MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFβ. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity.

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Sister-chromatid disjunction in anaphase requires the resolution of DNA catenanes by topoisomerase II together with Plk1-interacting checkpoint helicase (PICH) and Bloom's helicase (BLM). We here identify Rif1 as a factor involved in the resolution of DNA catenanes that are visible as ultrafine DNA bridges (UFBs) in anaphase to which PICH and BLM localize. Rif1, which during interphase functions downstream of 53BP1 in DNA repair, is recruited to UFBs in a PICH-dependent fashion, but independently of 53BP1 or BLM. Similar to PICH and BLM, Rif1 promotes the resolution of UFBs: its depletion increases the frequency of nucleoplasmic bridges and RPA70-positive UFBs in late anaphase. Moreover, in the absence of Rif1, PICH, or BLM, more nuclear bodies with damaged DNA arise in ensuing G1 cells, when chromosome decatenation is impaired. Our data reveal a thus far unrecognized function for Rif1 in the resolution of UFBs during anaphase to protect genomic integrity.

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore–microtubule (KT–MT) attachments and for the stabilization of bi-oriented KT–MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation.

To understand how the chromosomal passenger complex ensures chromosomal stability, it is crucial to identify its substrates and to find ways to specifically inhibit the enzymatic core of the complex, Aurora B. We therefore developed a chemical genetic approach to selectively inhibit human Aurora B. By mutating the gatekeeper residue Leu-154 in the kinase active site, the ATP-binding pocket was enlarged, but kinase function was severely disrupted. A unique second site suppressor mutation was identified that rescued kinase activity in the Leu-154 mutant and allowed the accommodation of bulky N 6 -substituted adenine analogs. Using this analog-sensitive Aurora B kinase, we found that retention of the chromosomal passenger complex at the centromere depends on Aurora B kinase activity. Furthermore, analog-sensitive Aurora B was able to use bulky ATP␥S analogs and could thiophosphorylate multiple proteins in cell extracts. Utilizing an unbiased approach for kinase substrate mapping, we identified several novel substrates of Aurora B, including the nucleosomal-binding protein HMGN2. We confirmed that HMGN2 is a bona fide Aurora B substrate in vivo and show that its dynamic association to chromatin is controlled by Aurora B. Molecular & Cellular