Concurrent Sessions

Concurrent Sessions RNA:DNA hybrids as modulators of chromatin structure and genome instability A. Aguilera Sevilla, Spain Abstract not available.

The architecture of Tetrahymena telomerase holoenzyme J. Feigon Los Angeles, United States Abstract not available.

P02-003-SH Signal regulated localisation of a mutagenic protein complex at the Igh locus J. Hauser, C. Grundstr€ om, R. Kumar, T. Grundstr€om Ume a University, Molecular Biology, Ume a, Sweden Our system to produce antibodies is critical for our survival against numerous infections, but it causes also many tumors. Blymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates these processes by deaminating cytosine to uracil. How somatic hypermutation (SH) and class switch recombination (CSR) are targeted is key to understanding the defect DNA integrity in lymphomas and also in other tumors where inflammatory signals aberrantly induces AID. The trans-acting factors mediating specific targeting of AID and thereby SH and CSR have remained elusive. Here we show that mutant E2A with defect inhibition by the Ca2+-sensor protein calmodulin results in reduced B cell receptor- (BCR-), IL4plus CD40 ligand-stimulated CSR to IgE and instead aberrant CSR. AID is shown to be together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus in activated mouse splenic B cells. Calmodulin shows proximity with each of them after BCR stimulation. Direct proteinprotein interactions enable formation of the complexes. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks reduction of binding to these target sites and increases binding to other target sites. Thus, E2A, AID, PAX5 and IRF4 are components of a CSR and SH complex that is redistributed on the IgH gene by BCR signaling through calmodulin binding.

P02-004-SH The sequence requirements for base J in DNA P. Borst1, H. G. A. M. Van Luenen2, J. Korlach3, L. Baugh4, P. Myler4 1 Div. of Mol. Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands, 2Netherlands Cancer Institute, Amsterdam, the Netherlands, 3Pacific Biosciences, Menlo Park, United States, 4 Seattle Biomedical Research Institute, Seattle, United States Base J (b-glucosyl-hydroxymethyluracil) is a DNA-base present in kinetoplastid flagellates and in Euglena. It replaces 1% of thymine in nuclear DNA and it acts as an indispensable transcription termination signal for RNA polymerase II in Leishmania (Van Luenen et al., Cell 2012; 150:909). The initial step of J biosynthesis, the hydroxylation of selected T-residues in DNA, yields hydroxymethyluracil and is catalyzed by two enzymes, J-binding Protein (JPB) 1 (which also binds to J-DNA) or JBP2 (which does not bind to J-DNA). To determine the DNA sequences rec-

FEBS Journal 282 (Suppl. 1) (2015) 6–52 ª 2015 The Authors. FEBS Journal ª 2015 FEBS

Sunday 5 July ognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. Only cloned DNA segments naturally containing J are able to pick up J in plasmids. J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and de novo hydroxylation of T is likely catalysed by JBP2, since it does not occur in JBP2-null cells. Glucosylation of the resultant HOMedU forms J that can be bound by JBP1, which hydroxylates another T-residue 13 bp downstream (but not upstream) on the complementary strand (Genest et al., NAR., 2015, 43:2102). This explains why in the telomeric sequence (GGGTTA)n only the second T is replaced by J as the only T on the complementary strand (CCCAAT)n is then exactly 13 nucleotides downstream. We are using mutagenesis to precisely define the sequences required for de novo J insertion and its spreading into neighboring sequences.

Sunday 5 July 15:00–17:00, ECC Room 2 Molecular Clocks (Part II) Transcriptional regulatory logic of the diurnal cycle F. Naef Lausanne, Switzerland Abstract not available.

Transcriptional refractoriness is dependent on core promoter architecture M. Brunner Heidelberg, Germany Abstract not available.

P27-003-SH Feedback loops of the mammalian circadian clock constitute repressilator P. Pett1, A. Korencic2, H. Herzel1,3 1 Institute for Theoretical Biology, Berlin, Germany, 2Institute of Biochemistry, Ljubljana, Slovenia, 3Charite - Universit€ atsmedizin, Berlin, Germany An autonomous circadian clock controls daily rhythms in physiology and behaviour. Circadian rhythms are generated by intracellular transcriptional feedback loops involving cis-regulatory elements such as E-boxes, D-boxes, and ROR-elements. Clock genes assemble a complex gene regulatory network with multiple negative and positive feedback loops. Most studies consider the E-box mediated negative feedback via Period and Cryptochrome as the major driver of circadian rhythms. More recent studies suggest that another negative feedback loop with the nuclear receptors Rev-Erba and Rev-Erbb is not merely an auxiliary loop, but capable of generating self-sustained oscillations. Indeed, double-knockouts of Rev-Erb genes destroy rhythmicity. In order to explore the role of core clock genes and their interactions we analyze a recently published gene regulatory network model. This network includes Bmal1 as a driver of E-box mediated transcription, Per2 and Cry1 as early and late E-box targets, the D-box regulator Dbp and the nuclear receptor Rev-Erba. The model is based on experimentally verified regulatory interactions, degradation rates and post-transcriptional delays. The unknown parame-

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