Winfried Krueger

Activation of Human Monocytes by Live Borrelia burgdorferi Generates TLR2-Dependent and -Independent Responses Which Include Induction of IFN-β

It is widely believed that innate immune responses to Borrelia burgdorferi (Bb) are primarily triggered by the spirochetes outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs) elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-β and a number of interferon-stimulated genes (ISGs), which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-α, IL-6, IL-10 and IL-1β in monocytes than did lysates. Secreted IL-18, which, like IL-1β, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-β and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs.

RNA Trafficking Signals in Human Immunodeficiency Virus Type 1

ABSTRACT Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr andtat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelledgag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently asvpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

Epigenetic regulatory mechanisms during preimplantation development

Following fertilization, the newly formed zygote faces several critical decisions regarding cell fate and lineage commitment. First, the parental genomes must be reprogrammed and reset for the zygotic genome to assume responsibility for gene expression. Second, blastomeres must be committed to form either the inner cell mass or trophectoderm before implantation. A variety of epigenetic mechanisms underlies each of these steps, allowing for proper activation of transcriptional circuits which function to specify a cells identity and maintain or adjust that state as developmental and environmental conditions dictate. These epigenetic mechanisms encompass DNA methylation, post-translational histone modification, chromatin remodeling, and alterations in nuclear architecture. In recent years, stem cells derived from the inner cell mass have been used to examine the epigenetic pathways that regulate pluripotency, differentiation, and lineage commitment. From a technical standpoint, embryonic stem cells provide an easier system to work with compared to preimplantation embryos; however, it is currently unknown how closely the epigenetic mechanisms of cultured stem cells resemble their counterparts in the intact embryo. Furthermore, it remains unclear how similar the reprogramming pathways in artificially created systems, such as nuclear transfer-derived embryos and induced pluripotent stem cells, are to those in naturally created embryos. In this review, we summarize the current knowledge of epigenetic influences during preimplantation development and shed light on the extent to which these pathways are conserved in cultured pluripotent cells in vitro. In doing so, we demonstrate the critical role that epigenetic mechanisms play in the establishment of cell fate during the earliest stages of mammalian development.

Genome-Wide Reprogramming in Hybrids of Somatic Cells and Embryonic Stem Cells

Recent experiments demonstrate that somatic nuclei can be reprogrammed to a pluripotent state when fused to ESCs. The resulting hybrids are pluripotent as judged by developmental assays, but detailed analyses of the underlying molecular‐genetic control of reprogrammed transcription in such hybrids are required to better understand fusion‐mediated reprogramming. We produced hybrids of mouse ESCs and fibroblasts that, although nearly tetraploid, exhibit characteristics of normal ESCs, including apparent immortality in culture, ESC‐like colony morphology, and pluripotency. Comprehensive analysis of the mouse embryonic fibroblast/ESC hybrid transcriptome revealed global patterns of gene expression reminiscent of ESCs. However, combined analysis of variance and hierarchical clustering analyses revealed at least seven distinct classes of differentially regulated genes in comparisons of hybrids, ESCs, and somatic cells. The largest class includes somatic genes that are silenced in hybrids and ESCs, but a smaller class includes genes that are expressed at nearly equivalent levels in hybrids and ESCs that contain many genes implicated in pluripotency and chromatin function. Reprogrammed genes are distributed throughout the genome. Reprogramming events include both transcriptional silencing and activation of genes residing on chromosomes of somatic origin. Somatic/ESC hybrid cell lines resemble their pre‐fusion ESC partners in terms of behavior in culture and pluripotency. However, they contain unique expression profiles that are similar but not identical to normal ESCs. ESC fusion‐mediated reprogramming provides a tractable system for the investigation of mechanisms of reprogramming.

ES Cell Cycle Progression and Differentiation Require the Action of the Histone Methyltransferase Dot1L

Mouse embryonic stem cells (ESCs) proliferate with rapid cell cycle kinetics but without loss of pluripotency. The histone methyltransferase Dot1L is responsible for methylation of histone H3 at lysine 79 (H3K79me). We investigated whether ESCs require Dot1L for proper stem cell behavior. ESCs deficient in Dot1L tolerate a nearly complete loss of H3K79 methylation without a substantial impact on proliferation or morphology. However, shortly after differentiation is induced, Dot1L‐deficient cells cease proliferating and arrest in G2/M‐phase of the cell cycle, with increased levels of aneuploidy. In addition, many aberrant mitotic spindles occur in Dot1L‐deficient cells. Surprisingly, these mitotic and cell cycle defects fail to trigger apoptosis, indicating that mouse ESCs lack stringent cell cycle checkpoint control during initial stages of differentiation. Transcriptome analysis indicates that Dot1L deficiency causes the misregulation of a select set of genes, including many with known roles in cell cycle control and cellular proliferation as well as markers of endoderm differentiation. The data indicate a requirement for Dot1L function for early stages of ESC differentiation where Dot1L is necessary for faithful execution of mitosis and proper transcription of many genes throughout the genome. STEM CELLS 2009;27:1538–1547

