Single molecule characterization of the roles of long non-coding RNAs in eukaryotic transcription regulation

Récemment, des analyses dans divers organismes eucaryotes ont révélé que l'ensemble du génome est transcrit et produit en plus des ARNs messagers, une grande variété d’ARNs non codants de différentes longueurs. Les ARNs non codants de plus de 200 nucleotides, classés comme longs ARNs non codants (LARNnc), représentent la classe la plus abondante de transcripts non codants. Les études des fonctions des LARNnc suggèrent que beaucoup d'entre eux seraient impliqués dans la régulation de la transcription. L'objectif de ma thèse de doctorat était d'élucider les mécanismes de la régulation transcriptionnelle médiée par des LARNnc dans différents systèmes eucaryotes. Dans mon premier projet, j'ai étudié le rôle d'un long ARN non codant antisens dans la régulation transcriptionnelle du gène PHO84, codant un transporteur de phosphate à haute affinité, chez S. cerevisiae. Des études antérieures ont montré que la suppression d’une proteine de l’exosome Rrp6 entraîne une augmentation de l'expression antisens et la répression de PHO84. Il a été suggéré que la perte de Rrp6 entraîne une stabilisation antisens au locus PHO84, entraînant le recrutement de l'histone de-acétylase Hda1 et la répression de PHO84. Cependant, le mécanisme par lequel Rrp6p régule la transcription de PHO84 n’était pas connu. En combinant des méthodes à l’échelle de cellule unique, des approches biochimiques et génétiques, nous avons montré que les niveaux d'ARN antisens sont régulés principalement lors de l'élongation par le complexe Nrd1-Nab3-Sen1, qui nécessite Rrp6 pour un recrutement efficace à l`extrémité 3`de PHO84. De plus, nous révélons l'expression anticorrelé du sens et de l'antisens, En résumé, nos données suggèrent que la transcription antisens régule le seuil d'activation du promoteur PHO84. Dans mon second projet, j'ai étudié les rôles des ARNs dérivés des amplificateurs (ARNa) dans la regulation de la transcription. En utilisant les cellules de cancer du sein MCF7 comme système modèle, nous avons cherché à déterminer comment les ARNa induits par l'oestrogène (E2) participent à la régulation de la transcription médiée par le recepteur d’oestrogène (ERα) au niveau de l'allèle unique. À l'aide de l’hybridation fluorescente à l’échelle de molécule unique (smFISH), nous avons révélé qu`après induction d'E2, les ARNa sont induits avec une cinétique similaire à celle des ARNm cibles, sont localisés exclusivement dans le noyau, principalement associés à la chromatine, et sont moins abondants que les ARNm. De manière surprenante, nous avons constaté que les ARNa sont rarement co-transcrits avec leurs loci cibles, indiquant que la transcription active des gènes ne nécessite pas la synthèse continue ou l'accumulation d'ARNa sur l'amplificateur. En outre, en utilisant des mesures de la distance à sous-diffraction, nous avons démontré que la cotranscription des ARNa et des ARNm se produit rarement dans une boucle amplificateurpromoteur. De plus, nous avons révélé que la transcription basale d'ARNa n'exige pas ERα ou l'histone méthyltransférase MLL1 qui active l'amplificateur par la mono-méthylation H3K4. Dans l'ensemble, nos résultats ont montré que les ARNa peuvent jouer un rôle lors de l'activation du promoteur, mais ne sont pas nécessaires pour maintenir la transcription de l'ARNm ou pour stabiliser les interactions amplificateur-promoteur.Transcription is the initial step in gene expression and is subject to extensive regulation. Recently, analyses in diverse eukaryotes have revealed that in addition to protein coding genes, transcription occurs throughout the noncoding genome, producing non-coding RNAs of various lengths. Non-coding RNAs longer than 200 nucleotides, classified as long non-coding RNAs (lncRNAs), represent the most abundant class of non-coding transcripts, whose functions however are poorly understood. Recent studies suggest that many lncRNAs might have roles in transcription regulation. The goal of my PhD thesis was to elucidate the mechanisms of lncRNA mediated transcription regulation in different eukaryotic systems. For my first project, I investigated the role of an antisense long noncoding RNA in transcription regulation of the high-affinity phosphate transporter gene PHO84 in the unicellular eukaryote S. cerevisiae. Previous studies showed that deletion of the nuclear exosome component Rrp6 results in increased antisense expression and repression of PHO84. It was suggested that the loss of Rrp6 results in antisense stabilization at the PHO84 locus, leading to recruitment of the histone de-acetylase Hda1 and repression of PHO84. However, most of the mechanistic details of how Rrp6p functions in regulating PHO84 transcription were not understood. Combining single cell methods with biochemical and genetic approaches, we showed that antisense RNA levels are regulated primarily during transcriptional elongation by the Nrd1-Nab3-Sen1 complex, which requires Rrp6 for efficient recruitment to the 3’end of PHO84. Furthermore, we reveal anti-correlated expression of sense and antisense, which have distinct modes of transcription. In summary, our data suggest a model whereby antisense transcriptional read-through into the PHO84 promoter regulates the activation threshold of the gene. For my second project, I investigated the roles of enhancer derived RNAs (eRNAs). eRNAs are lncRNAs transcribed from enhancers that have been suggested to regulate transcription through different mechanisms, including enhancer-promoter looping, RNA polymerase elongation, and chromatin remodeling. However, no coherent model of eRNA function has yet emerged. Using MCF7 breast cancer cells as a model system, we sought to determine how estrogen (E2) induced eRNAs participate in estrogen receptor alpha (ERα) mediated transcription regulation at the single allele level. Using single molecule fluorescent in situ hybridization (smFISH), we revealed that upon E2 induction eRNAs are induced with similar kinetics as target mRNAs, but are localized exclusively in the nucleus, mostly chromatin associated, and are less abundant than mRNAs. Surprisingly, we found that eRNAs are rarely co-transcribed with their target loci, indicating that active gene transcription does not require the continuous synthesis or accumulation of eRNAs at the enhancer. Furthermore, using sub-diffraction-limit distance measurements, we demonstrated that co-transcription of eRNAs and mRNAs rarely occurs within a closed enhancer-promoter loop. Moreover, we revealed that basal eRNA transcription does not require ERα or the histone methyltransferase MLL1, which activates the enhancer through H3K4 mono-methylation. Altogether, our findings showed that eRNAs may play a role during promoter activation, but are not required to sustain mRNA transcription or stabilize enhancer-promoter looping interactions

Chromatin regulation by the histone acetyltransferase MOF in Drosphila

Like in mammals, sex determination in Drosophila melanogaster involves an unequal distribution of sex chromosomes, with male flies carrying an X and a Y chromosome, as compared to two Xs in females. To prevent the deleterious effects of chromosomal aneuploidy, flies have evolved a dosage compensation system, which upregulates transcription from the single male X chromosome to match transcript levels produced from the two female Xs. This transcriptional activation is achieved by the dosage compensation complex (DCC), a ribonucleoprotein complex consisting of five male specific lethal proteins (MSL) and two non coding RNAs on the X (roX). The DCC is physically tethered to hundreds of target loci along the male X chromosome, where it promotes hyper acetylation of X-linked chromatin at Lysine 16 of histone H4 (H4K16ac). This histone mark is associated with an open, permissive chromatin structure, and its enrichment on the male X chromosome is thought to be required for the twofold increase in X-linked transcription during dosage compensation. However, the exact mechanism by which X-linked transcription is activated in males is still unknown. Responsible for hyper-acetylation of the male X chromosome is the histone acetyltransferase males absent on the first (MOF), which is part of the DCC. Recent studies have shown that MOF plays an additional role in autosomal gene regulation, as it has been found at thousands of autosomal gene promoters as part of the non specific lethal (NSL) complex. However, to what extent H4K16ac at autosomal genes is MOF-dependent, and how MOF is differentially distributed between the two complexes is currently unknown. During the course of my PhD, I used genetic, biochemical, and genomewide approaches to address a wide range of questions, concerning MOF functions in autosomal gene regulation and dosage compensation; the DCC recruitment process to X-linked target genes; and the mechanism of transcriptional upregulation of X-linked genes during dosage compensation. Besides other contributions, investigating the role of the H3K36 specific methyltransferase HypB/Set2 during MSL targeting and dosage compensation, as well as the role of MOF for NSL function at autosomal promoters, I was addressing these questions in the context of two main projects. During the first one of these, I have been able to show that MOF is responsible for genomewide H4K16ac in male and female flies, and that MOF is an essential gene in females. I demonstrated that the Drosophila specific unstructured N-terminus of the MOF protein is required for assembly of the DCC on the male X chromosome, and at the same time constrains MOFs HAT activity. The N-terminus therefore controls MOFs function in X chromosome compensation. I was furthermore able to reveal the biological role of the chromobarrel domain, which is conserved from yeast to human. Unexpectedly, disruption of the MOF chromobarrel domain, which has been shown previously to be required for MOF interaction with roX RNAs, led to a dramatic loss of H4K16ac from all chromosomes. Accordingly, I showed that the chromobarrel domain serves to trigger H4K16ac after the recruitment of MOF to its chromatin targets, revealing for the first time a biological role of this domain in vivo. In a parallel project, to work towards unraveling of the dosage compensation mechanism, I wanted to identify the step in the RNA PolII transcription cycle at which dosage compensation operates in flies. To this end, I generated genomewide profiles of RNA PolII in 3rd instar larva salivary glands from male and female flies, and from male flies with disrupted dosage compensation. Strikingly, we find that the density of PolII is approximately twofold elevated on the male X chromosome as compared to autosomes, including X-linked promoters. This data suggests that dosage compensation operates via enhanced transcription initiation, which constitutes a major advance in our understanding of the dosage compensation process

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

Functionally Relevant Macromolecular Interactions of Disordered Proteins

Disordered proteins are relatively recent newcomers in protein science. They were first described in detail by Wright and Dyson, in their J. Mol. Biol. paper in 1999. First, it was generally thought for more than a decade that disordered proteins or disordered parts of proteins have different amino acid compositions than folded proteins, and various prediction methods were developed based on this principle. These methods were suitable for distinguishing between the disordered (unstructured) and structured proteins known at that time. In addition, they could predict the site where a folded protein binds to the disordered part of a protein, shaping the latter into a well-defined 3D structure. Recently, however, evidence has emerged for a new type of disordered protein family whose members can undergo coupled folding and binding without the involvement of any folded proteins. Instead, they interact with each other, stabilizing their structure via “mutual synergistic folding” and, surprisingly, they exhibit the same residue composition as the folded protein. Increasingly more examples have been found where disordered proteins interact with non-protein macromolecules, adding to the already large variety of protein–protein interactions. There is also a very new phenomenon when proteins are involved in phase separation, which can represent a weak but functionally important macromolecular interaction. These phenomena are presented and discussed in the chapters of this book

The evolutionary genomics of CTCF binding and functional signatures in mouse.

Genetic differences within and between species predominantly lie in the noncoding sequence of the regulatory regions of the genome whose function and significance largely remain poorly understood. Despite significant progress in the field of genomics and the rapid progress in sequencing methods and the subsequent explosion of genomic data, our understanding of the role of the non- coding genetic sequence in the regulation of tissue- and species-specific gene expression is still lagging behind, limiting our comprehension of the evolutionary mechanisms and pressures that shape those expression profiles, and their involvement in the health and disease. The CTCF protein demarcates mammalian genomes into discrete transcriptionally active domains, providing the platform for complex spatial and temporal regulatory processing of genetic information that govern biological processes. In this thesis, I investigate the dynamics and functional implications of evolutionarily novel CTCF binding sites in two Mus genus mouse subspecies, Mus musculus domesticus and Mus musculus castaneus, separated by a short evolutionary time of only one million years. The project investigated the subspecies-specific binding of CTCF in terms of the repeat content, evolution, functional impact and involvement in chromatin conformation. The key findings of this investigation are: (1) the incorporation of young CTCF sites into the non-coding genome via action of transposable elements is followed rapidly with the exhibition of various characteristics of biological function; (2) Unlike other tissue-specific transcription factors, allele- specific CTCF occupancy is affected by cis- and trans-acting regulatory mechanisms that exhibit similar functional characteristics; (3) CTCF evolutionary dynamics support both maintenance of pre-existing structures and functions and provide template for novel ones. In summary, this thesis discusses the evolutionary dynamics of CTCF genomic occupancy and functional signatures in short evolutionary time, and illustrates how either novel species-specific CTCF sites, or common sites with newly-acquired genotypic variants integrate into existing genomic architecture and begin to exert their effects

