Chromosome compaction is triggered by an autonomous DNA-binding module within condensin.

The compaction of chromatin into mitotic chromosomes is essential for faithful transmission of the genome during cell division. In eukaryotes, chromosome morphogenesis is regulated by the condensin complex, though the exact mechanism used to target condensin to chromatin and initiate condensation is not understood. Here, we reveal that condensin contains an intrinsically disordered region (IDR) that modulates its association with chromatin in early mitosis and exhibits phase separation. We describe DNA-binding motifs within the IDR that, upon deletion, inflict striking defects in chromosome condensation and segregation, ill-timed condensin turnover on chromatin, and cell death. Importantly, we demonstrate that the condensin IDR can impart cell cycle regulatory functions when transferred to other subunits within the complex, indicating its autonomous nature. Collectively, our study unveils the molecular basis for the initiation of chromosome condensation in early mitosis and how this process ultimately promotes genomic stability and faultless cell division.

The SMC5/6 complex: folding chromosomes back into shape when genomes take a break.

High-level folding of chromatin is a key determinant of the shape and functional state of chromosomes. During cell division, structural maintenance of chromosome (SMC) complexes such as condensin and cohesin ensure large-scale folding of chromatin into visible chromosomes. In contrast, the SMC5/6 complex plays more local and context-specific roles in the structural organization of interphase chromosomes with important implications for health and disease. Recent advances in single-molecule biophysics and cryo-electron microscopy revealed key insights into the architecture of the SMC5/6 complex and how interactions connecting the complex to chromatin components give rise to its unique repertoire of interphase functions. In this review, we provide an integrative view of the features that differentiates the SMC5/6 complex from other SMC enzymes and how these enable dramatic reorganization of DNA folding in space during DNA repair reactions and other genome transactions. Finally, we explore the mechanistic basis for the dynamic targeting of the SMC5/6 complex to damaged chromatin and its crucial role in human health.

Phenotypic heterogeneity associated with KIF21A: Two new cases and review of the literature.

Our understanding of genetic and phenotypic heterogeneity associated with the clinical spectrum of rare diseases continues to expand. Thorough phenotypic descriptions and model organism functional studies are valuable tools in dissecting the biology of the disease process. Kinesin genes are well known to be associated with specific disease phenotypes and a subset of kinesin genes, including KIF21A, have been associated with more than one disease. Here we report two patients with KIF21A variants identified by exome sequencing; one with biallelic variants, supporting a novel KIF21A related syndrome with recessive inheritance and the second report of this condition, and another with a heterozygous de novo variant allele representing a phenotypic expansion of the condition described to date. We provide detailed phenotypic information on both families, including a novel neuropathology finding of neuroaxonal dystrophy associated with biallelic variants in KIF21A. Additionally, we studied the dominant variant in Saccharomyces cerevisiae to assess variant pathogenicity and found that this variant appears to impair protein function. KIF21A associated disease has mounting evidence for phenotypic heterogeneity; further patients and study of an allelic series are required to define the phenotypic spectrum and further explore the molecular etiology for each of these conditions.

Large-scale phenogenomic analysis of human cancers uncovers frequent alterations affecting SMC5/6 complex components in breast cancer.

Cancer cells often experience large-scale alterations in genome architecture because of DNA damage and replication stress. Whether mutations in core regulators of chromosome structure can also lead to cancer-promoting loss in genome stability is not fully understood. To address this question, we conducted a systematic analysis of mutations affecting a global regulator of chromosome biology -the SMC5/6 complex- in cancer genomics cohorts. Analysis of 64 959 cancer samples spanning 144 tissue types and 199 different cancer genome studies revealed that the SMC5/6 complex is frequently altered in breast cancer patients. Patient-derived mutations targeting this complex associate with strong phenotypic outcomes such as loss of ploidy control and reduced overall survival. Remarkably, the phenotypic impact of several patient mutations can be observed in a heterozygous context, hence providing an explanation for a prominent role of SMC5/6 mutations in breast cancer pathogenesis. Overall, our findings suggest that genes encoding global effectors of chromosome architecture can act as key contributors to cancer development in humans.

PGC-1s shape epidermal physiology by modulating keratinocyte proliferation and terminal differentiation.

Skin plays central roles in systemic physiology, and it undergoes significant functional changes during aging. Members of the peroxisome proliferator-activated receptor-gamma coactivator (PGC-1) family (PGC-1s) are key regulators of the biology of numerous tissues, yet we know very little about their impact on skin functions. Global gene expression profiling and gene silencing in keratinocytes uncovered that PGC-1s control the expression of metabolic genes as well as that of terminal differentiation programs. Glutamine emerged as a key substrate promoting mitochondrial respiration, keratinocyte proliferation, and the expression of PGC-1s and terminal differentiation programs. Importantly, gene silencing of PGC-1s reduced the thickness of a reconstructed living human epidermal equivalent. Exposure of keratinocytes to a salicylic acid derivative potentiated the expression of PGC-1s and terminal differentiation genes and increased mitochondrial respiration. Overall, our results show that the PGC-1s are essential effectors of epidermal physiology, revealing an axis that could be targeted in skin conditions and aging.

The Polo kinase Cdc5 is regulated at multiple levels in the adaptation response to telomere dysfunction.

Telomere dysfunction activates the DNA damage checkpoint to induce a cell cycle arrest. After an extended period of time, however, cells can bypass the arrest and undergo cell division despite the persistence of the initial damage, a process called adaptation to DNA damage. The Polo kinase Cdc5 in Saccharomyces cerevisiae is essential for adaptation and for many other cell cycle processes. How the regulation of Cdc5 in response to telomere dysfunction relates to adaptation is not clear. Here, we report that Cdc5 protein level decreases after telomere dysfunction in a Mec1-, Rad53- and Ndd1-dependent manner. This regulation of Cdc5 is important to maintain long-term cell cycle arrest but not for the initial checkpoint arrest. We find that both Cdc5 and the adaptation-deficient mutant protein Cdc5-ad are heavily phosphorylated and several phosphorylation sites modulate adaptation efficiency. The PP2A phosphatases are involved in Cdc5-ad phosphorylation status and contribute to adaptation mechanisms. We finally propose that Cdc5 orchestrates multiple cell cycle pathways to promote adaptation.

Moonlighting at the Poles: Non-Canonical Functions of Centrosomes.

Centrosomes are best known as the microtubule organizing centers (MTOCs) of eukaryotic cells. In addition to their classic role in chromosome segregation, centrosomes play diverse roles unrelated to their MTOC activity during cell proliferation and quiescence. Metazoan centrosomes and their functional doppelgängers from lower eukaryotes, the spindle pole bodies (SPBs), act as important structural platforms that orchestrate signaling events essential for cell cycle progression, cellular responses to DNA damage, sensory reception and cell homeostasis. Here, we provide a critical overview of the unconventional and often overlooked roles of centrosomes/SPBs in the life cycle of eukaryotic cells.

Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II.

Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

The Smc5/6 Core Complex Is a Structure-Specific DNA Binding and Compacting Machine.

The structural organization of chromosomes is a crucial feature that defines the functional state of genes and genomes. The extent of structural changes experienced by genomes of eukaryotic cells can be dramatic and spans several orders of magnitude. At the core of these changes lies a unique group of ATPases-the SMC proteins-that act as major effectors of chromosome behavior in cells. The Smc5/6 proteins play essential roles in the maintenance of genome stability, yet their mode of action is not fully understood. Here we show that the human Smc5/6 complex recognizes unusual DNA configurations and uses the energy of ATP hydrolysis to promote their compaction. Structural analyses reveal subunit interfaces responsible for the functionality of the Smc5/6 complex and how mutations in these regions may lead to chromosome breakage syndromes in humans. Collectively, our results suggest that the Smc5/6 complex promotes genome stability as a DNA micro-compaction machine.

Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division.

Polo-like kinases (Plks) are key cell cycle regulators. They contain a kinase domain followed by a polo-box domain that recognizes phosphorylated substrates and enhances their phosphorylation. The regulatory subunit of the Dbf4-dependent kinase complex interacts with the polo-box domain of Cdc5 (the sole Plk in Saccharomyces cerevisiae) in a phosphorylation-independent manner. We have solved the crystal structures of the polo-box domain of Cdc5 on its own and in the presence of peptides derived from Dbf4 and a canonical phosphorylated substrate. The structure bound to the Dbf4-peptide reveals an additional density on the surface opposite to the phospho-peptide binding site that allowed us to propose a model for the interaction. We found that the two peptides can bind simultaneously and non-competitively to the polo-box domain in solution. Furthermore, point mutations on the surface opposite to the phosphopeptide binding site of the polo-box domain disrupt the interaction with the Dbf4 peptide in solution and cause an early anaphase arrest phenotype distinct from the mitotic exit defect typically observed in cdc5 mutants. Collectively, our data illustrates the importance of non-canonical interactions mediated by the polo-box domain and provide key mechanistic insights into the combinatorial recognition of substrates by Polo-like kinases.

Interphase Microtubules Safeguard Mitotic Progression by Suppressing an Aurora B-Dependent Arrest Induced by DNA Replication Stress.

