Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific ...events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.
Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been ...described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.
miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. ...Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes - endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.
In the last years miRNAs have increasingly been recognised as potent posttranscriptional regulators of gene expression. Possibly, miRNAs exert their action on virtually any biological process by ...simultaneous regulation of numerous genes. The importance of miRNA-based regulation in health and disease has inspired research to investigate diverse aspects of miRNA origin, biogenesis, and function. Despite the recent rapid accumulation of experimental data, and the emergence of functional models, the complexity of miRNA-based regulation is still far from being well understood. In particular, we lack comprehensive knowledge as to which cellular processes are regulated by which miRNAs, and, furthermore, how temporal and spatial interactions of miRNAs to their targets occur. Results from large-scale functional analyses have immense potential to address these questions. In this review, we discuss the latest progress in application of high-content and high-throughput functional analysis for the systematic elucidation of the biological roles of miRNAs.
The understanding of miRNA target interactions is still limited due to conflicting data and the fact that high-quality validation of targets is a time-consuming process. Faster methods like ...high-throughput screens and bioinformatics predictions are employed but suffer from several problems. One of these, namely the potential occurrence of downstream (i.e. secondary) effects in high-throughput screens has been only little discussed so far. However, such effects limit usage for both the identification of interactions and for the training of bioinformatics tools. In order to analyse this problem more closely, we performed time-dependent microarray screening experiments overexpressing human miR-517a-3p, and, together with published time-dependent datasets of human miR-17-5p, miR-135b and miR-124 overexpression, we analysed the dynamics of deregulated genes. We show that the number of deregulated targets increases over time, whereas seed sequence content and performance of several miRNA target prediction algorithms actually decrease over time. Bioinformatics recognition success of validated miR-17 targets was comparable to that of data gained only 12 h post-transfection. We therefore argue that the timing of microarray experiments is of critical importance for detecting direct targets with high confidence and for the usability of these data for the training of bioinformatics prediction tools.
As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering ...(si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
We applied 14-mer 2'-OMe RNAs as inhibitors of selected micro RNAs. To improve their properties, we introduced a trimethoxystilbene residue at the 5'-terminus and three 2'-fluoro-2'-deoxynucleotides ...at the 3'-terminus to obtain potent inhibitors, whose mismatch discrimination is substantially better than that of typically applied >18-mers.
Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a ...major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called 'Capture and Amplification by Tailing and Switching' (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2-3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples.
The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from ...diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner.
It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes.
Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL.
Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program.
Kipps:Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.
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