FRET analysis of the hypoxia-inducible-factor (HIF) interacting with tissue specific transcription factors during erythropoietin (EPO) gene expression

Die Expression des Gens für Erythropoietin (EPO) wird über die gewebespezifische Zusammensetzung des Transkriptionsfaktorkomplexes HIF (hypoxia-inducible factor) reguliert. In der Arbeit wurde untersucht welche Rolle HIF-1α, HIF-2α, HNF-4α (hepatocyte nuclear factor-4alpha) und RXRα (retinoic X receptor alpha) bei der EPO-Expression in den unterschiedlichen Zelltypen spielt. Aus der Arbeit geht hervor, dass für eine hypoxische Induktion des Erythropietin (EPO)-Gens ein aktiver HIF-Komplex vorhanden sein muss. Die β-Untereinheit (ARNT, aryl hydrocarbon receptor nuclear translocator) alleine ist nicht transkriptionell aktiv am EPO-Gen. Durch siHIF-1α- und siHIF-2α-Experimente zeigte sich, dass sowohl in den Neuroblastomzellen der Linie SY5Y als auch in den Nierenzellen der Linie HK120 die EPO-mRNA stärker durch HIF-2α als HIF-1α kontrolliert wird. Des Weiteren fanden sich in beiden Zelltypen Hinweise darauf, dass HIF-2α ein Zielgen von HIF-1α ist. Ein aktiver HIF-Komplex allein ist jedoch nicht ausreichend für eine hypoxische Aktivierung des EPO-Gens. Innerhalb des hypoxia-responsive element (HRE) im 3´ Enhancer des EPO-Gens befinden sich außer der HIF binding site (HBS) auch ein DR-2 (direct repeat of two hexanucleotides separated by two base pairs)-Element, an das spezifisch nuclear hormone receptors (NHRs) binden können. Es hat sich gezeigt, dass diese NHRs meist gewebespezifisch vorliegen. Die Neuroblastomzellen der Linie SY5Y scheint unter anderem der RXRα mit all-trans-retinoic-acid (at-RA) als stimulierendem Liganden für die EPO-mRNA Induktion zu rekrutieren. Noch ungeklärt ist, ob RXRα als Homodimer oder als Heterodimer mit unbekanntem Partner bindet. Für die Nierenzellen der Linie HK120 konnte HNF-4α, das in der Regel als Homodimer bindet, als genregulierender NHR identifiziert werden. Obwohl die HK120-Zellen RXRα enthalten, konnte durch at-RA keine Stimulation der EPO-mRNA-Expression erreicht werden. Im Gegensatz zu den Neuroblastom-Zellen zeigte sich in den HK120-Zellen sogar, dass durch at-RA die RXRα-mRNA und HNF-4α-mRNA und in Folge die EPO-mRNA reduziert wurden. Weiterhin konnte gezeigt werden, dass HNF-4α ein Zielgen von HIF-2α und HIF-1α ist, da die HNF-4α-mRNA unter Hypoxie vermindert war. Dieser Effekt ließ sich durch den Einsatz von siHIF-2α und HIF-1α wieder aufheben. Die Transfektion von Kelly Zellen mit HNF-4α verminderte die Hypoxie-induzierte EPO-Expression. Mit Hilfe der FRET-Technik konnte bewiesen werden, dass HIF-1α einen Komplex mit ARNT bildet, der jedoch einen kürzeren Abstand zueinander aufweist als für HIF-2α mit ARNT. Dabei musste HIF-2α im Gegensatz zu HIF-1α für diese Komplexbildung an die DNA gebunden sein, was sich mit Hilfe von Deletionsmutanten der DNA-bindenden Domäne von HIF-2α und HIF-1α nachweisen ließ. Eine direkte Interaktion von HNF-4α mit den einzelnen HIF-Untereinheiten (HIF-2α,-1α und ARNT) konnte nicht festgestellt werden, auch nicht, wenn ein aktiver endogener HIF-Komplex zwischen HIF-1α und ARNT bestand. Eine Interaktion muss somit über ein Adaptorprotein wie CBP zustande kommen, wobei die Interaktion des gesamten CBP mit HNF-4α offen bleibt, weil nur zwei Domänen von CBP, die CBP-CH1- und die CBP-CH3-Domäne, für die Messungen zur Verfügung standen. Es stellte sich heraus, dass nur die CH3-Domäne direkt mit HNF-4α interagieren kann. Verwendet man eine Mutante von HNF-4α (HNF-4α-C106R), die nicht an DNA binden kann, war ein engerer Komplex zur CH3-Domäne möglich. Zusammenfassend lässt sich festhalten, dass das EPO-Gen durch den HIF-Komplex mit HNF-4α über das CBP-Adaptorprotein in Nierenzellen hypoxisch induziert werden kann. Gewebespezifisch bedingt kann RXRα statt HNF-4α in neuronalen Zellen agieren. Eine Interaktion von RXRα mit CBP müsste mittels FRET noch untersucht werden

Rev Proteins of Human and Simian Immunodeficiency Virus Enhance RNA Encapsidation

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5′ end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA

USP4 Auto-Deubiquitylation Promotes Homologous Recombination.

Repair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.Research in the S.P.J. laboratory is funded by CRUK Program Grant C6/A11224, CRUK Project Grant C6/A14831 and the European Community Seventh Framework Program grant agreement no. HEALTH-F2-2010-259893 (DDResponse). R.N. was funded by the Daiichi Sankyo Foundation of Life Sciences fellowship. Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096 provided core infrastructure funding. S.P.J receives his salary from the University of Cambridge, supplemented by CRUK. The John Fell Fund 133/075 and the Wellcome Trust grant 097813/Z/11/Z funded research performed by B.M.K and R.K..This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.molcel.2015.09.01

The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage.

