Oliver Pusch

A high-throughput screen to identify enhancers of ADAR-mediated RNA-editing

Adenosine to inosine deamination of RNA is widespread in metazoa. Inosines are recognized as guanosines and, therefore, this RNA-editing can influence the coding potential, localization and stability of RNAs. Therefore, RNA editing contributes to the diversification of the transcriptome in a flexible manner. The editing reaction is performed by adenosine deaminases that act on RNA (ADARs), which are essential for normal life and development in many organisms. Changes in editing levels are observed during development but also in neurological pathologies like schizophrenia, depression or tumors. Frequently, changes in editing levels are not reflected by changes in ADAR levels suggesting a regulation of enzyme activity. Until now, only a few factors are known that influence the activity of ADARs. Here we present a two-stage in vivo editing screen aimed to isolate enhancers of editing. A primary, high-throughput yeast-screen is combined with a more accurate secondary screen in mammalian cells that uses a fluorescent read-out to detect minor differences in RNA-editing. The screen was successfully employed to identify DSS1/SHFM1, the RNA binding protein hnRNP A2/B1 and a 3′ UTR as enhancers of editing. By varying intracellular DSS1/SHFM1 levels, we can modulate A to I editing by up to 30%. Proteomic analysis indicates an interaction of DSS1/SHFM1 and hnRNP A2/B1 suggesting that both factors may act by altering the cellular RNP landscape. An extension of this screen to cDNAs from different tissues or developmental stages may prove useful for the identification of additional enhancers of RNA-editing.

A single allele of Hdac2 but not Hdac1 is sufficient for normal mouse brain development in the absence of its paralog

The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells. Ablation of Hdac1 or Hdac2 by Nestin-Cre had no obvious consequences on brain development and architecture owing to compensation by the paralog. By contrast, combined deletion of Hdac1 and Hdac2 resulted in impaired chromatin structure, DNA damage, apoptosis and embryonic lethality. To dissect the individual roles of HDAC1 and HDAC2, we expressed single alleles of either Hdac1 or Hdac2 in the absence of the respective paralog in neural cells. The DNA-damage phenotype observed in double knockout brains was prevented by expression of a single allele of either Hdac1 or Hdac2. Strikingly, Hdac1-/-Hdac2+/- brains showed normal development and no obvious phenotype, whereas Hdac1+/-Hdac2-/- mice displayed impaired brain development and perinatal lethality. Hdac1+/-Hdac2-/- neural precursor cells showed reduced proliferation and premature differentiation mediated by overexpression of protein kinase C, delta, which is a direct target of HDAC2. Importantly, chemical inhibition or knockdown of protein kinase C delta was sufficient to rescue the phenotype of neural progenitor cells in vitro. Our data indicate that HDAC1 and HDAC2 have a common function in maintaining proper chromatin structures and show that HDAC2 has a unique role by controlling the fate of neural progenitors during normal brain development.

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti‐cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild‐type and HDAC1‐deficient embryonic stem cells. HDAC1‐deficient teratomas showed no significant difference in size compared with wild‐type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E‐cadherin localization and displayed up‐regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1‐deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress

The selectivity of transcriptional responses to extracellular cues is reflected by the deposition of stimulus-specific chromatin marks. Although histone H3 phosphorylation is a target of numerous signaling pathways, its role in transcriptional regulation remains poorly understood. Here, for the first time, we report a genome-wide analysis of H3S28 phosphorylation in a mammalian system in the context of stress signaling. We found that this mark targets as many as 50% of all stress-induced genes, underlining its importance in signal-induced transcription. By combining ChIP-seq, RNA-seq, and mass spectrometry we identified the factors involved in the biological interpretation of this histone modification. We found that MSK1/2-mediated phosphorylation of H3S28 at stress-responsive promoters contributes to the dissociation of HDAC corepressor complexes and thereby to enhanced local histone acetylation and subsequent transcriptional activation of stress-induced genes. Our data reveal a novel function of the H3S28ph mark in the activation of mammalian genes in response to MAP kinase pathway activation.

Expression of class I histone deacetylases during chick and mouse development

Histone deacetylases (HDACs) are a family of enzymes which regulate the acetylation state of nucleosomal histones, as well as non-histone proteins. By altering local chromatin architecture, HDACs play important roles in shaping cell differentiation and morphogenesis. Expression of class I HDACs during early chick development has so far not been analyzed. Here, we report the expression profile of chick class I HDACs from the onset of gastrulation (HH2) to day 4 of development and compare it to relevant stages during mouse development. Visualized by in situ hybridization to whole mount embryos and tissue sections, we found tissue-specific overlapping temporal and spatial expression domains for all four class I HDACs in chick and mouse, although species-specific differences could be identified. All class I HDACs in both species are highly expressed in the developing brain. In particular, HDAC1 is expressed at sites of anterior and posterior neural tube closure most obvious in the hot spot-like expression of HDAC1 in HH12 chicken embryos. A significant species-specific spatio-temporal expression pattern was observed for HDAC8. Whereas HDAC8 is exclusively found in fore- and midbrain regions during early mouse embryogenesis, the chick ortholog shows an expanded expression pattern, suggesting a more diversified role of HDAC8 in the chick system. Our results present a basis for further functional analysis of class I HDACs in chick development.

