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See detailIdentification of genes under dynamic post-transcriptional regulation from time-series epigenomic data
Becker, Julia Christina UL; Gerard, Déborah UL; Ginolhac, Aurélien UL et al

in Epigenomics (2019)

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 ... [more ▼]

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. [less ▲]

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See detailImpaired serine metabolism complements LRRK2-G2019S pathogenicity in PD patients
Nickels, Sarah UL; Walter, Jonas; Bolognin, Silvia UL et al

in Parkinsonism and Related Disorders (2019)

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See detailTemporal enhancer profiling of parallel lineages identifies AHR and GLIS1 as regulators of mesenchymal multipotency
Gerard, Déborah UL; Schmidt, Florian; Ginolhac, Aurélien UL et al

in Nucleic Acids Research (2018)

Temporal data on gene expression and context-specific open chromatin states can improve identification of key transcription factors (TFs) and the gene regulatory networks (GRNs) controlling cellular ... [more ▼]

Temporal data on gene expression and context-specific open chromatin states can improve identification of key transcription factors (TFs) and the gene regulatory networks (GRNs) controlling cellular differentiation. However, their integration remains challenging. Here, we delineate a general approach for data-driven and unbiased identification of key TFs and dynamic GRNs, called EPIC-DREM. We generated time-series transcriptomic and epigenomic profiles during differentiation of mouse multipotent bone marrow stromal cell line (ST2) toward adipocytes and osteoblasts. Using our novel approach we constructed time-resolved GRNs for both lineages and identifed the shared TFs involved in both differentiation processes. To take an alternative approach to prioritize the identified shared regulators, we mapped dynamic super-enhancers in both lineages and associated them to target genes with correlated expression profiles. The combination of the two approaches identified aryl hydrocarbon receptor (AHR) and Glis family zinc finger 1 (GLIS1) as mesenchymal key TFs controlled by dynamic cell type-specific super-enhancers that become repressed in both lineages. AHR and GLIS1 control differentiation-induced genes and their overexpression can inhibit the lineage commitment of the multipotent bone marrow-derived ST2 cells. [less ▲]

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See detailTREM2 triggers microglial density and age‐related neuronal loss
Linnartz-Gerlach, Bettina; Bodea, Liviu-Gabriel; Klaus, Christine et al

in Glia (2018)

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked to ... [more ▼]

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked to inflammatory neurodegenerative diseases associated with aging. The typical aging process goes along with microglial changes and mild neuronal loss, but the exact contribution of TREM2 is still unclear. Aged TREM2 knock‐out mice showed decreased age‐related neuronal loss in the substantia nigra and the hippocampus. Transcriptomic analysis of the brains of 24 months old TREM2 knock‐out mice revealed 211 differentially expressed genes mostly downregulated and associated with complement activation and oxidative stress response pathways. Consistently, 24 months old TREM2 knock‐out mice showed lower transcription of microglial (Aif1 and Tmem119), oxidative stress markers (Inos, Cyba, and Cybb) and complement components (C1qa, C1qb, C1qc, C3, C4b, Itgam, and Itgb2), decreased microglial numbers and expression of the microglial activation marker Cd68, as well as accumulation of oxidized lipids. Cultured microglia of TREM2 knock‐out mice showed reduced phagocytosis and oxidative burst. Thus, microglial TREM2 contributes to age‐related microglial changes, phagocytic oxidative burst, and loss of neurons with possible detrimental effects during physiological aging. [less ▲]

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See detailAnalysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture
Bouvy-Liivrand, Maria; Hernandez de Sande, Ana; Pölönen, Petri et al

in Nucleic Acids Research (2017)

Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA ... [more ▼]

Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29a∼29b-1, mir-100∼let-7a-2∼125b-1 and miR-221∼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci. [less ▲]

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See detailIntegrated metabolic modelling reveals cell-type specific epigenetic control points of the macrophage metabolic network
Pacheco, Maria UL; John, Elisabeth UL; Kaoma, Tony et al

in BMC Genomics (2015), 16(809),

Background: The reconstruction of context-specific metabolic models from easily and reliably measurable features such as transcriptomics data will be increasingly important in research and medicine ... [more ▼]

