Ulrich Siebenlist

E. coli RNA polymerase interacts homologously with two different promoters.

We present and review experiments that identify points of close approach of the RNA polymerase to two promoters, lac UV5 and T7 A3. We identify the contacts to the phosphates along the DNA backbone, to the N7s of guanines in the major groove and the N3s of adenines in the minor groove, and to the methyl groups of thymines. These contacts to the two promoters are strikingly homologous in space, as shown on three-dimensional models, and identify major regions of interactions lying on one side of the DNA molecule (at -35 and -16), as well as further areas extending through the Pribnow box. Both promoters are unwound similarly by the polymerase, across a region of about twelve bases extending from the middle of the Pribnow box to just beyond the RNA start site. We discuss the areas of interaction in the context of promoter homologies and promoter mutations. The disposition of the contacts in space suggests a model for the pathway along which the RNA polymerase binds to promoters.

Chromatin structure and protein binding in the putative regulatory region of the c-myc gene in burkitt lymphoma

A chromosomal myc gene displays one of three patterns of activity depending upon the arrangement of the gene and its allelic partner. In nonmalignant B cells both myc alleles are normally expressed. In Burkitt lymphoma cells carrying both a translocated and a nontranslocated myc allele, the translocated allele is inappropriately expressed, while the nontranslocated allele is virtually inactive. Here we examine the chromatin structure of these genes using DNAase I hypersensitivity in nonmalignant lymphoblastoid cells and in the Burkitt lymphoma, BL31 . Three hypersensitivity patterns emerge that correlate with the state of the gene and reveal sites associated with putative regulatory structures. One region is associated with the two myc promoters, one with a specific nuclear protein binding site, and one--which is markedly enhanced in the inactive germline gene in the Burkitt cell--with a putative negative control region. The perturbation of the normal pattern in this particular Burkitt cell may be due to the action of an immunoglobulin enhancer.

BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonizes BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb.

Cell Cycle Arrest and Reversion of Ras-Induced Transformation by a Conditionally Activated Form of Mitogen-Activated Protein Kinase Kinase Kinase 3

ABSTRACT Signal-induced proliferation, differentiation, or stress responses of cells depend on mitogen-activated protein kinase (MAPK) cascades, the core modules of which consist of members of three successively acting kinase families (MAPK kinase kinase [MAP3K], MAPK kinase, and MAPK). It is demonstrated here that the MEKK3 kinase inhibits cell proliferation, a biologic response not commonly associated with members of the MAP3K family of kinases. A conditionally activated form of MEKK3 stably expressed in fibroblasts arrests these cells in early G1. MEKK3 critically blocks mitogen-driven expression of cyclin D1, a cyclin which is essential for progression of fibroblasts through G1. The MEKK3-induced block of cyclin D1 expression and of cell cycle progression may be mediated via p38 MAPK, a downstream effector of MEKK3. The MEKK3-mediated block of proliferation also reverses Ras-induced cellular transformation, suggesting possible tumor-suppressing functions for this kinase. Together, these results suggest an involvement of the MEKK3 kinase in negative regulation of cell cycle progression, and they provide the first insights into biologic activities of this kinase.

IL-17-induced NF-κB Activation via CIKS/Act1: PHYSIOLOGIC SIGNIFICANCE AND SIGNALING MECHANISMS*

Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and activation of transcription factor NF-κB; it is not known whether CIKS and/or NF-κB are required for all gene induction events. Here we report that CIKS is essential for all IL-17-induced immediate-early genes in primary mouse embryo fibroblasts, whereas NF-κB is profoundly involved. We also identify a novel subdomain in the N terminus of CIKS that is essential for IL-17-mediated NF-κB activation. This domain is both necessary and sufficient for interaction between CIKS and TRAF6, an adaptor required for NF-κB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases.Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and activation of transcription factor NF-κB; it is not known whether CIKS and/or NF-κB are required for all gene induction events. Here we report that CIKS is essential for all IL-17-induced immediate-early genes in primary mouse embryo fibroblasts, whereas NF-κB is profoundly involved. We also identify a novel subdomain in the N terminus of CIKS that is essential for IL-17-mediated NF-κB activation. This domain is both necessary and sufficient for interaction between CIKS and TRAF6, an adaptor required for NF-κB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases.

Lipopolysaccharide-mediatedInterferonRegulatoryFactor Activation Involves TBK1-IKK-dependent Lys 63 -linked Polyubiquitination and Phosphorylation of TANK/I-TRAF *

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKϵ-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/ITRAF is required for the TBK1- and IKKϵ-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKϵ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide- and TBK1-IKKϵ-mediated Lys63-linked polyubiquitination, a mechanism that does not require TBK1-IKKϵ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKϵ complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys63-linked polyubiquitination of their scaffold protein, TANK.

