MicroRNAs regulate "de novo" DNA methylation and histone mRNA 3' end formation in mammalian cells

MicroRNAs (miRNAs) are known to have many important functions in mammalian cells. They can influence the expression of their target genes and in this way regulate the function of not only their primary targets, but also of the pathways and mechanisms acting downstream of the primary targets. There are several key proteins that are required for the biogenesis of miRNAs and for mediating the repressive functions of miRNAs in mammals, the most critical being the ribonuclease (RNase) III enzyme Dicer. Since Dicer is required for generation of all known mammalian miRNAs, depletion of Dicer is an appealing strategy to identify and study the pathways under miRNA-mediated control. Deletion of Dicer in mouse embryonic stem cells (ESCs) is rendering the cells to slow growth rate and inability to differentiate, and thus, to loose their most important feature i.e. pluripotency. We aimed to understand in further detail the causes behind these critical defects. We have performed transcriptional profiling of Dicer-deficient ESCs and through bioinformatic analysis we identified miRNAs of the ESC-specific miR-290 cluster to be functionally most important for mouse ESCs. These miRNAs were found to directly control the expression of several hundred primary targets and through their regulation influence many features of the ESCs. We found the miR-290 miRNAs to contribute to the growth rate of the ESCs and to influence also expression of many secondary target genes. Among their secondary targets we identified de novo DNA methyltrasferases (DNMT3s) that were significantly downregulated in Dicer-deficient mouse ESCs. The downregulation was due to an increased expression of Retinoblastomalike2 (RBL2), a transcriptional repressor and primary target miR-290 miRNAs. As a consequence of lowered DNMT3 expression the cells were unable to methylate DNA at the promoter of pluripotency genes such as Oct-4 (Octamer-binding transcription factor-4, also known as Pou5f1 for POU-domain, class 5, transcription factor 1), and thus, incapable of fully silencing these genes during differentiation. Hence, regulation of DNMT3s by miR-290 miRNAs is contributing to the maintenance of mouse ESC pluripotency. Further analysis of the promoter of primary miR-290 transcript (pri-miR-290) showed that the ESC specific expression and subsequent silencing of the transcript during neuronal differentiation is regulated by the chromatin status of the promoter. During neuronal differentiation the pri-miR-290 promoter looses histone modifications characteristic of active genes and gains typical marks of silenced chromatin. This is followed by de novo DNA methylation of the pri-miR-290 promoter. It is likely that the silencing of pri-miR-290 depends on DNA methylation of its promoter, thus allowing an auto-regulatory loop between the miRNAs and DNMT3 enzymes. In addition to Dicer-deficient mouse ESCs, we have studied the importance of Dicer as well as Argonaute proteins for the function of human cell lines by inducibly depleting these proteins in human HEK293T-REx cells. We observed that an intact RNA silencing pathway is needed for normal expression of many of the replication-dependent histone genes. We found up to 25% of all histone mRNAs to be upregulated upon loss of RNAi machinery and more detailed analysis of one of the histone genes, HIST1H3H, demonstrated that the upregulation was due to enhanced polyadenylation of the histone mRNA. This is in contrast to the normal 3’ end processing of replication-dependent histone mRNAs that takes place at the 3’ end-proximal stem-loop and is not followed by polyadenylation. The analysis of RNA from Dicer- or Dgcr8-deficient ESCs showed that this type of regulation of 3’ end formation by RNA silencing pathway is conserved in mice and depends on the generation of miRNAs. Thus, miRNAs seem to regulate the 3’ end processing of replication-dependent histone mRNAs. Future work will be needed to identify specific miRNAs and processing factors involved

Missing heritability in Parkinson’s disease: the emerging role of non‑coding genetic variation