Background: The reconstruction of context-specific metabolic models from easily and reliably measurable features such as transcriptomics data will be increasingly important in research and medicine. Current reconstruction methods suffer from high computational effort and arbitrary threshold setting. Moreover, understanding the underlying epigenetic regulation might allow the identification of putative intervention points within metabolic networks. Genes under high regulatory load from multiple enhancers or super-enhancers are known key genes for disease and cell identity. However, their role in regulation of metabolism and their placement within the metabolic networks has not been studied. Methods: Here we present FASTCORMICS, a fast and robust workflow for the creation of high-quality metabolic models from transcriptomics data. FASTCORMICS is devoid of arbitrary parameter settings and due to its low computational demand allows cross-validation assays. Applying FASTCORMICS, we have generated models for 63 primary human cell types from microarray data, revealing significant differences in their metabolic networks. Results: To understand the cell type-specific regulation of the alternative metabolic pathways we built multiple models during differentiation of primary human monocytes to macrophages and performed ChIP-Seq experiments for histone H3 K27 acetylation (H3K27ac) to map the active enhancers in macrophages. Focusing on the metabolic genes under high regulatory load from multiple enhancers or super-enhancers, we found these genes to show the most cell type-restricted and abundant expression profiles within their respective pathways. Importantly, the high regulatory load genes are associated to reactions enriched for transport reactions and other pathway entry points, suggesting that they are critical regulatory control points for cell type-specific metabolism. Conclusions: By integrating metabolic modelling and epigenomic analysis we have identified high regulatory load as a common feature of metabolic genes at pathway entry points such as transporters within the macrophage metabolic network. Analysis of these control points through further integration of metabolic and gene regulatory networks in various contexts could be beneficial in multiple fields from identification of disease intervention strategies to cellular reprogramming. [less ▲]

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See detailCell type-selective disease-association of genes under high regulatory load
Galhardo, Mafalda Sofia UL; Berninger, Philipp; Nguyen, Thanh Phuong UL et al

in Nucleic Acids Research (2015), 43(18), 8839-8855

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 ... [more ▼]

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. [less ▲]

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See detailTranscriptomics profiling of human SGBS adipogenesis
Galhardo, Mafalda Sofia UL; Sinkkonen, Lasse UL; Berninger, Philipp et al

in Genomics Data (2014), 2

Obesity is an ever-growing epidemic where tissue homeostasis is influenced by the differentiation of adipocytes that function in lipid metabolism, endocrine and inflammatory processes. While this ... [more ▼]

Obesity is an ever-growing epidemic where tissue homeostasis is influenced by the differentiation of adipocytes that function in lipid metabolism, endocrine and inflammatory processes. While this differentiation process has been well-characterized in mice, limited data is available from human cells. Applying microarray expression profiling in the human SGBS pre-adipocyte cell line, we identified genes with differential expression during differentiation in combination with constraint-based modeling of metabolic pathway activity. Here we describe the experimental design and quality controls in detail for the gene expression and related results published by Galhardo et al. in Nucleic Acids Research 2014 associated with the data uploaded to NCBI Gene Expression Omnibus (). [less ▲]

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See detailChIP-seq profiling of the active chromatin marker H3K4me3 and PPARγ, CEBPα and LXR target genes in human SGBS adipocytes
Galhardo, Mafalda Sofia UL; Sinkkonen, Lasse UL; Berninger, Philipp et al

in Genomics Data (2014), 2

Transcription factors (TFs) represent key factors to establish a cellular phenotype. It is known that several TFs could play a role in disease, yet less is known so far how their targets overlap. We ... [more ▼]

Transcription factors (TFs) represent key factors to establish a cellular phenotype. It is known that several TFs could play a role in disease, yet less is known so far how their targets overlap. We focused here on identifying the most highly induced TFs and their putative targets during human adipogenesis. Applying chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) in the human SGBS pre-adipocyte cell line, we identified genes with binding sites in their vicinity for the three TFs studied, PPARγ, CEBPα and LXR. Here we describe the experimental design and quality controls in detail for the deep sequencing data and related results published by Galhardo et al. in Nucleic Acids Research 2014 [1] associated with the data uploaded to NCBI Gene Expression Omnibus (). [less ▲]

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See detailNeurodegeneration by Activation of the Microglial Complement–Phagosome Pathway
Bodea, Liviu-Gabriel; Wang, Yiner; Linnartz-Gerlach, Bettina et al

in Journal of Neuroscience (2014), 34(25), 8546-8556

Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation. We now demonstrate in vivo that repeated systemic challenge of mice over four ... [more ▼]

Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation. We now demonstrate in vivo that repeated systemic challenge of mice over four consecutive days with bacterial LPS maintained an elevated microglial inflammatory phenotype and induced loss of dopaminergic neurons in the substantia nigra. The same total cumulative LPS dose given within a single application did not induce neurodegeneration. Whole-genome transcriptome analysis of the brain demonstrated that repeated systemic LPS application induced an activation pattern involving the classical complement system and its associated phagosome pathway. Loss of dopaminergic neurons induced by repeated systemic LPS application was rescued in complement C3-deficient mice, confirming the involvement of the complement system in neurodegeneration. Our data demonstrate that a phagosomal inflammatory response of microglia is leading to complement-mediated loss of dopaminergic neurons. [less ▲]