Mechanism of valproic acid induced dysmorphogenesis via oxidative stress and epigenetic regulation at the Hoxa2 gene promoter

Valproic acid (2-propylpentanoic acid, VPA) is a clinically used anti-epileptic drug and an effective mood stabilizer. VPA is also a histone deacetylase inhibitor and can induce embryonic malformations in both humans and mice. The mechanism(s) of VPA-induced teratogenicity are not well characterized. The objectives of my study were three fold, to: (i) investigate the effect of VPA on mouse embryonic development, (ii) characterize the putative mechanism(s) of VPA-induced teratogenicity and, (iii) investigate VPA associated epigenetic regulation of Hoxa2 gene in cell lines and in developing embryos. Whole mouse embryo cultures were treated with VPA at doses of 0, 50 (0.35 mM), 100 (0.70 mM), 200 (1.4 mM), and 400 µg/mL (2.8 mM), encompassing the therapeutic range of 0.35 mM to 0.70 mM. Van Maele-Fabry’s morphologic scoring system was used to quantitatively assess embryonic organ differentiation and development. Hoxa2 gene expression was measured by quantitative real-time RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). To assess epigenetic changes on the Hoxa2 gene promoter, DNA methylation was determined by bisulfite (BSP) sequencing and pyrosequencing. Histone “bivalent domains” H3K4me3 (histone 3 lysine 4 trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation) associated with gene activation repression, respectively, analyzed qChIP-PCR (quantitative chromatin immunoprecipitation-PCR). Telomere length and telomerase activity were analyzed in mouse embryos and in NIH3T3 cell line treated with VPA. Results indicate significantly increased incidence of dysmorphogenesis in embryos (11.8%, 35.3%, 47.0% and 88.3%) exposed to increasing doses of VPA (0.35 mM, 0.70 mM, 1.4 mM and 2.8 mM respectively). Van Maele-Fabry’s quantitative differentiation assessment of developing embryos demonstrated a significantly lower score for the circulation system, central nervous system, craniofacial development and limb development in VPA treated embryos (0.35 mM to 2.8 mM) compared to the untreated control group. Glutathione homeostasis was altered as indicated by decreased total glutathione content and increased GSSG/GSH ratio in all VPA treatment groups. In addition, a dose-dependent inhibition of Hoxa2 gene expression was observed in embryos and in the NIH3T3 cell line exposed to VPA. Pre-treatment with ascorbic acid [1000 µg/mL (5 mM)] restored glutathione level and normalized Hoxa2 gene expression in embryos exposed to VPA. DNA methylation status was characterized on the Hoxa2 gene promoter at the three CpG islands; CpG island 1 (-277 to -620 bp), CpG island 2 (-919 to -1133 bp), and CpG island 3 (-1176 to -1301 bp) in the two cells lines (NIH3T3 and EG7) and in developing embryos. CpG sites remained unmethylated on the Hoxa2 gene promoter in the NIH3T3 cell line which expresses the Hoxa2 gene, whereas these same CpG sites were methylated in EG7 cells that did not express Hoxa2. CpG island 1 is closest to Hoxa2 transcription start site and its methylation status was most affected. In developing embryos, CpG island 1 was found to be highly methylated at E6.5 when Hoxa2 is not expressed, whereas the methylation status of CpG sites on the CpG island 1 declined between E8.5 and E10.5 when Hoxa2 expression is present. VPA induced methylation of several CpG sites on CpG island 1 in NIH3T3 cell line and in E10.5 embryos when Hoxa2 expression was down regulated following VPA exposure. In addition, embryos and the NIH3T3 cell line treated with VPA impacted the “bivalent domains” resulting in increased H3K27me3 enrichment and decreased H3K4me3 enrichment on Hoxa2 promoter. Pre-treatment with ascorbic acid normalized Hoxa2 expression and histone bivalent domain changes and prevented increased DNA methylation following VPA exposure. Moreover, the telomerase activity and telomere length were both impacted by changes in glutathione redox potential induced by VPA. Oxidative stress following VPA treatment reduced telomerase activity and accelerated telomere shortening. These results are the first to demonstrate: (i) a correlation between VPA dose and total morphologic score in the developing mouse embryos. VPA impacted embryonic tissue differentiation and neural system development in the dose range of 0.35 mM to 2.8 mM; (ii) VPA altered glutathione homeostasis in cultured mouse embryos and inhibited Hoxa2 gene expression; (iii) Histone bivalent domains of H3K27 and H3K4 trimethylation and DNA methylation status at the Hoxa2 gene promoter region were altered following treatment with VPA. This appears to be the epigenetic event in transcriptional silencing of Hoxa2 gene expression after VPA exposure; and (iv) Ascorbic acid normalizes glutathione homeostasis, H3K27 and H3K4 trimethylation and DNA methylation status, restoring Hoxa2 gene expression following VPA exposure. Taken together our results show VPA- induced altered glutathione homeostasis, telomere shortening and telomerase dysfunction, and an inhibition of Hoxa2 gene expression leads to developmental abnormalities. Exposure to ascorbic acid had a protective effect on developing embryos exposed to VPA

Epigenetic regulation of enhancer activity in the mammalian genome

Cell types are defined by their spatiotemporal gene expression patterns and their differential activity of promoters and enhancers. Enhancers are cis-regulatory elements in the DNA critical for the acquisition and maintenance of cellular identities by regulating the expression of key genes. Enhancers serve as landing pads for transcription factors (TFs) which are DNA-binding proteins that interpret the genomic code and enhance gene expression upon their binding. However, the underlying DNA sequence does not solely convey binding specificity, and therefore it is still largely elusive what additional factors regulate TF binding. An important regulatory layer in gene expression are dynamic and reversible epigenetic modifications of chromatin including DNA and histone proteins. To date, dozens of histone modifications have been identified that are associated with different genomic contexts and transcriptional states. For instance, histone H3 lysine acetylation has been generally associated with active chromatin as active enhancers and promoters, while histone H3 tri-methylation at lysine 23 (H3K27me3) is coupled to transcription repression. Yet, the causal contribution of such histone modifications to the regulation of enhancer activity and TF binding is still large unknown. To address this question, I developed a technical approach to analyse TF binding at DNA molecules where a certain histone modification of interest is present. For this, I combined a genomic enrichment technique with a single molecule footprinting (SMF) approach that allows to detect TF binding at single DNA molecule resolution. However, this experimental set-up paired with different optimization approaches did not produce high enough enrichments of DNA molecules harboring certain histone modifications to suffice the required statistical power. Therefore, the focus was laid on investigating the causal role of DNA methylation. DNA methylation in CpG context is the most common epigenetic modification in the mammalian genome that covers 70-80% of all CpG dinucleotides. Despite its prevalence, DNA methylation can be highly dynamic, especially at enhancer elements that exhibit reduced methylation levels during their activation. Previous studies have identified that the binding of TFs to enhancers is correlated with the partial loss in DNA methylation and it has been suggested that DNA methylation regulates enhancer activity. This hypothesis has remained elusive up to date, which has multiple reasons. First, the relationship between TFs and DNA methylation is bidirectional. Previous studies have identified many methyl-sensitive TFs in vitro whose binding is reduced upon methylation of their DNA binding motif. Some of those have been confirmed by in vivo studies, which showed that DNA methylation prevents the spurious binding of those TFs in the genome. Opposingly, TFs have also been identified to be directly responsible for the demethylation of enhancers. In consequence, the bidirectional regulation between DNA methylation and TF binding has prevented the establishment of a causal relationship between them. Second, the cell-to-cell epigenetic variability observed as intermediate methylation at enhancers elements makes common bulk-cell genomics approaches ineffective to identify a direct correlation between DNA methylation and TF binding and to determine whether DNA methylation generally contributes to the regulation of enhancer activity. In the here presented PhD project, I overcame these issues and limitation by advancing the single molecule footprinting (SMF) approach to resolve chromatin accessibility, TF binding, and simultaneously quantify the presence of DNA methylation on the same DNA molecules. By applying this technology across the murine genome, I demonstrate that TFs can bind most (>90%) enhancers irrespective of the underlying DNA methylation, suggesting that presence of DNA methylation does not generally impede enhancer activity. Yet, for stem cells and three somatic cell types, I identified active enhancers where TF occupancy is directly repressed by DNA methylation, including enhancers involved in the control of key cell identity genes. Using global perturbation assays and orthogonal enhancer activity measurements, I was able to show that at these active sites, DNA methylation directly controls the occupancy levels of TFs such as Max-Myc, that play a key role in the control of stem cell identity and proliferation. In the end, my data suggest a model where the function of DNA methylation extends beyond protecting the genome from spurious TF binding, by directly regulating the activation of cell-type specific enhancers. This detailed analysis is an important addition to our general knowledge on gene regulation and suggest that while epigenetic factors may have largely redundant functions, their individual contributions can play important and instructive roles in tuning the quantitative expression of key cell- specific genes. Understanding the regulation of such genes involved in cell identity will have important implications in the comprehension of development and disease