Non-homologous end-joining (NHEJ), which can promote genomic instability when dysfunctional, is a major DNA double-strand break (DSB) repair pathway. Although ubiquitylation of the core NHEJ factor, Ku (Ku70-Ku80), which senses broken DNA ends, is important for its removal from sites of damage upon completion of NHEJ, the mechanism regulating Ku ubiquitylation remains elusive. We provide evidence showing that the ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) interacts with and directly deubiquitylates one of the Ku heterodimer subunits, Ku80. Additionally, depleting UCHL3 resulted in reduced Ku80 foci formation, Ku80 binding to chromatin after DSB induction, moderately sensitized cells to ionizing radiation and decreased NHEJ efficiencies. Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity. Collectively, these data suggest that UCHL3 facilitates cellular viability after DSB induction by antagonizing Ku80 ubiquitylation to enhance Ku80 retention at sites of damage.This work was funded by Grant-in-Aid for Research Activity start-up 15H06738 (R.N.), Grant-in Aid for Young Scientists (A) 16H05888 (R.N.), Daiichi Sankyo Foundation of Life Science (R.N.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (R.N.), Cancer Research UK (CRUK) Grant C6/A11224 and C6/A18796 (P.W.), CRUK Project Grant C6/A14831 (R.N.). T.L.B. and Q.W. are funded by Wellcome Trust Investigator Award (200814_Z_16_Z). Research in the B.M.K. laboratory is supported by a John Fell Fund 133/075, the Wellcome Trust (097813/Z/11/Z) and the Engineering and Physical Sciences Research Council (EP/N034295/1). Research in the S.P.J. laboratory is funded by CRUK Program Grant C6/A18796, and Wellcome Trust Grant WT206388/Z/17/Z. Cancer Research UK Grant C6946/A24843 and Wellcome Trust Grant WT203144 provided core infrastructure funding

p97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation

Funding Information: This work was funded by Cancer Research UK (CRUK) program grant C5255/A23755 to A.E.K. Medical Research Council UK (MRC) program grant MC_PC 12001/1 (MC_UU_00001/1) and Breast Cancer Now (Grant No. 2019DecPR1406) to K.R. S.K. was supported by the MRC Oxford Institute of Radiation Oncology (OIRO) CRUK studentship. We thank Dr. Sovan Sarkar (Department of Oncology, University of Oxford) for generously providing DR-GFP U2OS cells. We thank Diogo Dias (Ludwig Cancer Research Institute, University of Oxford) for his technical advice on HR and SSA assays and assistance with the analysis. We thank Dr. Lisa Folkes and Alix Hampson for the high-performance liquid chromatography (HPLC) analysis of CB-5083 concentration in tissue extracts from CD-1 nude mice bearing subcutaneous RT112 tumors. We also thank the Oxford Radcliffe Biobank for providing us with human tissue sections.Peer reviewedPublisher PD

Male reproductive aging arises via multifaceted mating-dependent sperm and seminal proteome declines, but is postponable in Drosophila

I.S. and S.W. were supported by a Biotechnology and Biological Sciences Research Council (BBSRC) Fellowship to S.W. (BB/K014544/1) and S.W. additionally by a Dresden Senior Fellowship. B.M.K., P.D.C., and R.F. were supported by the Kennedy Trust and John Fell Funds. R.D. was supported by Marie Curie Actions (Grant 655392). B.R.H. was funded by the EP Abraham Cephalosporin-Oxford Graduate Scholarship with additional support from the BBSRC Doctoral Training Programme. M.F.W. was supported by a NIH Grant R01HD038921. Work in the J.S. Laboratory was supported by NIH Grant R15HD080511.Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, be-tween reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending inter-ventions could ameliorate the declining performance of the ejacu-late as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration con-tributes to male reproductive decline via mating-dependent mech-anisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproduc-tive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling re-sults in improved late-life ejaculate performance, indicating simul-taneous amelioration of both somatic and reproductive aging.Publisher PDFPeer reviewe

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.Fil: Markolovic, Suzana. University of Oxford; Reino UnidoFil: Zhuang, Qinqin. University Of Birmingham; Reino UnidoFil: Wilkins, Sarah E.. University of Oxford; Reino UnidoFil: Eaton, Charlotte D.. University Of Birmingham; Reino UnidoFil: Abboud, Martine I.. University of Oxford; Reino UnidoFil: Katz, Maximiliano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: McNeil, Helen E.. University Of Birmingham; Reino UnidoFil: Leśniak, Robert K.. University of Oxford; Reino UnidoFil: Hall, Charlotte. University Of Birmingham; Reino UnidoFil: Struwe, Weston B.. University of Oxford; Reino UnidoFil: Konietzny, Rebecca. University of Oxford; Reino UnidoFil: Davis, Simon. University of Oxford; Reino UnidoFil: Yang, Ming. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Ge, Wei. University of Oxford; Reino UnidoFil: Benesch, Justin L. P.. University of Oxford; Reino UnidoFil: Kessler, Benedikt M.. University of Oxford; Reino UnidoFil: Ratcliffe, Peter J.. University of Oxford; Reino Unido. The Francis Crick Institute; Reino UnidoFil: Cockman, Matthew E.. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Fischer, Roman. University of Oxford; Reino UnidoFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Chowdhury, Rasheduzzaman. University of Stanford; Estados Unidos. University of Oxford; Reino UnidoFil: Coleman, Mathew L.. University Of Birmingham; Reino UnidoFil: Schofield, Christopher J.. University of Oxford; Reino Unid