Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo‐oligomerization of DnaA via its N‐terminus (amino acid residues 1–86) is also essential for initiation. Results from solid‐phase protein‐binding assays indicate that residues 1–86 (or 1–77) of DnaA are necessary and sufficient for self interaction. Using a ‘one‐hybrid‐system’ we found that the DnaA N‐terminus can functionally replace the dimerization domain of coliphage lambda cI repressor: a λcI‐DnaA chimeric protein inhibits λ plasmid replication as efficiently as λcI repressor. DnaA derivatives with deletions in the N‐terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N‐terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N‐termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross‐talk with the nucleotide‐binding domain III or the DNA‐binding domain IV. We propose that E. coli DnaA is composed of largely independent domains — or modules — each contributing a partial, though essential, function to the proper functioning of the ‘holoprotein’.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by bandshift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory DnaA box.

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The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

Abstract. We registered 170 relapses in 1392 children with nephroblastoma in the SIOP/GPOH trials. The study aimed to evaluate prognostic factors for outcome in relapsed patients. Age, gender, initial stage, metastatic disease, local stage, histology, time to relapse and tumour volume were analysed for their prognostic relevance. Overall survival after relapse was 48% (median follow-up 5 years). Relapses were local in 28%, metastatic in 57% and combined in 15%. The median age of the cohort was 4.5 years whereas patients in complete continuous remission were significantly younger (3.1 years, p=0.001). Patients with initial stage I and II showed a significantly better prognosis than children with stage III (57 vs. 31%, p=0.008). Patients with high-risk tumours had a much poorer prognosis than those with intermediate and lowrisk tumours (58 vs. 31%, p=0.003). Children with recurrence within 6 months after diagnosis had a poorer outcome than children relapsing later on (54 vs. 22%, p=0.0001). The tumour volume initially and after preoperative chemotherapy did not have any influence on outcome. Patients with isolated distant metastasis had a significantly better outcome than those with local and combined relapses (p=0.001). In conclusion, factors for poor prognosis after relapse are early relapse, local stage III, high-risk histology and combined relapse.