Parkinson's disease (PD) is a neurodegenerative disorder caused by a complex interplay of genetic and environmental factors. For the stratification of PD patients and the development of advanced clinical trials, including causative treatments, a better understanding of the underlying genetic architecture of PD is required. Despite substantial efforts, genome-wide association studies have not been able to explain most of the observed heritability. The majority of PD-associated genetic variants are located in non-coding regions of the genome. A systematic assessment of their functional role is hampered by our incomplete understanding of genotype-phenotype correlations, for example through differential regulation of gene expression. Here, the recent progress and remaining challenges for the elucidation of the role of non-coding genetic variants is reviewed with a focus on PD as a complex disease with multifactorial origins. The function of gene regulatory elements and the impact of non-coding variants on them, and the means to map these elements on a genome-wide level, will be delineated. Moreover, examples of how the integration of functional genomic annotations can serve to identify disease-associated pathways and to prioritize disease- and cell type-specific regulatory variants will be given. Finally, strategies for functional validation and considerations for suitable model systems are outlined. Together this emphasizes the contribution of rare and common genetic variants to the complex pathogenesis of PD and points to remaining challenges for the dissection of genetic complexity that may allow for better stratification, improved diagnostics and more targeted treatments for PD in the future

Regulation of the human cyclin C gene via multiple vitamin D(3)-responsive regions in its promoter

The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)], but binding sites for the 1α,25(OH)(2)D(3) receptor (VDR), so-called 1α,25(OH)(2)D(3) response elements (VDREs), have not yet been identified in the promoter of this gene. We screened various cancer cell lines by quantitative PCR and found that the 1α,25(OH)(2)D(3) inducibility of cyclin C mRNA expression, in relationship with the 24-hydroxylase (CYP24) gene, was best in MCF-7 human breast cancer cells. To characterize the molecular mechanisms, we analyzed 8.4 kb of the cyclin C promoter by using chromatin immunoprecipitation assays (ChIP) with antibodies against acetylated histone 4, VDR and its partner receptor, retinoid X receptor (RXR). The histone 4 acetylation status of all 23 investigated regions of the cyclin C promoter did not change significantly in response to 1α,25(OH)(2)D(3), but four independent promoter regions showed a consistent, 1α,25(OH)(2)D(3)-dependent association with VDR and RXR over a time period of 240 min. Combined in silico/in vitro screening identified in each of these promoter regions a VDRE and reporter gene assays confirmed their functionality. Moreover, re-ChIP assays monitored simultaneous association of VDR with RXR, coactivator, mediator and RNA polymerase II proteins on these regions. Since cyclin C protein is associated with those mediator complexes that display transcriptional repressive properties, this study contributes to the understanding of the downregulation of a number of secondary 1α,25(OH)(2)D(3)-responding genes

Time-Resolved Expression Profiling of the Nuclear Receptor Superfamily in Human Adipogenesis

Background: The differentiation of fibroblast-like pre-adipocytes to lipid-loaded adipocytes is regulated by a network of transcription factors, the most prominent one being the nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma. However, many of the other 47 members of the nuclear receptor superfamily have an impact on adipogenesis, which in human cells has not been investigated in detail. Methodology/Principal Findings: We analyzed by quantitative PCR all human nuclear receptors at multiple time points during differentiation of SGBS pre-adipocytes. The earliest effect was the down-regulation of the genes RARG, PPARD, REVERBA, REV-ERBB, VDR and GR followed by the up-regulation of PPARG, LXRA and AR. These observations are supported with data from 3T3-L1 mouse pre-adipocytes and primary human adipocytes. Investigation of the effects of the individual differentiation mix components in short-term treatments and of their omission from the full mix showed that the expression levels of the early-regulated nuclear receptor genes were most affected by the glucocorticoid receptor (GR) ligand cortisol and the phosophodiesterase inhibitor IBMX. Interestingly, the effects of both compounds converged to repress the genes PPARD, REV-ERBA, REV-ERBB, VDR and GR, whereas cortisol and IBMX showed antagonistic interaction for PPARG, LXRA and AR causing a time lag in their up-regulation. We hypothesize that the well-known auto-repression of GR fine-tunes the detected early responses. Consistently, chromatin immunoprecipitation experiments showed that GR association increased on the transcription start sites of the genes RARG, REV-ERBB, VDR and GR. Conclusions/Significance: Adipocyte differentiation is a process, in which many members of the nuclear receptor superfamily change their mRNA expression. The actions of cortisol and IBMX converged to repress several nuclear receptors early in differentiation, while up-regulation of other nuclear receptor genes showed a time lag due to antagonisms of the signals. Our results place GR and its ligand cortisol as central regulatory factors controlling early regulatory events in human adipogenesis that precedes the regulation of the later events by PPARG

Cell type-selective disease-association of genes under high regulatory load

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manne

Integrated analysis of transcript-level regulation of metabolism reveals disease-relevant nodes of the human metabolic network

Metabolic diseases and comorbidities represent an ever-growing epidemic where multiple cell types impact tissue homeostasis. Here, the link between the metabolic and gene regulatory networks was studied through experimental and computational analysis. Integrating gene regulation data with a human metabolic network prompted the establishment of an open-sourced web portal, IDARE (Integrated Data Nodes of Regulation), for visualizing various gene-related data in context of metabolic pathways. Motivated by increasing availability of deep sequencing studies, we obtained ChIP-seq data from widely studied human umbilical vein endothelial cells. Interestingly, we found that association of metabolic genes with multiple transcription factors (TFs) enriched disease-associated genes. To demonstrate further extensions enabled by examining these networks together, constraint-based modeling was applied to data from human preadipocyte differentiation. In parallel, data on gene expression, genome-wide ChIP-seq profiles for peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding protein (CEBP) α, liver X receptor (LXR) and H3K4me3 and microRNA target identification for miR-27a, miR-29a and miR-222 were collected. Disease-relevant key nodes, including mitochondrial glycerol-3-phosphate acyltransferase (GPAM), were exposed from metabolic pathways predicted to change activity by focusing on association with multiple regulators. In both cell types, our analysis reveals the convergence of microRNAs and TFs within the branched chain amino acid (BCAA) metabolic pathway, possibly providing an explanation for its downregulation in obese and diabetic condition

Identification of genes under dynamic post-transcriptional regulation from time-series epigenomic data

Aim: Prediction of genes under dynamic post-transcriptional regulation from epigenomic data. Materials & methods: We used time-series profiles of chromatin immunoprecipitation-seq data of histone modifications from differentiation of mesenchymal progenitor cells toward adipocytes and osteoblasts to predict gene expression levels at five time points in both lineages and estimated the deviation of those predictions from the RNA-seq measured expression levels using linear regression. Results & conclusion: The genes with biggest changes in their estimated stability across the time series are enriched for noncoding RNAs and lineage-specific biological processes. Clustering mRNAs according to their stability dynamics allows identification of post-transcriptionally coregulated mRNAs and their shared regulators through sequence enrichment analysis. We identify miR-204 as an early induced adipogenic microRNA targeting Akr1c14 and Il1rl1

Cell type-selective disease-association of genes under high regulatory load

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3'UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner

2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

peer reviewed2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target

Normal and Pathological NRF2 Signalling in the Central Nervous System

The nuclear factor erythroid 2-related factor 2 (NRF2) was originally described as a master regulator of antioxidant cellular response, but in the time since, numerous important biological functions linked to cell survival, cellular detoxification, metabolism, autophagy, proteostasis, inflammation, immunity, and differentiation have been attributed to this pleiotropic transcription factor that regulates hundreds of genes. After 40 years of in-depth research and key discoveries, NRF2 is now at the center of a vast regulatory network, revealing NRF2 signalling as increasingly complex. It is widely recognized that reactive oxygen species (ROS) play a key role in human physiological and pathological processes such as ageing, obesity, diabetes, cancer, and neurodegenerative diseases. The high oxygen consumption associated with high levels of free iron and oxidizable unsaturated lipids make the brain particularly vulnerable to oxidative stress. A good stability of NRF2 activity is thus crucial to maintain the redox balance and therefore brain homeostasis. In this review, we have gathered recent data about the contribution of the NRF2 pathway in the healthy brain as well as during metabolic diseases, cancer, ageing, and ageing-related neurodegenerative diseases. We also discuss promising therapeutic strategies and the need for better understanding of cell-type-specific functions of NRF2 in these different fields