acted as negative regulators. Autoreactive helper T cells specifically lacking GM-CSF failed to initiate neuroinflammation despite expression of IL-17A or IFN-γ, whereas GM-CSF secretion by Ifng−/−Il17a−/− helper T cells was sufficient to induce experimental autoimmune encephalomyelitis (EAE). During the disease effector phase, GM-CSF sustained neuroinflammation via myeloid cells that infiltrated the central nervous system. Thus, in contrast to all other known helper T cell–derived cytokines, GM-CSF serves a nonredundant function in the initiation of autoimmune inflammation regardless of helper T cell polarization.

Scanning the human genome with combinatorial transcription factor libraries.

Despite the critical importance of transcription factors in mediating gene regulation, there exists no general, genome-wide tool that uses transcription factors to induce or silence a target gene or select for a particular phenotype. In the strategy described here, we prepared large combinatorial libraries of artificial transcription factors comprising three or six zinc-finger domains, and selected transcription factor–DNA interactions able to upregulate several genes in human cells. Selected transcription factors either induced the expression of an endothelial-specific differentiation marker, VE-cadherin, in non-endothelial cell lines or, when combined with a repression domain, knocked down expression. Potential binding sites for a number of these transcription factors were mapped along the promoter of CDH5, the gene encoding VE-cadherin. Transcription factor libraries represent a useful approach for studying and modulating gene function in cells and potentially in whole organisms.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

Chemically inducible gene switches that regulate expression of endogenous genes have multiple applications for basic gene expression research and gene therapy. Single-chain zinc-finger transcription factors that utilize either estrogen receptor homodimers or retinoid X receptor-α/ecdysone receptor heterodimers are shown here to be effective regulators of ICAM-1 and ErbB-2 transcription. Using activator (VP64) and repressor (Krüppel-associated box) domains to impart regulatory directionality, ICAM-1 was activated by 4.8-fold and repressed by 81% with the estrogen receptor-inducible transcription factors. ErbB-2 was activated by up to threefold and repressed by 84% with the retinoid X receptor-α/ecdysone receptor-inducible transcription factors. The dynamic range of these proteins was similar to the constitutive system and showed negligible basal regulation when ligand was not present. We have also demonstrated that the regulation imposed by these inducible transcription factors is dose dependent, sustainable for at least 11 days and reversible upon cessation of drug treatment. Importantly, these proteins can be used in conjunction with each other with no detectable overlap of activity enabling concurrent and temporal regulation of multiple genes within the same cell. Thus, these chemically inducible transcription factors are valuable tools for spatiotemporal control of gene expression that should prove valuable for research and gene therapy applications.

Zinc Finger Transcription Factors Designed for Bispecific Coregulation of ErbB2 and ErbB3 Receptors: Insights into ErbB Receptor Biology

ABSTRACT Signaling through the ErbB family of tyrosine kinase receptors in normal and cancer-derived cell lines contributes to cell growth and differentiation. In this work, we altered the levels of ErbB2 and ErbB3 receptors, individually and in combination, by using 6-finger and 12-finger synthetic zinc finger protein artificial transcription factors (ATFs) in an epidermoid squamous cell carcinoma line, A431. We successfully designed 12-finger ATFs capable of coregulating ErbB3 and ICAM-1 or ErbB2 and ErbB3. With ATFs, the effects of changes in ErbB2 and ErbB3 receptor levels were evaluated by using cell proliferation, cell migration, and cell signaling assays. Cell proliferation was increased when ErbB2 and ErbB3 were both overexpressed. Cell migration on collagen was decreased when ErbB2 was down-regulated, yet migration on laminin was significantly increased with ErbB3 overexpression. ErbB2 and ErbB3 overexpression also stimulated the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Our ATF approach has elucidated differences in ErbB receptor-mediated proliferation, migration, and intracellular signaling that cannot be explained merely by the presence or absence of particular ErbB receptors and emphasizes the dynamic nature of the ErbB signaling system. The transcription factor approach developed here provides a gene-economical route to the regulation of multiple genes and may be important for complex gene therapies.

Negative regulation of the RTBV promoter by designed zinc finger proteins

The symptoms of rice tungro disease are caused by infection by a DNA-containing virus, rice tungro bacilliform virus (RTBV). To reduce expression of the RTBV promoter, and to ultimately reduce virus replication, we tested three synthetic zinc finger protein transcription factors (ZF-TFs), each comprised of six finger domains, designed to bind to sequences between −58 and +50 of the promoter. Two of these ZF-TFs reduced expression from the promoter in transient assays and in transgenic Arabidopsis thaliana plants. One of the ZF-TFs had significant effects on plant regeneration, apparently as a consequence of binding to multiple sites in the A. thaliana genome. Expression from the RTBV promoter was reduced by ~45% in transient assays and was reduced by up to 80% in transgenic plants. Co-expression of two different ZF-TFs did not further reduce expression of the promoter. These experiments suggest that ZF-TFs may be used to reduce replication of RTBV and thereby offer a potential method for control of an important crop disease.