RNA Trafficking in Oligodendrocytes

A2RE and hnRNP A2 have been identified as important cis/trans determinants for MBP RNA trafficking in oligodendrocytes. Since A2RE-like sequences are found in several different transported RNAs, and since hnRNP A2 is expressed in most cell types, this may represent a general RNA trafficking pathway shared by a variety of different RNAs in different cell types. In oligodendrocytes, A2RE/hnRNP A2 determinants are involved in at least four steps in the RNA trafficking pathway: (1) export from the nucleus to the cytoplasm, (2) granule assembly in the perikaryon, (3) transport along microtubules in the processes, and (4) translation activation in the myelin compartment. The components of the cellular machinery mediating each of these steps are known. How A2RE/hnRNP A2 determinants interact with these components to mediate RNA trafficking is being investigated by a combination of: biochemistry to analyze molecular interactions in vitro, imaging to visualize molecular interactions in living cells, and computational modeling to simulate molecular interactions in the Virtual Cell.

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes

Abstract : Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane‐directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP‐2/synaptobrevin‐2, syntaxin‐2 and ‐4, nsec‐1/munc‐18‐1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP‐2, the small GTP‐binding protein Rab3a, and the target SNARE syntaxin‐4 were up‐regulated. VAMP‐2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network ; in situ anti‐VAMP‐2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane‐directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.

Conserved Chromosome 2q31 Conformations Are Associated with Transcriptional Regulation of GAD1 GABA Synthesis Enzyme and Altered in Prefrontal Cortex of Subjects with Schizophrenia

Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression, including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here, we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus, and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS-50kbLoop was enriched with nucleosomes epigenetically decorated with the transcriptional mark, histone H3 trimethylated at lysine 4, and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia, GAD1-TSS-50kbLoop was decreased compared with controls, in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS-55kbLoop, the murine homolog to GAD1-TSS-50kbLoop, is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture, Gad1-TSS-55kbLoop and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures, including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression, are conserved between the rodent and primate brain, and subject to developmental and activity-dependent regulation, and disordered in some cases with schizophrenia. More broadly, the findings presented here draw a connection between noncoding DNA, spatial genome architecture, and neuronal plasticity in development and disease.

Isoniazid-induced cell death is precipitated by underlying mitochondrial complex I dysfunction in mouse hepatocytes

Isoniazid (INH) is an antituberculosis drug that has been associated with idiosyncratic liver injury in susceptible patients. The underlying mechanisms are still unclear, but there is growing evidence that INH and/or its major metabolite, hydrazine, may interfere with mitochondrial function. However, hepatic mitochondria have a large reserve capacity, and minor disruption of energy homeostasis does not necessarily induce cell death. We explored whether pharmacologic or genetic impairment of mitochondrial complex I may amplify mitochondrial dysfunction and precipitate INH-induced hepatocellular injury. We found that INH (≤ 3000 μM) did not induce cell injury in cultured mouse hepatocytes, although it decreased hepatocellular respiration and ATP levels in a concentration-dependent fashion. However, coexposure of hepatocytes to INH and nontoxic concentrations of the complex I inhibitors rotenone (3 μM) or piericidin A (30 nM) resulted in massive ATP depletion and cell death. Although both rotenone and piericidin A increased MitoSox-reactive fluorescence, Mito-TEMPO or N-acetylcysteine did not attenuate the extent of cytotoxicity. However, preincubation of cells with the acylamidase inhibitor bis-p-nitrophenol phosphate provided protection from hepatocyte injury induced by rotenone/INH (but not rotenone/hydrazine), suggesting that hydrazine was the cell-damaging species. Indeed, we found that hydrazine directly inhibited the activity of solubilized complex II. Hepatocytes isolated from mutant Ndufs4(+/-) mice, although featuring moderately lower protein expression levels of this complex I subunit in liver mitochondria, exhibited unchanged hepatic complex I activity and were therefore not sensitized to INH. These data indicate that underlying inhibition of complex I, which alone is not acutely toxic, can trigger INH-induced hepatocellular injury.

Developmental effects of tobacco smoke exposure during human embryonic stem cell differentiation are mediated through the transforming growth factor-β superfamily member, Nodal.

While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs.