SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase.

Optimization of STR locus enrichment for STR profiling of fragmented DNA.

DNA degradation is a major obstacle in gaining an accurate profile with standard DNA typing technology. Although alternative genotyping strategies such as mini-STRs and SNPs have proven to be more successful in profiling degraded DNA, these approaches also have limitations. Here, we show that locus enrichment by hybridization of degraded genomic DNA with an STR locus-specific biotinylated oligonucleotide is a powerful approach to overcome problems in STR typing of highly degraded DNA. An experimental investigation of factors affecting the efficiency of this method indicates that the choice of primer and molar ratio of primers to genomic DNA are critical factors in improving enrichment of the STR locus before genotyping with multiplex kits. In addition, we find that indirect capture rather than direct capture with magnetic beads yields better enrichment efficiency for STR locus enrichments. Using these strategies, we demonstrate an improvement in STR typing of DNA from cultured cells damaged by exposure to sunlight or UV. We suggest that this approach could be applied to highly degraded forensic samples alone or in combination with mini-STRs.

Two ß-strands of RAGE participate in the recognition and transport of amyloid-ß peptide across the blood brain barrier.

Amyloid-ß (Aß) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aß peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two ß-strands of RAGE participate in the interaction with Aß peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth ß-strands are required for interaction with Aß peptide. Site-directed mutagenesis of amino acids located in the third and eighth ß-strands abolish the interaction of RAGE with Aß peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aß peptide treatment, while a RAGE mutant defective in Aß binding does not. Furthermore, use of peptide for the third ß-strand or a RAGE monoclonal antibody that targets the RAGE-Aß interaction interface inhibited transport of the Aß peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.

WIP1, a homeostatic regulator of the DNA damage response, is targeted by HIPK2 for phosphorylation and degradation.

WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR.

Mdm2 associates with Ras effector NORE1 to induce the degradation of oncoprotein HIPK1.

The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2-NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.

WDR11, a WD protein that interacts with transcription factor EMX1, is mutated in idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

Covalent conjugation of Groucho with SUMO-1 modulates its corepressor activity.

Groucho is a corepressor that forms a macromolecular complex for its corepressor activity, in which HDAC1 is an essential component for the modulation of chromatin structure and transcriptional repression of target genes. Here, we show that Groucho is covalently conjugated with small ubiquitin-related modifier-1 (SUMO-1) in vitro and in vivo. SUMO conjugations of Groucho occur at four different lysine residues. Substitutions of all these residues abolished sumoylation of Groucho and inhibited its corepressor activity. In addition, Groucho corepressor activity was reduced by inhibition of SUMO-1 conjugation via Ubc9 knockdown through expression of short-hairpin RNA against Ubc9. Furthermore, interactions between Groucho and HDAC1 are enhanced by sumoylation of Groucho, which is mediated by the SUMO-interaction motif of HDAC1. Taken together, these findings indicate that Groucho sumoylation increases its corepressor activity by enhancing the recruitment of HDAC1 to Groucho corepressor complex.

Ubiquitination and degradation of homeodomain-interacting protein kinase 2 by WD40 repeat/SOCS box protein WSB-1.

Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53- and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses.