Defective transcription of ATF3 responsive genes, a marker for Cockayne Syndrome.

Cockayne syndrome (CS) is a rare genetic disorder caused by mutations (dysfunction) in CSA and CSB. CS patients exhibit mild photosensitivity and severe neurological problems. Currently, CS diagnosis is based on the inefficiency of CS cells to recover RNA synthesis upon genotoxic (UV) stress. Indeed, upon genotoxic stress, ATF3, an immediate early gene is activated to repress up to 5000 genes encompassing its responsive element for a short period of time. On the contrary in CS cells, CSA and CSB dysfunction impairs the degradation of the chromatin-bound ATF3, leading to a permanent transcriptional arrest as observed by immunofluorescence and ChIP followed by RT-PCR. We analysed ChIP-seq of Pol II and ATF3 promoter occupation analysis and RNA sequencing-based gene expression profiling in CS cells, as well as performed immunofluorescence study of ATF3 protein stability and quantitative RT-PCR screening in 64 patient cell lines. We show that the analysis of few amount (as for example CDK5RAP2, NIPBL and NRG1) of ATF3 dependent genes, could serve as prominent molecular markers to discriminate between CS and non-CS patient's cells. Such assay can significantly simplify the timing and the complexity of the CS diagnostic procedure in comparison to the currently available methods.

AMPKß1 and AMPKß2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, ß, and γ. AMPKα and ß are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKß1 and AMPKß2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKß2 was associated with a higher number of differentially regulated genes than the deletion of AMPKß1, suggesting that AMPKß2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKß2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKß1 impairs, whereas deficiency of AMPKß2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

Cockayne syndrome (CS) is caused by mutations in CSA and CSB. The CSA and CSB proteins have been linked to both promoting transcription-coupled repair and restoring transcription following DNA damage. We show that UV stress arrests transcription of approximately 70% of genes in CSA- or CSB-deficient cells due to the constitutive presence of ATF3 at CRE/ATF sites. We found that CSB, CSA/DDB1/CUL4A, and MDM2 were essential for ATF3 ubiquitination and degradation by the proteasome. ATF3 removal was concomitant with the recruitment of RNA polymerase II and the restart of transcription. Preventing ATF3 ubiquitination by mutating target lysines prevented recovery of transcription and increased cell death following UV treatment. Our data suggest that the coordinate action of CSA and CSB, as part of the ubiquitin/proteasome machinery, regulates the recruitment timing of DNA-binding factors and provide explanations about the mechanism of transcription arrest following genotoxic stress.

Comparative performance analysis of human iPSC-derived and primary neural progenitor cells (NPC) grown as neurospheres in vitro.

Developmental neurotoxicity (DNT) testing performed in rats is resource-intensive (costs, time, animals) and bears the issue of species extrapolation. Thus, reliable alternative human-based approaches are needed for predicting neurodevelopmental toxicity. Human induced pluripotent stem cells (hiPSCs) represent a basis for an alternative method possibly being part of an alternative DNT testing strategy. Here, we compared two hiPSC neural induction protocols resulting in 3D neurospheres: one using noggin and one cultivating cells in neural induction medium (NIM protocol). Performance of Nestin+/SOX2+ hiPSC-derived neural progenitor cells (NPCs) was compared to primary human NPCs. Generally, primary hNPCs first differentiate into Nestin+ and/or GFAP+ radial glia-like cells, while the hiPSC-derived NPCs (hiPSC-NPC) first differentiate into ßIII-Tubulin+ neurons suggesting an earlier developmental stage of hiPSC-NPC. In the 'Neurosphere Assay', NIM generated hiPSC-NPC produced neurons with higher performance than with the noggin protocol. After long-term differentiation, hiPSC-NPC form neuronal networks, which become electrically active on microelectrode arrays after 85days. Finally, methylmercury chloride inhibits hiPSC-NPC and hNPC migration with similar potencies. hiPSC-NPCs-derived neurospheres seem to be useful for DNT evaluation representing early neural development in vitro. More system characterization by compound testing is needed to gain higher confidence in this method.

MED12-related XLID disorders are dose-dependent of immediate early genes (IEGs) expression.

Mediator occupies a key role in protein coding genes expression in mediating the contacts between gene specific factors and the basal transcription machinery but little is known regarding the role of each Mediator subunits. Mutations in MED12 are linked with a broad spectrum of genetic disorders with X-linked intellectual disability that are difficult to range as Lujan, Opitz-Kaveggia or Ohdo syndromes. Here, we investigated several MED12 patients mutations (p.R206Q, p.N898D, p.R961W, p.N1007S, p.R1148H, p.S1165P and p.R1295H) and show that each MED12 mutations cause specific expression patterns of JUN, FOS and EGR1 immediate early genes (IEGs), reflected by the presence or absence of MED12 containing complex at their respective promoters. Moreover, the effect of MED12 mutations has cell-type specificity on IEG expression. As a consequence, the expression of late responsive genes such as the matrix metalloproteinase-3 and the RE1 silencing transcription factor implicated respectively in neural plasticity and the specific expression of neuronal genes is disturbed as documented for MED12/p.R1295H mutation. In such case, JUN and FOS failed to be properly recruited at their AP1-binding site. Our results suggest that the differences between MED12-related phenotypes are essentially the result of distinct IEGs expression patterns, the later ones depending on the accurate formation of the transcription initiation complex. This might challenge clinicians to rethink the traditional syndromes boundaries and to include genetic criterion in patients' diagnostic.