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See detailCombinatorial regulation of lipoprotein lipase by microRNAs during mouse adipogenesis
Liivrand, Maria UL; Heinäniemi, Merja UL; John, Elisabeth UL et al

in RNA Biology (2014), 11(1), 76-91

MicroRNAs (miRNAs) regulate gene expression directly through base pairing to their targets or indirectly through participating in multi-scale regulatory networks. Often miRNAs take part in feed-forward ... [more ▼]

MicroRNAs (miRNAs) regulate gene expression directly through base pairing to their targets or indirectly through participating in multi-scale regulatory networks. Often miRNAs take part in feed-forward motifs where a miRNA and a transcription factor act on shared targets to achieve accurate regulation of processes such as cell differentiation. Here we show that the expression levels of miR-27a and miR-29a inversely correlate with the mRNA levels of lipoprotein lipase (Lpl), their predicted combinatorial target, and its key transcriptional regulator peroxisome proliferator activated receptor gamma (Pparg) during 3T3-L1 adipocyte differentiation. More importantly, we show that Lpl, a key lipogenic enzyme, can be negatively regulated by the two miRNA families in a combinatorial fashion on the mRNA and functional level in maturing adipocytes. This regulation is mediated through the Lpl 3′UTR as confirmed by reporter gene assays. In addition, a small mathematical model captures the dynamics of this feed-forward motif and predicts the changes in Lpl mRNA levels upon network perturbations. The obtained results might offer an explanation to the dysregulation of LPL in diabetic conditions and could be extended to quantitative modeling of regulation of other metabolic genes under similar regulatory network motifs. [less ▲]

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See detailIntegrated analysis of transcript-level regulation of metabolism reveals disease-relevant nodes of the human metabolic network
Galhardo, Mafalda Sofia UL; Sinkkonen, Lasse UL; Berninger, Philippe et al

in Nucleic Acids Research (2013)

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 ... [more ▼]

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, constraintbased 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) c, CCAAT/enhancer binding protein (CEBP) a, 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-phosphateacyltransferase (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 conditions. [less ▲]

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See detailGene-pair expression signatures reveal lineage control
Heinäniemi, Merja UL; Nykter, Matti; Kramer, Roger et al

in Nature Methods (2013)

The distinct cell types of multicellular organisms arise due to constraints imposed by gene regulatory networks on the collective change of gene expression across the genome, creating self-stabilizing ... [more ▼]

The distinct cell types of multicellular organisms arise due to constraints imposed by gene regulatory networks on the collective change of gene expression across the genome, creating self-stabilizing expression states, or attractors. We compiled a resource of curated human expression data comprising 166 cell types and 2,602 transcription regulating genes and developed a data driven method built around the concept of expression reversal defined at the level of gene pairs, such as those participating in toggle switch circuits. This approach allows us to organize the cell types into their ontogenetic lineage-relationships and to reflect regulatory relationships among genes that explain their ability to function as determinants of cell fate. We show that this method identifies genes belonging to regulatory circuits that control neuronal fate, pluripotency and blood cell differentiation, thus offering a novel large-scale perspective on lineage specification. [less ▲]

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See detailDataset integration identifies transcriptional regulation of microRNA genes by PPARgamma in differentiating mouse 3T3-L1 adipocytes
John, Elisabeth UL; Wienecke-Baldacchino, Anke UL; Liivrand, Maria UL et al

in Nucleic Acids Research (2012), 40(10), 4446-4460

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPARgamma binding sites in mouse ... [more ▼]

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPARgamma binding sites in mouse adipocytes and PPARgamma upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPARgamma-targets. By integration of four separate datasets of genome-wide PPARgamma binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPARgamma binding within 50 kb from miRNA transcription start sites. Nineteen mature miRNAs were upregulated >/=2-fold during adipogenesis and for six of these miRNA loci the PPARgamma binding sites were confirmed by at least three datasets. The upregulation of five miRNA genes miR-103-1 (host gene Pank3), miR-148b (Copz1), miR-182/96/183, miR-205 and miR-378 (Ppargc1b) followed that of Pparg. The PPARgamma-dependence of four of these miRNA loci was demonstrated by PPARgamma knock-down and the loci of miR-103-1 (Pank3), miR-205 and miR-378 (Ppargc1b) were also responsive to the PPARgamma ligand rosiglitazone. Finally, chromatin immunoprecipitation analysis validated in silico predicted PPARgamma binding sites at all three loci and H3K27 acetylation was analyzed to confirm the activity of these enhancers. In conclusion, we identified 22 putative PPARgamma target miRNA genes, showed the PPARgamma dependence of four of these genes and demonstrated three as direct PPARgamma target genes in mouse adipogenesis. [less ▲]

