Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program

The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA–microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis.Leukemia & Lymphoma Society of America (Scholar Award)National Institute of General Medical Sciences (U.S.) (Grant GM088313)American Cancer Society (Research Scholar Grant 121776)National Science Foundation (U.S.). Graduate Research Fellowshi

Mechanisms for the propagation and recognition of human centromeres

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.Each time a cell divides, the genome must be segregated equally between the two new daughter cells. To accomplish this, a specific region of each chromosome, termed the centromere, recruits the macromolecular kinetochore structure to mediate attachments to spindle microtubules. In vertebrates, each chromosome must establish a single site of microtubule attachment. The failure to maintain this site or the generation of multiple distinct microtubule attachment sites on a single chromosome can have profoundly deleterious effects on cell and organismal viability. My graduate work has used cell biological analyses in tissue culture cells and biochemical reconstitutions to define the molecular mechanisms by which human cells maintain one and only one site of microtubule attachment on each chromosome. First, I defined the regulatory paradigms that ensure the faithful propagation of the centromere. I identified Polo-like kinase 1 as a key player in controlling the deposition of the epigenetic mark that specifies the centromere, the CENP-A nucleosome. I defined the molecular basis for this control, as well as an additional level of control downstream of the cyclin-dependent kinases. By identifying and dissecting the molecular features of this two-step regulatory paradigm, I developed a strategy to bypass the control of CENP-A deposition, which resulted in severe mitotic defects. In my second project, I defined the architecture and properties of the sixteen-protein assembly that connects CENP-A to the other proteins of the kinetochore. I analyzed the genetic relationships between these proteins in human cells through a combination of inducible knockouts and inducible protein degradation. I then reconstituted the sixteen proteins in vitro as five sub-complexes and defined their interactions biochemically. These analyses revealed an intricate meshwork of direct interactions between the proteins at the centromere-kinetochore interface, which is critical for ensuring assembly of the kinetochore at the correct site on the chromosome. Together, these findings provide new insights into the molecular mechanisms of centromere propagation and kinetochore assembly.by Kara Lavidge McKinley.Ph. D

Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects

Defining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, we generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. We performed single-cell microscopic analyses to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, we generated knockouts across cell lines of diverse origins. We demonstrate that p53 modulates the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression. Keywords: kinetochore; centromere; mitosis; DNA replication; spindle; microtubule; multipolarity; p53; CRISPR/Cas

Polo-like Kinase 1 Licenses CENP-A Deposition at Centromeres

To ensure the stable transmission of the genome during vertebrate cell division, the mitotic spindle must attach to a single locus on each chromosome, termed the centromere. The fundamental requirement for faithful centromere inheritance is the controlled deposition of the centromere-specifying histone, CENP-A. However, the regulatory mechanisms that ensure the precise control of CENP-A deposition have proven elusive. Here, we identify polo-like kinase 1 (Plk1) as a centromere-localized regulator required to initiate CENP-A deposition in human cells. We demonstrate that faithful CENP-A deposition requires integrated signals from Plk1 and cyclin-dependent kinase (CDK), with Plk1 promoting the localization of the key CENP-A deposition factor, the Mis18 complex, and CDK inhibiting Mis18 complex assembly. By bypassing these regulated steps, we uncoupled CENP-A deposition from cell-cycle progression, resulting in mitotic defects. Thus, CENP-A deposition is controlled by a two-step regulatory paradigm comprised of Plk1 and CDK that is crucial for genomic integrity.Leukemia & Lymphoma Society of America (Scholar Award)National Institute of General Medical Sciences (U.S.) (Grant GM088313)American Cancer Society (Research Scholar Grant 121776

The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface

During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.United States. National Institutes of Health (GM088313)United States. National Institutes of Health (GM108718)American Cancer Society (121776

Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4–6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7–12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a “wait anaphase” signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments. Rodriguez-Rodriguez et al. identify distinct roles for Bub1, KNL1, and RZZ in SAC signaling and fibrous corona formation. They also show that BUB1-disrupted clones re-express Bub1 and regain SAC function via nonsense-associated alternative splicing, an often-overlooked transcriptional response that can limit penetrance in genome editing experiments.National Institutes of Health (Grant R01GM094972)National Institutes of Health (Grant P30CA008748)National Institutes of Health (Grant R01GM059363)National Institutes of Health (Grant R35GM126930

Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the 'arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA. Keywords: centromeres; supramolecular assembl