Charakterisierung des Transkriptionsfaktors ZNF395/PBF im Rahmen der angeborenen Immunantwort

Genomweite Expressionsanalysen fanden eine erhöhte Expression des bisher wenig charakterisierten Transkriptionsfaktors ZNF395 in verschiedenen Krebsarten, u.a. im Rahmen einer Antwort des Tumors auf Hypoxie. Ein Mikroarray offenbarte, dass ZNF395 die Expression einer kleinen Gruppe Krebs-assoziierter Gene aktivierte, sowie einiger Interferon-regulierter Gene wie ISG56/IFIT1 und IFI16, die zur antiviralen Immunantwort beitragen. Diese Aktivierung benötigte den IKK-Signalweg, der gleichzeitig die verstärkte Degradation von ZNF395 induzierte. Das Ziel dieser Arbeit lag darin, den Beitrag von ZNF395 bei der angeborenen Immunantwort und dessen Regulation, v.a. durch Hypoxie und den IKK-Komplex, zu charakterisieren. Wie Immunfluoreszenz-Analysen zeigten, konnte eine Expression von ZNF395 im Kern von Tumorzellen in einzelnen Tumoren und weit häufiger im Zytoplasma von Immunzellen des Tumorstromas nachgewiesen werden. Diese konnten als CD79a+ B-Lymphozyten sowie CD14+ Monozyten identifiziert werden, hingegen war ZNF395 in CD68+ Makrophagen nur schwach detektierbar. Eine Verringerung der Expression von ZNF395 konnte im Zuge der Reifung von Monozyten zu Makrophagen am Beispiel der Monozyten-Zelllinie U937 mit Western Blot und qRT-PCR-Analysen bestätigt werden. C/EBP, ein Transkriptionsfaktor, der eine essenzielle Rolle für die Differenzierung von Monozyten spielt, aktivierte den ZNF395 Promotor, wie Reportergenanalysen offenbarten. Dies unterstützt die Annahme, dass ZNF395 eine funktionale Rolle in Monozyten hat und seine Expression im Rahmen der Makrophagen-Reifung abnimmt. Die Induktion von ZNF395 unter Hypoxie benötigt den Hypoxie-induzierten Transkriptionsfaktor HIF1, dessen Aktivität auch in Monozyten von Bedeutung ist. Es konnte zudem gezeigt werden, dass der IKK-Komplex, der durch Zytokine u.a. im Verlauf der Makrophagen Reifung aktiviert wird, für die Funktion von ZNF395 als Transkriptionsfaktor essenziell ist. Reportergenanalysen und Knockdown-Experimente zeigten, dass sowohl aktives IKK als auch IKK notwendig sind, dass ZNF395 den ISG56 Promotor aktivieren kann aber gleichzeitig auch dessen proteasomalen Abbau fördern. Bei diesem verstärkten IKK-stimulierten „turn over“ könnte es sich um einen negativen Feedback-Mechanismus handeln der dazu beiträgt, dass die Aktivierung der Zielgene durch ZNF395 gering und nur vorübergehend ist. SiRNA und qRT-PCR Analysen offenbarten, dass ZNF395 für die maximale IFN-vermittelte Induktion der IFN-regulierten Gene ISG56/IFIT1, ISG54/IFIT2, IFI44 und IFI16 notwendig ist. ZNF395 konnte die IFN-abhängige Induktion der hier untersuchten antiviralen Faktoren bis zu zehnfach modulieren. Dies zeigt, dass ZNF395 zur Etablierung eines antiviralen Status der Zelle maßgeblich beiträgt. Die in dieser Arbeit beschriebenen Ergebnisse belegen, dass im Rahmen der angeborenen Immunantwort und Entzündung die transkriptionelle Aktivität von ZNF395 über Stimulierung von HIF1 und IKK erhöht wird und somit die Effizienz der IFN-Antwort verstärken kann. HIF1 und IKK sind auch in Karzinomen regelmäßig konstitutiv aktiv, was zu erhöhten Mengen an aktivem ZNF395 führt, das dann über die Aktivierung seiner krebsassoziierten Zielgene die Karzinogenese unterstützen könnte

ZNF395 Is an Activator of a Subset of IFN-Stimulated Genes

Activation of the interferon (IFN) pathway in response to infection with pathogens results in the induction of IFN-stimulated genes (ISGs) including proinflammatory cytokines, which mount the proper antiviral immune response. However, aberrant expression of these genes is pathogenic to the host. In addition to IFN-induced transcription factors non-IFN-regulated factors contribute to the transcriptional control of ISGs. Here, we show by genome wide expression analysis, siRNA-mediated suppression and Doxycycline-induced overexpression that the cellular transcription factor ZNF395 activates a subset of ISGs including the chemokines CXCL10 and CXCL11 in keratinocytes. We found that ZNF395 acts independently of IFN but enhances the IFN-induced expression of CXCL10 and CXCL11. Luciferase reporter assays revealed a requirement of intact NFκB-binding sites for ZNF395 to stimulate the CXCL10 promoter. The transcriptional activation of CXCL10 and CXCL11 by ZNF395 was abolished after inhibition of IKK by BMS-345541, which increased the stability of ZNF395. ZNF395 encodes at least two motifs that mediate the enhanced degradation of ZNF395 in response to IKK activation. Thus, IKK is required for ZNF395-mediated activation of transcription and enhances its turn-over to keep the activity of ZNF395 low. Our results support a previously unrecognized role of ZNF395 in the innate immune response and inflammation