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See detailTime-Resolved Expression Profiling of the Nuclear Receptor Superfamily in Human Adipogenesis
Lahnalampi, Mari; Heinäniemi, Merja UL; Sinkkonen, Lasse UL et al

in PLoS ONE (2010), 5(9),

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 ... [more ▼]

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. [less ▲]

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See detailDicer is associated with ribosomal DNA chromatin in mammalian cells
Sinkkonen, Lasse UL; Hugenschmidt, Tabea; Filipowicz, Witold et al

in PLoS ONE (2010), 5(8), 1-2

BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different ... [more ▼]

BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays. [less ▲]

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See detailMicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells
Sinkkonen, Lasse UL; Hugenschmidt, Tabea; Berninger, Philipp et al

in Nature Structural & Molecular Biology (2008), 15(3), 259-67

Loss of microRNA (miRNA) pathway components negatively affects differentiation of embryonic stem (ES) cells, but the underlying molecular mechanisms remain poorly defined. Here we characterize changes in ... [more ▼]

Loss of microRNA (miRNA) pathway components negatively affects differentiation of embryonic stem (ES) cells, but the underlying molecular mechanisms remain poorly defined. Here we characterize changes in mouse ES cells lacking Dicer (Dicer1). Transcriptome analysis of Dicer-/- cells indicates that the ES-specific miR-290 cluster has an important regulatory function in undifferentiated ES cells. Consistently, many of the defects in Dicer-deficient cells can be reversed by transfection with miR-290 family miRNAs. We demonstrate that Oct4 (also known as Pou5f1) silencing in differentiating Dicer-/- ES cells is accompanied by accumulation of repressive histone marks but not by DNA methylation, which prevents the stable repression of Oct4. The methylation defect correlates with downregulation of de novo DNA methyltransferases (Dnmts). The downregulation is mediated by Rbl2 and possibly other transcriptional repressors, potential direct targets of miR-290 cluster miRNAs. The defective DNA methylation can be rescued by ectopic expression of de novo Dnmts or by transfection of the miR-290 cluster miRNAs, indicating that de novo DNA methylation in ES cells is controlled by miRNAs. [less ▲]

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See detailSpatio-temporal activation of chromatin on the human CYP24 gene promoter in the presence of 1alpha,25-Dihydroxyvitamin D3
Väisänen, Sami; Dunlop, Thomas W.; Sinkkonen, Lasse UL et al

in Journal of Molecular Biology (2005), 350(1), 65-77

The vitamin D3 24-hydroxylase gene (CYP24) is one of the most strongly induced genes known. Despite this, its induction by the hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3) has been characterized ... [more ▼]

The vitamin D3 24-hydroxylase gene (CYP24) is one of the most strongly induced genes known. Despite this, its induction by the hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3) has been characterized only partially. Therefore, we monitored the spatio-temporal, 1alpha,25OH2D3-dependent chromatin acetylation status of the human CYP24 promoter by performing chromatin immunoprecipitation (ChIP) assays with antibodies against acetylated histone 4. This was achieved by performing PCR on 25 contiguous genomic regions spanning the first 7.7 kb of the promoter. ChIP assays using antibodies against the 1alpha,25OH2D3 receptor (VDR) revealed that, in addition to the proximal promoter, three novel regions further upstream associated with VDR. Combined in silico/in vitro screening identified in three of the four promoter regions sequences resembling known VDREs and reporter gene assays confirmed the inducibility of these regions by 1alpha,25OH2D3)=. In contrast, the fourth VDR-associated promoter region did not contain any recognizable classical VDRE that could account for the presence of the protein on this region. However, re-ChIP assays monitored on all four promoter regions simultaneous association of VDR with retinoid X receptor, coactivator, mediator and RNA polymerase II proteins. These proteins showed a promoter region-specific association pattern demonstrating the complex choreography of the CYP24 gene promoter activation over 300 minutes. Thus, this study reveals new information concerning the regulation of the CYP24 gene by 1alpha,25OH2D3, and is a demonstration of the simultaneous participation of multiple, structurally diverse response elements in promoter activation in a living cell. [less ▲]

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See detailRegulation of the human cyclin C gene via multiple vitamin D3-responsive regions in its promoter
Sinkkonen, Lasse UL; Malinen, Marjo; Saavalainen, Katri et al

in Nucleic Acids Research (2005), 33(8), 2440-51

The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], but binding sites for the 1alpha,25(OH)2D3 ... [more ▼]

The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], but binding sites for the 1alpha,25(OH)2D3 receptor (VDR), so-called 1alpha,25(OH)2D3 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 1alpha,25(OH)2D3 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 1alpha,25(OH)2D3, but four independent promoter regions showed a consistent, 1alpha,25(OH)2D3-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 1alpha,25(OH)2D3-responding genes. [less ▲]

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