IL-17 drives psoriatic inflammation via distinct, target cell-specific mechanisms

Significance Psoriasis is an inflammatory disease affecting the skin, a barrier site. The disease is characterized by abnormal growth of keratinocytes and infiltration of inflammatory cells. Clinical trials targeting the IL-17 cytokine have shown remarkable efficacy, and IL-17 also has been strongly implicated in the imiquimod-induced mouse model of psoriasis. However why IL-17 cytokines should be so central is not known, because target cells and their functions have not been clearly delineated. Here we demonstrate that IL-17 signaling into nonkeratinocytes, specifically dermal fibroblasts, induces mediators that further increase IL-17 production by innate γδT cells and promote cellular infiltration, whereas IL-17 signaling into keratinocytes aids proliferation and blocks their differentiation. These findings reveal the circuitry underpinning critical disease-driving effects of IL-17. Psoriasis is a chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory cells. The mechanisms underlying psoriasis in humans and in mouse models are poorly understood, although evidence strongly points to crucial contributions of IL-17 cytokines, which signal via the obligatory adaptor CIKS/Act1. Here we identify critical roles of CIKS/Act1-mediated signaling in imiquimod-induced psoriatic inflammation, a mouse model that shares features with the human disease. We found that IL-17 cytokines/CIKS-mediated signaling into keratinocytes is essential for neutrophilic microabscess formation and contributes to hyperproliferation and markedly attenuated differentiation of keratinocytes, at least in part via direct effects. In contrast, IL-17 cytokines/CIKS-mediated signaling into nonkeratinocytes, particularly into dermal fibroblasts, promotes cellular infiltration and, importantly, leads to enhanced the accumulation of IL-17–producing γδT cells in skin, comprising a positive feed-forward mechanism. Thus, CIKS-mediated signaling is central in the development of both dermal and epidermal hallmarks of psoriasis, inducing distinct pathologies via target cell-specific effects. CIKS-mediated signaling represents a potential therapeutic target in psoriasis.

High-affinity binding site for a specific nuclear protein in the human IgM gene

Nature 314, 289–292 (1985) Lines 8 and 9 of Fig. 3 should read in the absence (lane 4) or presence (lane 3) of the.

The adaptor protein CIKS/ACT1 is necessary for collagen‐induced arthritis, and it contributes to the production of collagen‐specific antibody

OBJECTIVEnCIKS/ACT1 is an adaptor molecule that is necessary for signaling by members of the interleukin-17 cytokine family. The aim of this study was to determine whether this adaptor is required for the initiation of collagen-induced arthritis (CIA). If it is required, then CIKS-mediated signaling could be a potential target for therapeutic intervention in patients with rheumatoid arthritis (RA).nnnMETHODSnCIA model studies were performed with CIKS-deficient and CIKS-sufficient mice on an otherwise wild-type (WT) C57BL/6 background or on a C57BL/6 background lacking Fcγ receptor IIb (FcγRIIb). In addition, collagen antibody-induced arthritis (CAIA) studies were performed in WT and CIKS-deficient mice. Pathologic changes of arthritis were evaluated by visual inspection of the paws, by histochemical analysis of tissue sections, and by measurements of collagen-specific antibodies.nnnRESULTSnPathologic changes of CIA were readily induced in WT mice, with exacerbation of the changes in FcγRIIb-deficient mice. In contrast, CIKS-deficient mice were protected from all aspects of CIA pathology, even on an FcγRIIb-deficient background. The absence of CIKS completely prevented neutrophil infiltration into joints, bone erosion, and cartilage damage; furthermore, the production of type II collagen (CII)-specific antibodies was reduced. In contrast to the CIA model, CIKS-deficient mice in the CAIA model remained susceptible to arthritis.nnnCONCLUSIONnCIKS-mediated signaling is necessary for the pathogenesis of CIA, but not CAIA. These findings suggest critical functions of CIKS during the development of arthritis in the CIA model, including in the formation of CII antibodies, and they mark the CIKS adaptor as a potential therapeutic target in RA.

Cell-Autonomous Role for NF-κB in Immature Bone Marrow B Cells

The NF-κB transcription factors have many essential functions in B cells, such as during differentiation and proliferation of Ag-challenged mature B cells, but also during final maturation of developing B cells in the spleen. Among the various specific functions NF-κB factors carry out in these biologic contexts, their ability to assure the survival of mature and maturing B cells in the periphery stands out. Less clear is what if any roles NF-κB factors play during earlier stages of B cell development in the bone marrow. Using mice deficient in both NF-κB1 and NF-κB2, which are thus partially compromised in both the classical and alternative activation pathways, we demonstrate a B cell-autonomous contribution of NF-κB to the survival of immature B cells in the bone marrow. NF-κB1 and NF-κB2 also play a role during the earlier transition from proB to late preB cells; however, in this context these factors do not act in a B cell-autonomous fashion. Although NF-κB1 and NF-κB2 are not absolutely required for survival and progression of immature B cells in the bone marrow, they nevertheless make a significant contribution that marks the beginning of the profound cell-autonomous control these factors exert during all subsequent stages of B cell development. Therefore, the lifelong dependency of B cells on NF-κB-mediated survival functions is set in motion at the time of first expression of a full BCR.