IL-6, IL-8, MMP-2, MMP-9 are overexpressed in Fanconi anemia cells through a NF-κB/TNF-α dependent mechanism.

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder associated with a bone-marrow failure, genome instability, hypersensitivity to DNA crosslinking agents and a predisposition to cancer. Mutations have been documented in 16 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its symptoms. FA cells have been characterized by an overproduction of cytokines, MAPKs, and Interleukins. Through this study we have identified the overexpression of additional secretory factors such as IL-6, IL-8, MMP-2, and MMP-9 in FA cells and in cells depleted of FANCA or FANCC and proved that their expression is under the control of NF-κB/TNF-α signaling pathways. We also demonstrated that these overexpressed secretory factors were effective in promoting the proliferation, migration, and invasion of surrounding tumor cells a fundamental event in the process of epithelial mesenchymal transition (EMT) and that they also modulated the expression of EMT markers such as E-cadherin and SNAIL. Overall our data suggest that the upregulation of EMT promoting factors in FA may contribute to predisposing FA patients to cancer, thereby providing new insights into possible therapeutic interventions.

Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress.

Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.

Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.

Recently, we and others identified the microRNA miR-34a as a target of the tumor suppressor gene product p53. Ectopic miR-34a induces a G(1) cell cycle arrest, senescence and apoptosis. Here we report that miR-34a expression is silenced in several types of cancer due to aberrant CpG methylation of its promoter. 19 out of 24 (79.1%) primary prostate carcinomas displayed CpG methylation of the miR-34a promoter and concomitant loss of miR-34a expression. CpG methylation of the miR-34a promoter was also detected in breast (6/24; 25%), lung (7/24; 29.1%), colon (3/23; 13%), kidney (3/14; 21.4%), bladder (2/6; 33.3%) and pancreatic (3/19; 15.7%) carcinoma cell lines, as well as in melanoma cell lines (19/44; 43.2%) and primary melanoma (20/32 samples; 62.5%). Silencing of miR-34a was dominant over its transactivation by p53 after DNA damage. Re-expression of miR-34a in prostate and pancreas carcinoma cell lines induced senescence and cell cycle arrest at least in part by targeting CDK6. These results show that miR-34a represents a tumor suppressor gene which is inactivated by CpG methylation and subsequent transcriptional silencing in a broad range of tumors.

Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest.

In a genome-wide screen for microRNAs regulated by the transcription factor encoded by the p53 tumor suppressor gene we found that after p53-activation the abundance of thirty-four miRNAs was significantly increased, whereas sixteen miRNAs were suppressed. The induction of miR-34a was most pronounced among all differential regulations. Also expression of the primary miR-34a transcript was induced after p53 activation and by DNA damage in a p53-dependent manner. p53 occupied an evolutionarily conserved binding site proximal to the first non-coding exon of miR-34a. Ectopic miR-34a induced apoptosis and a cell cycle arrest in the G1-phase, thereby suppressing tumor cell proliferation. Other p53-induced miRNAs identified here may also have tumor suppressive potential as they are known to suppress the anti-apoptotic factor Bcl2 (miR-15a/16) and the oncogenes RAS and HMGA2 (let-7a). Our results for the first time directly integrate the regulation of miRNA expression into the transcriptional network regulated by p53. siRNAs corresponding to p53-induced miRNAs may have potential as cancer therapeutic agents as RNA interference based therapies are currently emerging.

c-MYC delays prometaphase by direct transactivation of MAD2 and BubR1: identification of mechanisms underlying c-MYC-induced DNA damage and chromosomal instability.

