Epigenetic silencing of miR-181c by DNA methylation in glioblastoma cell lines.

BACKGROUND: Post-transcriptional regulation by microRNAs is recognized as one of the major pathways for the control of cellular homeostasis. Less well understood is the transcriptional and epigenetic regulation of genes encoding microRNAs. In the present study we addressed the epigenetic regulation of the miR-181c in normal and malignant brain cells. METHODS: To explore the epigenetic regulation of the miR-181c we evaluated its expression using RT-qPCR and the in vivo binding of the CCCTC-binding factor (CTCF) to its regulatory region in different glioblastoma cell lines. DNA methylation survey, chromatin immunoprecipitation and RNA interference assays were used to assess the role of CTCF in the miR-181c epigenetic silencing. RESULTS: We found that miR-181c is downregulated in glioblastoma cell lines, as compared to normal brain tissues. Loss of expression correlated with a notorious gain of DNA methylation at the miR-181c promoter region and the dissociation of the multifunctional nuclear factor CTCF. Taking advantage of the genomic distribution of CTCF in different cell types we propose that CTCF has a local and cell type specific regulatory role over the miR-181c and not an architectural one through chromatin loop formation. This is supported by the depletion of CTCF in glioblastoma cells affecting the expression levels of NOTCH2 as a target of miR-181c. CONCLUSION: Together, our results point to the epigenetic role of CTCF in the regulation of microRNAs implicated in tumorigenesis.

LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes.

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits-dL(3)mbt, dCoREST, and dLint-1-and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access.

Metabolic landscape of the male mouse gut identifies different niches determined by microbial activities.

Distinct niches of the mammalian gut are populated by diverse microbiota, but the contribution of spatial variation to intestinal metabolism remains unclear. Here we present a map of the longitudinal metabolome along the gut of healthy colonized and germ-free male mice. With this map, we reveal a general shift from amino acids in the small intestine to organic acids, vitamins and nucleotides in the large intestine. We compare the metabolic landscapes in colonized versus germ-free mice to disentangle the origin of many metabolites in different niches, which in some cases allows us to infer the underlying processes or identify the producing species. Beyond the known impact of diet on the small intestinal metabolic niche, distinct spatial patterns suggest specific microbial influence on the metabolome in the small intestine. Thus, we present a map of intestinal metabolism and identify metabolite-microbe associations, which provide a basis to connect the spatial occurrence of bioactive compounds to host or microorganism metabolism.

Zoonotic Chlamydiae as rare causes of severe pneumonia.

Zoonotic species of the Chlamydiaceae family should be considered as rare pathogenic agents of severe atypical pneumonia. A fatal case of a severe pneumonia due to Chlamydia psittaci was traced back to pet birds, and pneumonia in a pregnant woman was attributed to abortions in a sheep and goat flock, being the source of Chlamydia abortus. The two SARS­CoV­2-negative pneumonia cases presented here were investigated in an inter-disciplinary approach involving physicians and veterinarians. State-of-art molecular methods allowed the identification and genotyping of zoonotic Chlamydiae.

An Intronic Alu Element Attenuates the Transcription of a Long Non-coding RNA in Human Cell Lines.

Alu elements are primate-specific repeats and represent the most abundant type of transposable elements (TE) in the human genome. Genome-wide analysis of the enrichment of histone post-translational modifications suggests that human Alu sequences could function as transcriptional enhancers; however, no functional experiments have evaluated the role of Alu sequences in the control of transcription in situ. The present study analyses the regulatory activity of a human Alu sequence from the AluSx family located in the second intron of the long intergenic non-coding RNA Linc00441, found in divergent orientation to the RB1 gene. We observed that the Alu sequence acts as an enhancer element based on reporter gene assays while CRISPR-Cas9 deletions of the Alu sequence in K562 cells resulted in a marked transcriptional upregulation of Linc00441 and a decrease in proliferation. Our results suggest that an intragenic Alu sequence with enhancer activity can act as a transcriptional attenuator of its host lincRNA.

