Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex.

Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

The global campaign to eliminate HBV and HCV infection: International Viral Hepatitis Elimination Meeting and core indicators for development towards the 2030 elimination goals.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) affect more than 320 million people worldwide, which is more than HIV, tuberculosis (TB) and malaria combined. Elimination of HBV and HCV will, therefore, produce substantial public health and economic benefits and, most importantly, the prevention of 1.2 million deaths per year. In 2016, member states of the World Health Assembly unanimously adopted a resolution declaring that viral hepatitis should be eliminated by 2030. Currently, few countries have elimination programmes in place and even though the tools to achieve elimination are available, the right resources, commitments and allocations are lacking. During the fifth International Viral Hepatitis Elimination Meeting (IVHEM), 7-8 December 2018, Amsterdam, the Netherlands, an expert panel of clinicians, virologists and public health specialists discussed the current status of viral hepatitis elimination programmes across multiple countries, challenges in achieving elimination and the core indicators for monitoring progress, approaches that have failed and successful elimination plans.

Phylogenomics-guided discovery of a novel conserved cassette of short linear motifs in BubR1 essential for the spindle checkpoint.

The spindle assembly checkpoint (SAC) maintains genomic integrity by preventing progression of mitotic cell division until all chromosomes are stably attached to spindle microtubules. The SAC critically relies on the paralogues Bub1 and BubR1/Mad3, which integrate kinetochore-spindle attachment status with generation of the anaphase inhibitory complex MCC. We previously reported on the widespread occurrences of independent gene duplications of an ancestral 'MadBub' gene in eukaryotic evolution and the striking parallel subfunctionalization that lead to loss of kinase function in BubR1/Mad3-like paralogues. Here, we present an elaborate subfunctionalization analysis of the Bub1/BubR1 gene family and perform de novo sequence discovery in a comparative phylogenomics framework to trace the distribution of ancestral sequence features to extant paralogues throughout the eukaryotic tree of life. We show that known ancestral sequence features are consistently retained in the same functional paralogue: GLEBS/CMI/CDII/kinase in the Bub1-like and KEN1/KEN2/D-Box in the BubR1/Mad3-like. The recently described ABBA motif can be found in either or both paralogues. We however discovered two additional ABBA motifs that flank KEN2. This cassette of ABBA1-KEN2-ABBA2 forms a strictly conserved module in all ancestral and BubR1/Mad3-like proteins, suggestive of a specific and crucial SAC function. Indeed, deletion of the ABBA motifs in human BUBR1 abrogates the SAC and affects APC/C-Cdc20 interactions. Our detailed comparative genomics analyses thus enabled discovery of a conserved cassette of motifs essential for the SAC and shows how this approach can be used to uncover hitherto unrecognized functional protein features.

The E3 ligase CUL3/RDX controls centromere maintenance by ubiquitylating and stabilizing CENP-A in a CAL1-dependent manner.

Centromeres are defined by the presence of the histone H3 variant CENP-A in a subset of centromeric nucleosomes. CENP-A deposition to centromeres depends on a specialized loading factor from yeast to humans that is called CAL1 in Drosophila. Here, we show that CAL1 directly interacts with RDX, an adaptor for CUL3-mediated ubiquitylation. However, CAL1 is not a substrate of the CUL3/RDX ligase but functions as an additional substrate-specifying factor for the CUL3/RDX-mediated ubiquitylation of CENP-A. Remarkably, ubiquitylation of CENP-A by CUL3/RDX does not trigger its degradation but stabilizes CENP-A and CAL1. Loss of RDX leads to a rapid degradation of CAL1 and CENP-A and to massive chromosome segregation defects during development. Essentially, we identified a proteolysis-independent role of ubiquitin conjugation in centromere regulation that is essential for the maintenance of the centromere-defining protein CENP-A and its loading factor CAL1.