The Transcription Factor ZNF395 Is Required for the Maximal Hypoxic Induction of Proinflammatory Cytokines in U87-MG Cells

Hypoxia activates the expression of proangiogenic and survival promoting factors as well as proinflammatory cytokines that support tissue inflammation. Hypoxia and inflammation are associated with tumor progression. The identification of the factors participating in the hypoxia associated inflammation is essential to develop strategies to control tumor hypoxia. The transcription factor ZNF395 was found to be overexpressed in various tumors including glioblastomas particularly in the network of a hypoxic response pointing to a functional role of ZNF395. On the other hand, ZNF395 was suggested to have tumor suppressor activities which may rely on its repression of proinflammatory factors. To address these conflictive observations, we investigated the role of ZNF395 in the expression of proinflammatory cytokines in the astrocytoma cell line U87-MG under hypoxia. We show that ZNF395 is a target gene of the hypoxia inducible factor HIF-1α. By gene expression analysis, RT-PCR and ELISA, we demonstrated that the siRNA-mediated suppression of ZNF395 impairs the hypoxic induction of IL-1β, IL-6, IL-8, and LIF in U87-MG cells. At ambient oxygen concentrations, ZNF395 had no enhancing effect, indicating that this transcriptional activation by ZNF395 is restricted to hypoxic conditions. Our results suggest that ZNF395 contributes to hypoxia associated inflammation by superactivating proinflammatory cytokines

The hypoxia-inducible transcription factor ZNF395 is controlled by IĸB kinase-signaling and activates genes involved in the innate immune response and cancer.

Activation of the hypoxia inducible transcription factor HIF and the NF-ĸB pathway promotes inflammation-mediated tumor progression. The cellular transcription factor ZNF395 has repeatedly been found overexpressed in various human cancers, particularly in response to hypoxia, implying a functional relevance. To understand the biological activity of ZNF395, we identified target genes of ZNF395 through a genome-wide expression screen. Induced ZNF395 expression led to the upregulation of genes known to play a role in cancer as well as a subset of interferon (IFN)-stimulated genes (ISG) involved in antiviral responses such as IFIT1/ISG56, IFI44 and IFI16. In cells that lack ZNF395, the IFN-α-mediated stimulation of these factors was impaired, demonstrating that ZNF395 is required for the full induction of these antiviral genes. Transient transfections revealed that ZNF395-mediated activation of the IFIT1/ISG56 promoter depends on the two IFN-stimulated response elements within the promoter and on the DNA-binding domain of ZNF395, a so-called C-clamp. We also show that IĸBα kinase (IKK)-signaling is necessary to allow ZNF395 to activate transcription and simultaneously enhances its proteolytic degradation. Thus, ZNF395 becomes activated at the level of protein modification by IKK. Moreover, we confirm that the expression of ZNF395 is induced by hypoxia. Our results characterize ZNF395 as a novel factor that contributes to the maximal stimulation of a subset of ISGs. This transcriptional activity depends on IKK signaling further supporting a role of ZNF395 in the innate immune response. Given these results it is possible that under hypoxic conditions, elevated levels of ZNF395 may support inflammation and cancer progression by activating the target genes involved in the innate immune response and cancer

ZNF395 activates innate immune response and cancer-associated genes.

<p>(<b>A</b>) Stably transfected RTS3b cells expressing the tet repressor and FLAG-tagged ZNF395 under control of the tet inducible promoter (lanes 3, 4) or the empty vector pcDNA4/TO (lanes 1, 2) were either grown in the absence (lanes 1, 3) or presence of Dox (lanes 2, 4) for 24h. Extracts were used for ImmunoBlot (IB) which was developed with the FLAG antibody and an anti-actin antibody as control. ns (non specific band) (<b>B</b>) Total RNA isolated from RTS3b TR-FLAG-ZNF395 cells, either grown with or without Dox was used for qRT-PCR to analyze the expression of the factors shown in the graph. The corresponding values were normalized to the values for the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (HPRT) and those obtained from cells grown in the absence of Dox were set as 1 for each factor. The graph represents the means of two independent experiments each performed in duplicate. The error bars represent the standard deviations. (<b>C</b>, <b>D</b>) RTS3b and U87-MG cells were transfected with control siRNA or siRNA targeting ZNF395 in duplicate. One set of samples was treated with solvent and the other with IFN-α, before total RNA was isolated. QRT-PCR was performed with the specific primer to amplify ISG56, IFI44, IFI16 and ZNF395 transcripts. CP-values obtained for the various factors were normalized against those for the housekeeping gene HPRT. The value with RNA from solvent treated cells transfected with siControl was set as 1 in each case. The fold activations were calculated according to the comparative threshold method described in Pfaffl et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074911#B64" target="_blank">64</a>]. QPCRs were performed four times and the standard deviations are given. The values provided in the figure reflect the non-induced basal expression level in the absence of ZNF395 (** p <0.01).</p

ZNF395 activates the ISG56 promoter and requires its DNA-binding domain and CR1.