Here we show that the human BubR1 and MAD2 genes, which encode inhibitors of the anaphase promoting complex (APC/C), are directly activated by the oncogenic transcription factor c-MYC via E-box sequences in their first introns. In colorectal cancer biopsies elevated expression of c-MYC correlated with increased MAD2 levels. Activation of a conditional c-MYC allele delayed progression through mitosis in pro-metaphase in a MAD2- and BubR1-dependent manner. A fraction of the daughter cells derived from extended mitotic events underwent synchronous apoptosis, which was in part mediated by BubR1. Furthermore, c-MYC activation resulted in CIN (chromosomal instability) in the diploid MIN (microsatellite instability) cell line DLD-1 and further enhanced CIN in the aneuploid CIN-line MCF7. Unexpectedly, c-MYC-induced CIN was independent of c-MYC-induced BubR1/MAD2 expression and mitotic delay. Therefore, c-MYC-induced CIN may be caused be alternative pathways. We observed that activation of c-MYC induced DNA double-strand breaks, as evidenced by formation of gamma-H2AX foci, which colocalized with foci of active DNA replication. Furthermore, c-MYC activation resulted in mitotic chromosomes exhibiting DNA damage. Therefore, oncogenic deregulation of c-MYC prevents repair of replication-stress induced DNA lesions in the G(2)-phase. We suggest that the c-MYC-mediated persistence of DNA lesions throughout mitosis leads to chromosomal missegregation and underlies c-MYC-induced CIN. The effects of deregulated c-MYC on progression through mitosis described here may have important implications for the origin of chromosomal instability in many tumor types and the sensitivity towards cancer therapeutic agents targeting DNA or the mitotic spindle.

Digital karyotyping reveals frequent inactivation of the dystrophin/DMD gene in malignant melanoma.

Malignant melanoma is still poorly understood at the genomic level. Recently, a new technique for the high-resolution analysis of copy number changes named digital karyotyping was introduced. This approach is derived from SAGE (serial analysis of gene expression) and allows the detection of genomic amplifications and deletions, which are indicative of oncogenes and tumor suppressor genes. Four human melanoma cell lines were subjected to analysis by digital karyotyping. 828,780 genomic tags were generated and analyzed quantitatively. Thereby, we identified a somatic, homozygous deletion of 570 kbp removing exons 3-29 of the dystrophin (DMD, Duchenne muscular dystrophy) gene. Analysis of 51 melanoma cell lines further revealed a homozygous and a hemizygous deletion in DMD. Furthermore, DMD mRNA expression was downregulated with respect to primary melanocytes and accompanied by loss of dystrophin protein expression in 38 of 55 (69%) and significantly reduced in 10 of 55 (18%) melanoma cell lines. Sequence analysis of DMD cDNAs in 37 melanoma cell lines revealed six new sequence variants with a significantly lower frequency than previously described DMD polymorphisms, which may affect dystrophin function. Knock-down of DMD enhanced migration and invasion, whereas re-expression of DMD attenuated migration and induced a senescent phenotype in melanoma cell lines. Therefore, loss of DMD may critically change the migratory and proliferative capacity of melanocytes. Taken together, our results suggest that inactivation of DMD is involved in the pathogenesis of malignant melanoma.

Inducible microRNA expression by an all-in-one episomal vector system.

Here we describe an episomal, one-vector system which allows the generation of cell populations displaying homogenous, inducible gene inactivation by RNA interference in a one step procedure. A dual tet-repressor/activator system tightly controls a bi-directional promoter, which simultaneously drives expression of microRNAs and a fluorescent marker protein. We demonstrate the effectiveness of this vector by knockdown of p53 expression in a human cell line which resulted in the expected loss of G1-arrest after DNA damage. The generation of a cell pool homogenously expressing the ectopic microRNAs was achieved in 1 week without the need for viral infections. Induction of microRNA expression did not elicit an interferon response. Furthermore, the vector was adapted for convenient ligation-free transfer of microRNA cassettes from public libraries. This conditional knockdown-system should prove useful for many research and gene therapeutic applications.

Functional epigenomics identifies genes frequently silenced in prostate cancer.

In many cases, silencing of gene expression by CpG methylation is causally involved in carcinogenesis. Furthermore, cancer-specific CpG methylation may serve as a tumor marker. In order to identify candidate genes for inactivation by CpG methylation in prostate cancer, the prostate cancer cell lines LNCaP, PC3, and Du-145 were treated with 5-aza-2' deoxycytidine and trichostatin A, which leads to reversion of epigenetic silencing. By microarray analysis of 18,400 individual transcripts, several hundred genes were found to be induced when compared with cells treated with trichostatin A. Fifty re-expressed genes were selected for further analysis based on their known function, which implied a possible involvement in tumor suppression. Twelve of these genes showed a significant degree of CpG methylation in their promoters. Six genes were silenced by CpG methylation in the majority of five analyzed prostate cancer cell lines, although they displayed robust mRNA expression in normal prostate epithelial cells obtained from four different donors. In primary prostate cancer samples derived from 41 patients, the frequencies of CpG methylation detected in the promoter regions of these genes were: GPX3, 93%; SFRP1, 83%; COX2, 78%; DKK3, 68%; GSTM1, 58%; and KIP2/p57, 56%. Ectopic expression of SFRP1 or DKK3 resulted in decreased proliferation. The expression of DKK3 was accompanied by attenuation of the mitogen-activated protein kinase pathway. The high frequency of CpG methylation detected in the promoters of the identified genes suggests a potential causal involvement in prostate cancer and may prove useful for diagnostic purposes.