dCas9-mediated dysregulation of gene expression in human induced pluripotent stem cells during primitive streak differentiation.

CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation.

Reference Genes for Expression Studies in Human CD8 Naïve and Effector Memory T Cells under Resting and Activating Conditions.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used for mRNA quantification. To accurately measure changing gene transcript levels under different experimental conditions, the use of appropriate reference gene transcripts is instrumental. In T cell immunology, suitable reference genes have been reported for bulk CD4 and CD8 T cells.

Priming exposures to lipopolysaccharides do not affect the induction of Polycomb target genes upon re-exposure.

The Polycomb group (PcG) proteins are chromatin factors underlying the process of transcriptional memory to preserve developmental decisions and keep cellular identities. However, not only developmental signals need to be memorized and thus maintained during the life of an organism. For host protection against pathogens, also a memory of previous exposures to an immunogenic stimulus is crucial to mount a more protective immune response upon re-exposure.

Distinct Transcriptional Responses across Tissue-Resident Macrophages to Short-Term and Long-Term Metabolic Challenge.

The innate immune system safeguards the organism from both pathogenic and environmental stressors. Also, physiologic levels of nutrients affect organismal and intra-cellular metabolism and challenge the immune system. In the long term, over-nutrition leads to low-grade systemic inflammation.

Bivalency in embryos is associated with strong inducibility of Polycomb target genes.

Polycomb group (PcG) and Trithorax group (TrxG) proteins orchestrate development of a multicellular organism by faithfully maintaining cell fate decisions made early in embryogenesis. An important chromatin mark connected to PcG/TrxG regulation is bivalent domains, the simultaneous presence of H3K27me3 and H3K4me3 on a given locus, originally identified in mammalian embryonic stem cells but considered to be absent in invertebrates. Here, we provide evidence for the existence of bivalency in fly embryos.

An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling.

Polycomb group proteins are epigenetic regulators maintaining transcriptional memory during cellular proliferation. In Drosophila larvae, malfunction of Polyhomeotic (Ph), a member of the PRC1 silencing complex, results in neoplastic growth. Here, we report an intrinsic tumour suppression mechanism mediated by the steroid hormone ecdysone during metamorphosis.

A switch in transcription and cell fate governs the onset of an epigenetically-deregulated tumor in .

Tumor initiation is often linked to a loss of cellular identity. Transcriptional programs determining cellular identity are preserved by epigenetically-acting chromatin factors. Although such regulators are among the most frequently mutated genes in cancer, it is not well understood how an abnormal epigenetic condition contributes to tumor onset.

Signalling crosstalk during early tumorigenesis in the absence of Polycomb silencing.

In response to stress and injury a coordinated activation of conserved signalling modules, such as JNK and JAK/STAT, is critical to trigger regenerative tissue restoration. While these pathways rebuild homeostasis and promote faithful organ recovery, it is intriguing that they also become activated in various tumour conditions. Therefore, it is crucial to understand how similar pathways can achieve context-dependent functional outputs, likely depending on cellular states.

Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus.

Background: Numerous target genes of the Polycomb group (PcG) are transiently activated by a stimulus and subsequently repressed. However, mechanisms by which PcG proteins regulate such target genes remain elusive.

Results: We employed the heat shock-responsive hsp70 locus in Drosophila to study the chromatin dynamics of PRC1 and its interplay with known regulators of the locus before, during and after heat shock.

Single vector non-leaky gene expression system for Drosophila melanogaster.

An ideal transgenic gene expression system is inducible, non-leaky, and well tolerated by the target organism. While the former has been satisfactorily realized, leakiness and heavy physiological burden imposed by the existing systems are still prominent hurdles in their successful implementation. Here we describe a new system for non-leaky expression of transgenes in Drosophila.

Stable Polycomb-dependent transgenerational inheritance of chromatin states in Drosophila.

Transgenerational epigenetic inheritance (TEI) describes the transmission of alternative functional states through multiple generations in the presence of the same genomic DNA sequence. Very little is known about the principles and the molecular mechanisms governing this type of inheritance. Here, by transiently enhancing 3D chromatin interactions, we established stable and isogenic Drosophila epilines that carry alternative epialleles, as defined by differential levels of Polycomb-dependent trimethylation of histone H3 Lys27 (forming H3K27me3).

A High-Density Map for Navigating the Human Polycomb Complexome.