<p>(<b>A</b>) RTS3b cells were seeded in six-well plates and transiently transfected with 500ng of the ISG56-Luc reporter construct and increasing amounts (5, 10, 20ng) of expression vector for FLAG-ZNF395 or the different mutants per well, as indicated. The structure of ZNF395 with its conserved regions CR1, CR2 and CR3 is depicted beneath the graphs including the sequence of the C-terminal 25 amino acids, which are conserved to the E-tail of TCF-1E and TCF-4E [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074911#B52" target="_blank">52</a>]. The M at pos. 169 and 172 were changed to A in mtNES while in ΔCR1 the amino acids from 165 to 188 were deleted. The amino acids that were mutated in mtCR3 and leading to loss of DNA-binding are indicated. (<b>B</b>) RTS3b and C33A cells were first transfected with siControl or siZNF395 and 24h later with the ISG56-Luc reporter construct. All graphs represent the results of at least three independent experiments. The standard deviations are given. (<b>C</b>) RTS3b cells were transiently transfected with the Luciferase reporter construct containing the IFI44-promoter including 560bp bases upstream of the initiation site. The segment harbors two overlapping ISREs, which have been shown to mediate the IFN-dependent induction of IFI44. The expression vector for ZNF395 and ZNF395mtNES were co-transfected as in A.</p

ZNF395 induced during hypoxia is modified by IKK.

<p>(<b>A</b>) U937 and U87-MG cells were either kept under normoxic or under hypoxic conditions (2% O₂ atmosphere) for 12h before preparing total cell extracts or RNA. RNA was used for qRT-PCR to analyze the expression of ZNF395, which was normalized to the expression of HPRT. The fold induction was calculated according the comparative threshold cycle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074911#B64" target="_blank">64</a>]. 50µg of protein extracts were used for an IB with the anti-ZNF395. Actin served as the loading control. (<b>B</b>) U87-MG cells were grown in ambient or 2% O₂ atmosphere and either treated with BMS-345541 or left untreated. Fifty µg of total cell extracts were used in an IB to detect ZNF395. In lanes 7, 9 and 10, these extracts were incubated with λ-phosphatase, as indicated. (<b>C</b>) 15µg of extracts from RTS3b-TR-FLAG-ZNF395 cells grown in the absence or presence of Dox, hypoxia and TNFα, as indicated in the figure, were analyzed in a IB for the expression of ZNF395, and actin. α.</p

Active IKK marks ZNF395 for degradation.

<p>(<b>A</b>) RTS3b cells were transiently transfected with 5ng (+) and 10ng (++) of the expression vector for FLAG-ZNF395 and in the experiments shown in the right graph, 5ng (+) or 10ng (++) of the vector for ZNF395mtNES was included. The transfected cells were treated either with IFN-α (left graph) or with polyI:C (right graph) as indicated. The bars represent the fold activations calculated from three independent experiments and the standard deviations are included. (<b>B</b>) Cells were transiently transfected with expression vector for FLAG-ZNF395 (lanes 4-8) or the empty vector (lanes 1-3) and treated either with polyI:C (lanes 2, 5), IFN-α (lanes 3, 6), TNFα (lane 8) or solvent (lanes 1, 4, 7). An IB with the anti-ZNF395 antibody and the anti-actin antibody was performed. (<b>C</b>) Cells transiently transfected with the FLAG-ZNF395 vector or the empty vector (in lanes 7, 10, 11) were treated with TNFα (lanes 1, 2), poly I:C (lanes 3, 4) or MG132 (lanes 9, 11, 13). BMS-345541 was added to the cells used in lanes 1, 3, 5 and the solvent DMSO in lanes 2, 4, 6, 7, 8. The analysis of ZNF395 expression was done by IB using the anti-FLAG and anti-actin antibody. In lanes 10–13, FLAG-ZNF395 was precipitated by M2-FLAG-agarose and the IB was performed with an antibody against ubiquitin. (<b>D</b>) RTS3b (lanes 2, 3), U937 (lanes 4, 5) or U87-MG cells (lanes 6, 7) were incubated in medium containing BMS-345541 (+) or DMSO (-) and analyzed for ZNF395 expression by an IB developed with the anti-ZNF395 antibody. In lane 1, extracts prepared from RTS3b TR-FLAG-ZNF395 cells used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074911#pone-0074911-g001" target="_blank">Figure 1</a> were used as a control.</p