Protein S100 as prognostic marker for gastrointestinal stromal tumors: a clinicopathological risk factor analysis.

Gastrointestinal stromal tumors (GISTs) are a heterogenous group of mesenchymal neoplasms ranging from semibenign tumors to highly aggressive neoplasms. Predicting their clinical behavior is challenging and criteria delineating benign from malignant cases are controversially discussed. The aims of the present study were to define the clinicopathological features of 35 GISTs and to determine whether any specific parameters were associated with the patient's outcome. In the present series, protein S100 (S100) expression was found in 13/35 (37%) patients with a varying staining intensity ranging between strong and moderate. The multivariate statistical analysis in the S100-positive group revealed a significantly poorer survival (p = .0058) and a tendency for higher recurrence (p = .052) compared to the negative GIST patients. Furthermore, the statistical analysis disclosed a correlation between the expression of CD117, CD34, desmin, and alpha-smooth muscle actin and survival or recurrence (p > .05). Positive immunoreactivity was seen for CD117 in 25 (71%) patients and for CD34 in 19 (54%) patients. In addition we confirmed the previously reported impact of initial tumor size on survival and recurrence rate (p = .034 and p = .039). The series was also analyzed according to established prognostic factors (tumor size and mitotic activity), which indicated that 77% of S100-positive cases were at high risk for malignant tumor behavior, 15% at intermediate risk, and 8% at low risk. Based on these findings, we suggest that protein S100 represents an additional prognostic factor to better define the malignant potential of GISTs and stratify the risk for each patient.

New insights on the role of DNA methylation from a global view.

In mammals, DNA methylation is a crucial epigenetic modification with key functions during development. Cellular processes that are regulated by DNA methylation comprise X chromosome inactivation, gene imprinting, genomic stability and transcriptional regulation. Generally, the methylation status of the majority of target sites is reliably propagated during mitosis. However, advances in genome-wide DNA methylation analysis at base-resolution have discovered a substantial amount of differential DNA methylation between normal cells of different tissue-origin. Moreover, aberrant DNA methylation changes are linked with a significant number of human diseases, particularly with cancer. Sites of differential and aberrant DNA methylation include regulatory DNA sequences, such as CpG islands in promoters and distal cis-regulatory elements, like enhancers. In this review, we will discuss novel aspects of DNA methylation dynamics, during normal development and in association with diseases.

Implications of DNA Methylation in Parkinson's Disease.

It has been 200 years since Parkinson's disease (PD) was first described, yet many aspects of its etiopathogenesis remain unclear. PD is a progressive and complex neurodegenerative disorder caused by genetic and environmental factors including aging, nutrition, pesticides and exposure to heavy metals. DNA methylation may be altered in response to some of these factors; therefore, it is proposed that epigenetic mechanisms, particularly DNA methylation, can have a fundamental role in gene-environment interactions that are related with PD. Epigenetic changes in PD-associated genes are now widely studied in different populations, to discover the mechanisms that contribute to disease development and identify novel biomarkers for early diagnosis and future pharmacological treatment. While initial studies sought to find associations between promoter DNA methylation and the regulation of associated genes in PD brain tissue, more recent studies have described concordant DNA methylation patterns between blood and brain tissue DNA. These data justify the use of peripheral blood samples instead of brain tissue for epigenetic studies. Here, we summarize the current data about DNA methylation changes in PD and discuss the potential of DNA methylation as a potential biomarker for PD. Additionally, we discuss environmental and nutritional factors that have been implicated in DNA methylation. Although the search for significant DNA methylation changes and gene expression analyses of PD-associated genes have yielded inconsistent and contradictory results, epigenetic modifications remain under investigation for their potential to reveal the link between environmental risk factors and the development of PD.

Transcriptome-wide identification of in vivo interactions between RNAs and RNA-binding proteins by RIP and PAR-CLIP assays.

