Hans-Rudolf Hotz

Noncoding RNAs prevent spreading of a repressive histone mark

Transcription of eukaryotic genomes is more widespread than was previously anticipated and results in the production of many non–protein-coding RNAs (ncRNAs) whose functional relevance is poorly understood. Here we demonstrate that ncRNAs can counteract the encroachment of heterochromatin into neighboring euchromatin. We have identified a long ncRNA (termed BORDERLINE) that prevents spreading of the HP1 protein Swi6 and histone H3 Lys9 methylation beyond the pericentromeric repeat region of Schizosaccharomyces pombe chromosome 1. BORDERLINE RNAs act in a sequence-independent but locus-dependent manner and are processed by Dicer into short RNAs referred to as brdrRNAs. In contrast to canonical centromeric short interfering RNAs, brdrRNAs are rarely loaded onto Argonaute. Our analyses reveal an unexpected regulatory activity of ncRNAs in demarcating an epigenetically distinct chromosomal domain that could also be operational in other eukaryotes.

RNAi keeps Atf1-bound stress response genes in check at nuclear pores.

RNAi pathways are prevalent throughout the eukaryotic kingdom and are well known to regulate gene expression on a post-transcriptional level in the cytoplasm. Less is known about possible functions of RNAi in the nucleus. In the fission yeast Schizosaccharomyces pombe, RNAi is crucial to establish and maintain centromeric heterochromatin and functions to repress genome activity by a chromatin silencing mechanism referred to as cotranscriptional gene silencing (CTGS). Mechanistic details and the physiological relevance of CTGS are unknown. Here we show that RNAi components interact with chromatin at nuclear pores to keep stress response genes in check. We demonstrate that RNAi-mediated CTGS represses stress-inducible genes by degrading mRNAs under noninduced conditions. Under chronic heat stress conditions, a Dicer thermoswitch deports Dicer to the cytoplasm, thereby disrupting CTGS and enabling expression of genes implicated in the acquisition of thermotolerance. Taken together, our work highlights a role for nuclear pores and the stress response transcription factor Atf1 in coordinating the interplay between the RNAi machinery and the S. pombe genome and uncovers a novel mode of RNAi regulation in response to an environmental cue.

An extended dsRBD with a novel zinc‐binding motif mediates nuclear retention of fission yeast Dicer

Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here, we report the solution structure of the C‐terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double‐stranded RNA binding domain (dsRBD) fold embedding a novel zinc‐binding motif that is conserved among dicers in yeast. Although the C‐terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleocytoplasmic trafficking and not substrate binding. The C‐terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.

Tenascin-C triggers fibrin accumulation by downregulation of tissue plasminogen activator

We explored novel functions of tenascin‐C by comparing mouse embryonic fibroblasts (MEFs) proficient or deficient in tenascin‐C expression. Transcript profiling analysis identified tissue plasminogen activator (tPA) as the most consistently over‐expressed gene in all tenascin‐C deficient MEFs. This was confirmed by real‐time PCR as well as by protein expression analysis. In agreement with these observations, tenascin‐C deficient MEFs had an increased capacity to digest fibrin in situ. Consistently, tenascin‐C expression in vivo was found to correlate with fibrin deposition in several diseases associated with tenascin‐C overexpression such as fibrosis, asthma and cancer. In conclusion, the present study suggests a new role of tenascin‐C as a regulator of the fibrinolytic system.

Erratum: Noncoding RNAs prevent spreading of a repressive histone mark

It was brought to our attention that the DNA sequence that was annotated as SPNCRNA.95 in PomBase (http://www.pombase.org/) is the same as a genomic element referred to as IRC1-R in Cam et al., Nat. Genet. 37, 809–819, 2005. Therefore, the BoRDeRlINe-encoding sequence and the IRC1-R element do partially overlap (Fig. 1). Correct annotations and coordinates have been updated in PomBase accordingly. We note that, when Swi6 is overexpressed, the IRC1 DNA sequence is required to stop heterochromatin spreading on the left side of centromere 1 (IRC1-l; Noma et al., Cell 125, 859–872, 2006), but it remains to be investigated whether the same applies to the cen1-R boundary when IRC1-R is deleted and Swi6 overexpressed. Because heterochromatin spreading in our paper was assessed under normal Swi6 expression levels, we conclude that noncoding RNA (ncRNA)–mediated boundary activity is sufficient to stop the spread of heterochromatin under physiological conditions, although overlapping or complementary mechanisms may exist.

Dynamic expression of chromatin modifiers during developmental transitions in mouse preimplantation embryos.

During mouse preimplantation development, major changes in cell fate are accompanied by extensive alterations of gene expression programs. Embryos first transition from a maternal to zygotic program and subsequently specify the pluripotent and the trophectodermal cell lineages. These processes are regulated by key transcription factors, likely in cooperation with chromatin modifiers that control histone and DNA methylation. To characterize the spatiotemporal expression of chromatin modifiers in relation to developmental transitions, we performed gene expression profiling of 156 genes in individual oocytes and single blastomeres of developing mouse embryos until the blastocyst stage. More than half of the chromatin modifiers displayed either maternal or zygotic expression. We also detected lineage-specific expression of several modifiers, including Ezh1, Prdm14, Scmh1 and Tet1 underscoring possible roles in cell fate decisions. Members of the SET-domain containing SMYD family showed differential gene expression during preimplantation development. We further observed co-expression of genes with opposing biochemical activities, such as histone methyltransferases and demethylases, suggesting the existence of a dynamic chromatin steady-state during preimplantation development.

Protein demethylation required for DNA methylation.

DNA methylation is an epigenetic mark directing stable, heritable gene silencing through development. A new study uncovers the importance of demethylation of the DNA methyltransferase-1 for maintenance of DNA methylation.

SYK inhibition blocks proliferation and migration of glioma cells and modifies the tumor microenvironment

Background Glioblastoma (GBM) is one of the most aggressive human brain tumors, with a median survival of 15-18 months. There is a desperate need to find novel therapeutic targets. Various receptor protein kinases have been identified as potential targets; however, response rates in clinical studies have been somewhat disappointing. Targeting the spleen tyrosine kinase (SYK), which acts downstream of a range of oncogenic receptors, may therefore show more promising results. Methods Kinase expression of brain tumor samples including GBM and low-grade tumors were compared with normal brain and normal human astrocytes by microarray analysis. Furthermore, SYK, LYN, SLP76, and PLCG2 protein expressions were analyzed by immunohistochemistry, western blot, and immunofluorescence of additional GBM patient samples, murine glioma samples, and cell lines. SYK was then blocked chemically and genetically in vitro and in vivo in 2 different mouse models. Multiphoton intravital imaging and multicolor flow cytometry were performed in a syngeneic immunocompetent C57BL/6J mouse GL261 glioma model to study the effect of these inhibitors on the tumor microenvironment. Results SYK, LYN, SLP76, and PLCG2 were found expressed in human and murine glioma samples and cell lines. SYK inhibition blocked proliferation, migration, and colony formation. Flow cytometric and multiphoton imaging imply that targeting SYK in vivo attenuated GBM tumor growth and invasiveness and reduced B and CD11b+ cell mobility and infiltration. Conclusions Our data suggest that gliomas express a SYK signaling network important in glioma progression, inhibition of which results in reduced invasion with slower tumor progression.

Community-Driven Data Analysis Training for Biology

The primary problem with the explosion of biomedical datasets is not the data, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences. This project is accessible at https://training.galaxyproject.org.