Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2–MND1

The HOP2–MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes and promotes the repair of DNA double-strand breaks. We investigated the conformational flexibility of HOP2–MND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL–MS). The final XL–MS workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC–MS/MS detection prior to data evaluation. We applied two different homobifunctional amine-reactive cross-linkers (DSS and BS²G) and one zero-length heterobifunctional cross-linker (EDC). Cross-linked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein–protein docking for cross-link-guided molecular modeling. Miniaturization of the size-exclusion enrichment step reduced the required starting material, led to a high amount of cross-linked peptides, and allowed the analysis of replicates. The major interaction site of HOP2–MND1 was identified in the central coiled-coil domains, and an open colinear parallel arrangement of HOP2 and MND1 within the complex was predicted. Moreover, flexibility of the C-terminal capping helices of both complex partners was observed, suggesting the coexistence of a closed complex conformation in solution

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression

Live-cell imaging RNAi screen identifies PP2A-B55 alpha and importin-beta 1 as key mitotic exit regulators in human cells

Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 12 (2010): 886-893, doi:10.1038/ncb2092.When vertebrate cells exit mitosis, they reorganize various cellular structures to build functional interphase cells1. This depends on Cdk1 inactivation and subsequent dephosphorylation of its substrates2-4. Members of PP1 and PP2A phosphatase families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5, 6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we used a live imaging assay to screen by RNAi a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identified a trimeric PP2A-B55α complex as a key factor for postmitotic reassembly of the nuclear envelope, the Golgi apparatus, and decondensed chromatin, as well as for mitotic spindle breakdown. Using a chemically-induced mitotic exit assay, we found that PP2A-B55α functions downstream of Cdk1 inactivation. PP2A-B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate histone H1 and it was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor Importin β1, and RNAi depletion of Importin β1 delayed mitotic exit synergistically with PP2A-B55α. This demonstrates that PP2A-B55α and Importin β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.This work was supported by SNF research grant 3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young Investigator (EURYI) award of the European Science Foundation to DWG, and a MBL Summer Research Fellowship by the Evelyn and Melvin Spiegel Fund to DWG, a Roche Ph.D. fellowship to MHAS, and a Mueller fellowship of the Molecular Life Sciences Ph.D. program Zurich to MH. MH and MHAS are fellows of the Zurich Ph.D. Program in Molecular Life Sciences. VJ and JG were supported by grants of the ‘Geconcerteerde OnderzoeksActies’ of the Flemish government, the ‘Interuniversitary Attraction Poles’ of the Belgian Science Policy P6/28 and the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’. AIL is a Wellcome Trust Principal Research Fellow. AAH acknowledges funding by the Max Planck Society, the EU-FP6 integrated project MitoCheck, and the BMBF grant DiGtoP [01GS0859]. Work in the groups of KM and JMP was supported by the EU-FP6 integrated project MitoCheck, Boehringer Ingelheim and by the GEN-AU programme of the Austrian Federal Ministry of Science and Research (Austrian Proteomics Platform III), by MeioSys within the Seventh Framework Programme of the European Commission, and by Chromosome Dynamics, which is funded by the Austrian Science Foundation (FWF)