Histone deacetylase inhibitor Trichostatin A induces neural tube defects and promotes neural crest specification in the chicken neural tube.

Epigenetic mechanisms serve as key regulatory elements during vertebrate embryogenesis. Histone acetylation levels, controlled by the opposing action of histone acetyl transferases (HATs) and histone deacetylases (HDACs), influence the accessibility of DNA to transcription factors and thereby dynamically regulate transcriptional programs. HDACs execute important functions in the control of proliferation, differentiation, and the establishment of cell identities during embryonic development. To investigate the global role of the HDAC family during neural tube development, we employed Trichostatin A (TSA) to locally block enzymatic HDAC activity in chick embryos in ovo. We found that TSA treatment induces neural tube defects at the level of the posterior neuropore, ranging from slight undulations to a complete failure of neural tube closure. This phenotype is accompanied by morphological changes in neuroepithelial cells and induction of apoptosis. As a molecular consequence of HDAC inhibition, we observed a timely deregulated cadherin switching in the dorsal neural tube, illustrated by induction of Cadherin 6B as well as reciprocal downregulation of N-Cadherin expression. Concomitantly, several neural crest specific markers, including Bmp4, Pax3, Sox9 and Sox10 are induced, causing a premature loss of epithelial characteristics. Our findings provide evidence that HDAC function is crucial to control the regulatory circuits operating during trunk neural crest development and neural tube closure.

Pore Exposed Tyrosine Residues of P-glycoprotein are Important Hydrogen Bonding Partners for Drugs

The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity

BackgroundShort interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels.ResultsHere, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels.ConclusionsOur study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.

Mathematical modelling reveals how MeCP2 restrains transcriptional elongation in human neurons

Gene expression patterns depend on the interaction of diverse transcription factors with their target genes. While many factors have a restricted number of targets, some appear to affect transcription globally. An example of the latter is MeCP2; an abundant chromatin-associated protein that is mutated in the neurological disorder Rett Syndrome. To understand how MeCP2 affects transcription, we integrated mathematical modelling with quantitative experimental analysis of human neurons expressing graded levels of MeCP2. We first used a model of MeCP2-DNA binding to demonstrate that changes in gene expression reflect MeCP2 density downstream of transcription initiation. We then tested five biologically plausible hypotheses for the effect of MeCP2 on transcription. The only model compatible with the data involved slowing down of RNA polymerase II by MeCP2, causing reduced transcript output due to polymerase queueing. Our general approach may prove fruitful in deciphering the mechanisms by which other global regulators choreograph gene expression.

PO-182 Examining the function of PDGFRB in anaplastic large cell lymphoma

Introduction Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin T-cell lymphoma (NHL) most commonly diagnosed in children and young adults. A majority of ALCL tumours harbour the translocation t(2;5(p23;q35), resulting in the fusion of Nucleophosmin (NPM) to the Anaplastic lymphoma kinase (ALK) gene. The oncogenic fusion protein (NPM-ALK) is constitutively expressed and consequently contributes to the pathogenesis and progression of about 70% of ALCL cases. Recent studies from our lab identified AP-1 transcription factors JUNB and cJUN as downstream effectors of NPM-ALK, which directly up-regulate platelet derived growth factor receptor beta (PDGFRB) expression in lymphoma cells. We demonstrated that besides increased receptor expression its ligand, PDGFB levels are also elevated in both mouse ALCL tumours and human ALCL patient plasma. Furthermore, therapeutic inhibition of PDGFRB with the kinase inhibitor imatinib resulted in rapid and sustained remission in late-stage therapy-resistant ALCL patients. Material and methods Despite these discoveries, the underlying mechanisms and the nature of PDGFRB signalling in lymphoma formation and progression still remain to be resolved. To elucidate the mechanisms of PDGFRB signalling in ALCL, we have crossed a murine ALCL model, which expresses NPM-ALK under the CD4 promoter, to PDGFRB floxed mice and a CD4 promoter driven Cre recombinase, to yield a specific deletion of PDGFRB in T cells (CD4-NPM-ALK-CD4ΔPDGFRB). Results and discussions Intriguingly, CD4-NPM-ALK-CD4ΔPDGFRB mice have significantly prolonged survival rates and reduced tumour growth, due to increased apoptosis. CD4-NPM-ALK-CD4ΔPDGFRB tumours exhibit a decrease in lymphatic vessels, which is accompanied by a decrease in tumour dissemination. Moreover, in vivo deletion of PDGFRB results in a decrease in the signal transducer and activator of transcription (STAT) and NPM-ALK signalling pathways dampening tumour aggressiveness. Manipulation of PDGFRB levels in vitro, via CRISPR/Cas9 mediated knockout or overexpression, in primary tumour cell lines isolated from CD4-NPM-ALK-CD4 and CD4-NPM-ALK-CD4ΔPDGFRB mirrors the changes in signalling pathway observed in the tumours in vivo. Conclusion In summary, loss of PDGFRB leads to decreased tumour development and dissemination in a CD4-NPM-ALK mouse model via modulation of the STAT-signalling pathway. Our data therefore supports the oncogenic role of PDGFRB and opens new avenues for therapeutic intervention in ALCL treatment.