A cytoskeletal function for PBRM1 reading methylated microtubules

Epigenetic effectors “read” marks “written” on chromatin to regulate function and fidelity of the genome. Here, we show that this coordinated read-write activity of the epigenetic machinery extends to the cytoskeleton, with PBRM1 in the PBAF chromatin remodeling complex reading microtubule methyl marks written by the SETD2 histone methyltransferase. PBRM1 binds SETD2 methyl marks via BAH domains, recruiting PBAF components to the mitotic spindle. This read-write activity was required for normal mitosis: Loss of SETD2 methylation or pathogenic BAH domain mutations disrupt PBRM1 microtubule binding and PBAF recruitment and cause genomic instability. These data reveal PBRM1 functions beyond chromatin remodeling with domains that allow it to integrate chromatin and cytoskeletal activity via its acetyl-binding BD and methyl-binding BAH domains, respectively. Conserved coordinated activity of the epigenetic machinery on the cytoskeleton opens a previously unknown window into how chromatin remodeler defects can drive disease via both epigenetic and cytoskeletal dysfunction.

Spindle assembly checkpoint satisfaction occurs through end-on but not lateral kinetochore-microtubule interactions under tension

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. What signals the SAC monitors remains unclear. We do not know the contributions of different microtubule attachment features, or tension from biorientation, to SAC satisfaction in normal mitosis - or how these possible cues change during attachment. Here, we quantify concurrent Mad1 intensity, reporting on SAC silencing, and real-time attachment geometry, occupancy, and tension at individual mammalian kinetochores. We show that Mad1 loss from the kinetochore occurs in switch-like events with robust kinetics, and that metaphase-like tension across sister kinetochores is established just before Mad1 loss events at the first sister. We demonstrate that CenpE-mediated lateral attachment of the second sister can persistently generate this metaphase-like tension prior to biorientation, likely stabilizing sister end-on attachment, yet cannot induce Mad1 loss from that kinetochore. Instead, Mad1 loss begins after several end-on microtubules attach. Thus, end-on attachment provides geometry-specific molecular cues, or force on specific kinetochore linkages, that other attachment geometries cannot provide.SummaryThe spindle assembly checkpoint (SAC) delays anaphase until kinetochores are properly attached to the spindle. The authors demonstrate that the SAC monitors geometry-specific molecular cues, or force on specific kinetochore linkages, that “end-on” but not “lateral” attachments generating persistent tension can provide.

The tumor suppressor CDKN3 controls mitosis

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2pThr-161 at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.

Cytokinesis Failure in Fanconi Anemia Pathway Deficient Murine Hematopoietic Stem Cells.

Abstract 495 Fanconi Anemia (FA) is a rare recessive chromosomal-instability disorder characterized by congenital malformations, a high predisposition to cancer, and progressive bone marrow failure. FA is genetically heterogeneous and, to date, thirteen FA genes have been identified (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N). The thirteen encoded FA proteins cooperate in a common DNA repair pathway active during the Synthesis (S) phase of the cell cycle. DNA damage detected during replication results in the monoubiquitination of two FA proteins, FANCD2 and FANCI, that translocate into chromatin-associated DNA repair foci where they colocalize with downstream components of the pathway. Partial colocalization with BLM, the RecQ helicase mutated in Bloom's syndrome, has also been described. How disruption of this pathway leads to bone marrow failure is a critical unanswered question. Interestingly, FA cells also have abnormalities that suggest a defect in mitosis, including micronuclei and multinucleation. The objectives of this study were to 1) investigate the role of the FA pathway in normal mitosis and 2) determine whether defects in this function underlie the bone marrow failure of FA patients. For this study, we used HeLa cells transiently or stably knocked down for FA genes, FA patient derived cell lines and hematopoietic stem cells from Fanconi mice models generated in our laboratory (Fancd2-/- and Fancg-/-). First, a polyclonal antibody was raised against FANCI and, together with an anti-FANCD2 antibody, used to investigate the localization of the FANCD2-I complex throughout the cell cycle by immunostaining. FANCI and FANCD2 colocalized to discrete foci on condensed chromosomes in a population of cells in Mitosis (M) phase, consistent with results of Chan et al. (Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol. 2009;11:753-760), Naim and Rosselli (The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat Cell Biol. 2009;11:761-768). These foci were dependent on an intact FA pathway, but did not localize at centromeres and did not increase when the spindle assembly checkpoint was challenged. By immunofluorescence, we showed an increase in the presence of Hoechst positive DNA bridges and PICH positive / BLM positive DNA bridges (Hoechst positive and negative) in anaphase and telophase of FA deficient cells compared to FA proficient cells. This increase of DNA bridges between separating sister chromatids in FA deficient cells correlated with an increase of multinucleated cells. Multinuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Furthermore, inhibition of apoptosis increased the number of binucleated cells, suggesting that cytokinesis failure led to apoptosis. Importantly, an increase in binucleated cells was also observed in the hematopoietic stem cells population from Fancd2-/- and Fancg-/- mice, compared to wild-type sibling mice, and this increase correlated with elevated apoptosis in those cells. Based on these new findings, we conclude that the Fanconi pathway is required for normal mitosis and hypothesize that apoptosis induced by cytokinesis failure of hematopoietic stem cells may cause the bone marrow failure commonly found in FA patients. Disclosures: No relevant conflicts of interest to declare.

Relative contributions of chromatin and kinetochores to mitotic spindle assembly

During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly.