Posttranslationale Kontrolle des zellulären Transkriptionsfaktors PBF/ZNF395

Genom-weite Expressionsanalysen fanden eine erhöhte Expression des bisher kaum charakterisierten Transkriptionsfaktors PBF (Papillomavirus Binding Factor, offizieller Genname ZNF395) in verschiedenen Krebstypen. Das Ziel dieser Arbeit war, die zellulären Gene von PBF zu identifizieren und die posttranslationale Kontrolle des Proteins näher zu untersuchen. Wie Microarray Analysen und qRT-PCR zeigten, induzierte die Überexpression von PBF die Transkription einer kleinen Gruppe von Interferon-stimulierten Genen, u.a. ISG56 und IFI44, sowie mehrerer Krebs-assoziierter Gene. Zur optimalen Aktivierung des ISG56 Promotors brauchte PBF beide ISREs (interferon stimulated response element), die im ISG56 Promotor vorliegen, wie mit Hilfe von transienten Transfektionen gezeigt wurde. Behandlung von Zellen mit TNF-α oder Poly I:C, einem doppelsträngigen RNA Analogon, die beide IκBα Kinase (IKK) aktivieren, resultierte in der Degradation von rekombinantem PBF. Phosphorylierung von IκBα durch IKK ist wichtig für die Aktivierung von NF-κB. IKK reguliert auch andere Substrate, die an der Immunantwort und der Karzinogenese beteiligt sind. BMS-345541, ein spezifischer IKK Inhibitor, verhinderte die proteasomale Degradation von rekombinantem PBF und stabilisierte auch endogenes PBF. Dies weist daraufhin, dass Phosphorylierung von PBF durch IKK als Signal für dessen Ubiquitinierung und den proteasomalen Abbau dient. Es konnte gezeigt werden, dass die Aminosäuren S212/214/215 und T216 bei der Degradation involviert sind. Allerdings ist mindestens eine weitere Region in PBF an den IKK-abhängigen Abbau beteiligt. Inhibition von IKK durch BMS-345541 verminderte die PBF-vermittelte Aktivierung des ISG56 Promotors in transienten Transfektionen. Dies impliziert, dass die IKK-abhängige Phosphorylierung nicht nur die Degradation von PBF, sondern auch die transkriptionelle Aktivität induziert. Es könnte sich dabei um einen negativen Feedback-Mechanismus handeln, um sicherzustellen, dass die Aktivierung der Zielgene durch PBF gering und nur vorübergehend ist. Die Stabilität von PBF wurde ebenfalls durch Überexpression von Komponenten des Histondeacetylase (HDAC) Komplexes, SAP30 und HDAC1, die beide direkt mit PBF interagieren, erhöht. HDAC Aktivität ist essentiell bei der Immunantwort, während HDAC Inhibitoren spezifisch das Wachstum von Krebszellen hemmen. Die hier identifizierten Zielgene von PBF und die Signalwege, die die Stabilität von PBF kontrollieren, sind essentielle Komponenten der angeborenen Immunantwort und der Krebsentstehung. Dies untermauert eine funktionelle Rolle von PBF bei Krebs und der angeborenen Immunantwort

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 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 acts through ISREI and ISREII to stimulate the ISG56 promoter.

<p>(<b>A</b>) Sequential deletions starting from the 5´ end of the ISG56 upstream regulatory region were introduced into the ISG56-Luc construct as indicated in the figure. The corresponding luciferase reporter constructs were co-transfected with 5, 10 and 20ng of the FLAG-ZNF395 expression vector. The relative luciferase activity of the full length ISG56-Luc (=ISG56-654) construct was set as 1. The numbers above each set represent the fold activations induced by ZNF395 with the relative activity of each mutated reporter construct set as 1. (<b>B</b>) The construct ISG56Δ117-93 contains a deletion of the two ISREs within the context of the full length ISG56-Luc construct which harbors the upstream region up to -654bp while in ISG56-mtISRE I/II a T in each ISRE has been changed into G, as indicated beneath the graph. All reporter constructs were co-transfected again with 5, 10 or 20ng of the expression vector for FLAG-ZNF395. The sequence of the two ISREs present in the ISG56 promoter with the point mutations that have been introduced is provided. (<b>C</b>) Transient transfections with ISG56-Luc reporter constructs that were modified to contain either two copies of ISRE I (ISG56-2x ISRE I) or two copies of ISRE II (ISG56-2x ISRE II) and 5ng of expression vector for ZNF395 or IRF3-5D, respectively. (<b>D</b>) ChIP-assay. RTS3b-TR-FLAG-ZNF395 cells were grown in the absence or presence of Dox, cross-linked and subjected to a ChIP assay with antibody against the FLAG-tag and control mouse IgG. The precipitated DNA segments were amplified with a LightCycler using primers flanking the ISREs of the ISG56 promoter. The ISG56-Luc reporter construct was included as standard to allow a quantification. The copy number obtained for the input was set as one for each extract and the fold enrichment was calculated. The PCRs were performed in triplicate (* p=0.05). (<b>E</b>) Gel shift analysis. Bacterially expressed his-tagged purified ΔN-ZNF395 (lacking amino acids 1-114) was incubated with 200pg ISREI-wt (lanes 1-4) or ISRE-mut oligonucleotide (lanes 5-7), both radioactively labeled with ³²P-γ-ATP and the binding was analyzed with a native PAA gel. In lanes 3 and 6, a 250-fold excess of unlabeled ISRE-wt and in lanes 4 and 7, of ISRE-mut oligonucleotide were added as competitors. In the gel shift shown on the right, nuclear extracts prepared from RTS3b-TR-FLAG-ZNF395 cells, either incubated in the absence or presence of Dox and polyI:C, as indicated, were incubated with labeled ISRE-wt oligonucleotide and a 250-fold excess of unlabeled ISRE-wt or ISRE-mut oligonucleotide. The position of the putative ZNF395-ISRE complex is indicated by an arrow.</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