Spatial And Temporal Control Of The Kinesin Motors Kif11 And Kif22 In Mitosis

Mitosis requires the control of mechanical forces generated by kinesin motors to ensure equal segregation of chromosomes into two daughter cells. Two kinesins contributing to these forces are KIF11 (or Eg5), which crosslinks and slides anti-parallel microtubules, and KIF22 (or Kid), which binds to microtubules and chromosome arms. This work addresses how post-translational modifications and pathogenic mutations alter the structure and function of KIF11 and KIF22. The homotetrameric kinesin KIF11 is subject to acetylation at lysine 146, a residue in the a2 helix of the motor domain. The effect of this acetylation was assessed at the single molecule and cellular levels using an acetylation mimetic mutant, KIF11 K146Q. In single molecule optical trapping assays, KIF11 K146Q dimers are more likely than wild type (WT) dimers to stall rather than dissociate from the microtubule under load. Based on these results, acetylated KIF11 motors would be predicted to stall microtubule sliding during spindle formation, acting as a brake and slowing pole separation. To test this prediction, mCherry (mCh) tagged KIF11 WT and K146Q motors were expressed at low levels in HeLa cells. To compare the functional activity of WT and K146Q KIF11, cells were treated with the KIF11 inhibitor monastrol, resulting in mitotic arrest and the formation of monopolar spindles. Bipolar spindle formation following monastrol washout was then imaged and measured. While spindle lengths at the completion of pole separation were similar in cells expressing mCh-KIF11 WT or K146Q, pole separation occurred at a significantly slower velocity in cells expressing mCh-KIF11 K146Q than in cells expressing mCh-KIF11 WT. This velocity difference is consistent with KIF11 acetylated at K146 stalling rather than dissociating from the microtubule and acting as a brake during pole separation. Acetylation at a2 helix lysine 146 represents a mechanism by which the activity of KIF11 may be controlled in mitotic cells. Structural changes in the a2 helix also affect the activity of KIF22. Point mutations at P148 and R149 in this domain, as well as at V475 in the tail of the motor, dominantly cause a skeletal developmental disorder. The effect of these pathogenic mutations on the function of KIF22 in mitosis was investigated. KIF22 uses plus end-directed motility and direct binding to chromosome arms to generate polar ejection forces, which contribute to chromosome congression and alignment in metaphase. Mutant KIF22 generated forces to move chromosomes toward microtubule plus ends in prometaphase, indicating that mutant motors are active. As cells proceeded through mitosis, however, mutations disrupted anaphase chromosome segregation and caused chromosome recongression, which resulted in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase, resulting in continued generation of polar ejection forces and impaired anaphase chromosome segregation. Consistent with this model, a phosphomimetic mutation in the tail of KIF22 which constitutively activates the motor phenocopied the effect of pathogenic mutations. Mimicking the phosphorylation of a2 helix residue T158 also prevented the inactivation of KIF22 in anaphase, demonstrating the importance of this region of the motor domain in controlling KIF22 activity