Polycomb group (PcG) proteins are major determinants of gene silencing and epigenetic memory in higher eukaryotes. Here, we systematically mapped the human PcG complexome using a robust affinity purification mass spectrometry approach. Our high-density protein interaction network uncovered a diverse range of PcG complexes.

Imaginal Disc Transplantation in Drosophila.

Since Ephrussi and Beadle introduced imaginal disc transplantation to Drosophila research in 1936, the method played an important part towards a better understanding of disc patterning, tissue regeneration, and reprogramming phenomena like transdetermination. Despite increasing usage of high-throughput approaches towards solving biological problems this classical manual method is still in use for studying disc development in a semi-physiological context. Here we describe in detail a protocol and provide recommendations on the procedure in particular for analyzing the regenerative potential of imaginal disks.

A Rapid TALEN Assembly Protocol.

Owing to their modular and highly specific DNA recognition mode, transcription activator-like effector nucleases (TALENs) have been rapidly adopted by the scientific community for the purpose of generating site-specific double-strand breaks (DSBs) on a DNA molecule. A pair of TALENs can be used to produce random insertions or deletions of various lengths via nonhomologous end-joining or together with a homologous donor DNA to induce precise sequence alterations by homologous recombination (HR). Here, we describe a method for TALEN assembly (easyT) and a strategy for genome engineering via HR.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance of stemness.

The legacy of Drosophila imaginal discs.

The study of Drosophila imaginal discs has contributed to a number of discoveries in developmental and cellular biology. In addition to the elucidation of the role of tissue compartments and organ-specific master regulator genes during development, imaginal discs have also become well established as models for studying cellular interactions and complex genetic pathways. Here, we review key discoveries resulting from investigations of these epithelial precursor organs, ranging from cell fate determination and transdetermination to tissue patterning.

DNAshapeR: an R/Bioconductor package for DNA shape prediction and feature encoding.

Unlabelled: DNAshapeR predicts DNA shape features in an ultra-fast, high-throughput manner from genomic sequencing data. The package takes either nucleotide sequence or genomic coordinates as input and generates various graphical representations for visualization and further analysis. DNAshapeR further encodes DNA sequence and shape features as user-defined combinations of k-mer and DNA shape features.

Ectopic expression of Msx2 in mammalian myotubes recapitulates aspects of amphibian muscle dedifferentiation.

In contrast to urodele amphibians and teleost fish, mammals lack the regenerative responses to replace large body parts. Amphibian and fish regeneration uses dedifferentiation, i.e.

Trithorax and Polycomb group-dependent regulation: a tale of opposing activities.

Intricate layers of regulation determine the unique gene expression profiles of a given cell and, therefore, underlie the immense phenotypic diversity observed among cell types. Understanding the mechanisms that govern which genes are expressed and which genes are silenced is a fundamental focus in biology. The Polycomb and Trithorax group chromatin proteins play important roles promoting the stable and heritable repression and activation of gene expression, respectively.

High-resolution profiling of Drosophila replication start sites reveals a DNA shape and chromatin signature of metazoan origins.

At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution.

During Drosophila disc regeneration, JAK/STAT coordinates cell proliferation with Dilp8-mediated developmental delay.

Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood.

Sensitive and highly resolved identification of RNA-protein interaction sites in PAR-CLIP data.

Background: PAR-CLIP is a recently developed Next Generation Sequencing-based method enabling transcriptome-wide identification of interaction sites between RNA and RNA-binding proteins. The PAR-CLIP procedure induces specific base transitions that originate from sites of RNA-protein interactions and can therefore guide the identification of binding sites. However, additional sources of transitions, such as cell type-specific SNPs and sequencing errors, challenge the inference of binding sites and suitable statistical approaches are crucial to control false discovery rates.

Transcriptional silencing by polycomb-group proteins.

Polycomb-group (PcG) genes encode chromatin proteins involved in stable and heritable transcriptional silencing. PcG proteins participate in distinct multimeric complexes that deposit, or bind to, specific histone modifications (e.g.

A deterministic analysis of genome integrity during neoplastic growth in Drosophila.

The development of cancer has been associated with the gradual acquisition of genetic alterations leading to a progressive increase in malignancy. In various cancer types this process is enabled and accelerated by genome instability. While genome sequencing-based analysis of tumor genomes becomes increasingly a standard procedure in human cancer research, the potential necessity of genome instability for tumorigenesis in Drosophila melanogaster has, to our knowledge, never been determined at DNA sequence level.