Comprehensive genomic and computational studies in the era of high-throughput sequencing revealed that the major proportion of the human genome is transcribed. This novel insight confronted the scientific community with new questions concerning the expanded role of RNA, especially noncoding RNA (ncRNA), in cellular pathways. In recent years, there has been mounting evidence that ncRNAs and RNA binding proteins (RBPs) are involved in a wide range of biological processes, such as developmental transitions, cell differentiation, stress response, genome organization, and regulation of gene expression. In particular, in the chromatin field long noncoding RNAs (lncRNAs) have drawn increasing attention to their function in epigenetic regulation due to the fact that they were found to interact with multiple chromatin regulators and modifiers. Recently, techniques to study the extent of RNA-protein interactions have been developed in many research laboratories. Here we describe protocols for RNA Immunoprecipitation-Sequencing (RIP-Seq) and Photoactivatable-Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation combined with deep sequencing (PAR-CLIP-Seq) to identify RNA targets of RNA-binding proteins (RBPs) on a transcriptome-wide level, discussing advantages and drawbacks.

Axis Patterning by BMPs: Cnidarian Network Reveals Evolutionary Constraints.

BMP signaling plays a crucial role in the establishment of the dorso-ventral body axis in bilaterally symmetric animals. However, the topologies of the bone morphogenetic protein (BMP) signaling networks vary drastically in different animal groups, raising questions about the evolutionary constraints and evolvability of BMP signaling systems. Using loss-of-function analysis and mathematical modeling, we show that two signaling centers expressing different BMPs and BMP antagonists maintain the secondary axis of the sea anemone Nematostella. We demonstrate that BMP signaling is required for asymmetric Hox gene expression and mesentery formation. Computational analysis reveals that network parameters related to BMP4 and Chordin are constrained both in Nematostella and Xenopus, while those describing the BMP signaling modulators can vary significantly. Notably, only chordin, but not bmp4 expression needs to be spatially restricted for robust signaling gradient formation. Our data provide an explanation of the evolvability of BMP signaling systems in axis formation throughout Eumetazoa.

Chromatin regulation: how complex does it get?

Gene transcription is tightly regulated at different levels to ensure that the transcriptome of the cell is appropriate for developmental stage and cell type. The chromatin state in which a gene is embedded determines its expression level to a large extent. Activation or repression of transcription is typically accomplished by the recruitment of chromatin-associated multisubunit protein complexes that combine several molecular tools, such as histone-binding and chromatin-modifying activities. Recent biochemical purifications of such complexes have revealed a substantial diversity. On the one hand, complexes that were thought to be unique have been revealed to be part of large complex families. On the other hand, protein subunits that were thought to only exist in separate complexes have been shown to coexist in novel assemblies. In this review we discuss our current knowledge of repressor complexes that contain MBT domain proteins and/or the CoREST co-repressor and use them as a paradigm to illustrate the unexpected heterogeneity and tool sharing of chromatin regulating protein complexes. These recent insights also challenge the ways we define and think about protein complexes in general.

Monomethylation of lysine 20 on histone H4 facilitates chromatin maturation.

Histone modifications play an important role in shaping chromatin structure. Here, we describe the use of an in vitro chromatin assembly system from Drosophila embryo extracts to investigate the dynamic changes of histone modifications subsequent to histone deposition. In accordance with what has been observed in vivo, we find a deacetylation of the initially diacetylated isoform of histone H4, which is dependent on chromatin assembly. Immediately after deposition of the histones onto DNA, H4 is monomethylated at K20, which is required for an efficient deacetylation of the H4 molecule. H4K20 methylation-dependent dl(3)MBT association with chromatin and the identification of a dl(3)MBT-dRPD3 complex suggest that a deacetylase is specifically recruited to the monomethylated substrate through interaction with dl(3)MBT. Our data demonstrate that histone modifications are added and removed during chromatin assembly in a highly regulated manner.