The segregation of chromosomes is a critical step during cell division. This process is driven by the elongation of spindle microtubules and is tightly regulated by checkpoint mechanisms. It is unknown whether microtubules affect checkpoint responses as passive contributors or active regulators of the process. We show here that interphase microtubules are essential to temporally restrict the effects of DNA replication stress to S phase in Saccharomyces cerevisiae. Tubulin mutants hypersensitive to DNA damage experience a strong but delayed mitotic checkpoint arrest after exposure to genotoxic stress in S phase. This untimely arrest is dependent on the Aurora B kinase but, surprisingly, not on the DNA damage checkpoint. Impaired microtubule-kinetochore interaction is the apparent cause for this unusual phenotype. Collectively, our results reveal that core components of microtubules potentiate the detection of DNA lesions created in S phase, thereby suppressing untimely activation of mitotic checkpoints after DNA replication stress.

Cell cycle-dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast.

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle-dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle-regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle-regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.

Combined Enrichment/Enzymatic Approach To Study Tightly Clustered Multisite Phosphorylation on Ser-Rich Domains.

The regulation of protein function through phosphorylation is often dominated by allosteric interactions and conformational changes. However, alternative mechanisms involving electrostatic interactions also regulate protein function. In particular, phosphorylation of clusters of Ser/Thr residues can affect protein-plasma membrane/chromatin interactions by electrostatic interactions between phosphosites and phospholipids or histones. Currently, only a few examples of such mechanisms are reported, primarily because of the difficulties of detecting highly phosphorylated proteins and peptides, due in part to the low ionization efficiency and fragmentation yield of multiphosphorylated peptides in mass spectrometry when using positive ion mode detection. This difficulty in detection has resulted in under-reporting of such modified regions, which can be thought of as phosphoproteomic dark matter. Here, we present a novel approach that enriches for multisite-phosphorylated peptides that until now remained inaccessible by conventional phosphoproteomics. Our technique enables the identification of multisite-phosphorylated regions on more than 300 proteins in both yeast and human cells and can be used to profile changes in multisite phosphorylation upon cell stimulation. We further characterize the role of multisite phosphorylation for Ste20 in the yeast mating pheromone response. Mutagenesis experiments confirmed that multisite phosphorylation of Ser/Thr-rich regions plays an important role in the regulation of Ste20 activity during mating pheromone signaling. The ability to detect protein multisite phosphorylation opens new avenues to explore phosphoproteomic dark matter and to study Ser-rich proteins that interact with binding partners through charge pairing mechanisms.

Condensin ATPase motifs contribute differentially to the maintenance of chromosome morphology and genome stability.

Effective transfer of genetic information during cell division requires a major reorganization of chromosome structure. This process is triggered by condensin, a conserved pentameric ATPase essential for chromosome condensation. How condensin harnesses the energy of ATP hydrolysis to promote chromatin reorganization is unknown. To address this issue, we performed a genetic screen specifically focused on the ATPase domain of Smc4, a core subunit of condensin. Our screen identified mutational hotspots that impair condensin's ability to condense chromosomes to various degrees. These mutations have distinct effects on viability, genome stability, and chromosome morphology, revealing unique thresholds for condensin enzymatic activity in the execution of its cellular functions. Biochemical analyses indicate that inactivation of Smc4 ATPase activity can result in cell lethality because it favors a specific configuration of condensin that locks ATP in the enzyme. Together, our results provide critical insights into the mechanism used by condensin to harness the energy of ATP hydrolysis for the compaction of chromatin.

Cdc48/VCP Promotes Chromosome Morphogenesis by Releasing Condensin from Self-Entrapment in Chromatin.

The morphological transformation of amorphous chromatin into distinct chromosomes is a hallmark of mitosis. To achieve this, chromatin must be compacted and remodeled by a ring-shaped enzyme complex known as condensin. However, the mechanistic basis underpinning condensin's role in chromosome remodeling has remained elusive. Here we show that condensin has a strong tendency to trap itself in its own reaction product during chromatin compaction and yet is capable of interacting with chromatin in a highly dynamic manner in vivo. To resolve this apparent paradox, we identified specific chromatin remodelers and AAA-class ATPases that act in a coordinated manner to release condensin from chromatin entrapment. The Cdc48 segregase is the central linchpin of this regulatory mechanism and promotes ubiquitin-dependent cycling of condensin on mitotic chromatin as well as effective chromosome condensation. Collectively, our results show that condensin inhibition by its own reaction product is relieved by forceful enzyme extraction from chromatin.

Oligomerization and ATP stimulate condensin-mediated DNA compaction.

Large-scale chromatin remodeling during mitosis is catalyzed by a heteropentameric enzyme known as condensin. The DNA-organizing mechanism of condensin depends on the energy of ATP hydrolysis but how this activity specifically promotes proper compaction and segregation of chromosomes during mitosis remains poorly understood. Purification of budding yeast condensin reveals that it occurs not only in the classical heteropentameric "monomer" form, but that it also adopts much larger configurations consistent with oligomerization. We use a single-DNA magnetic tweezers assay to study compaction of DNA by yeast condensin, with the result that only the multimer shows ATP-enhanced DNA-compaction. The compaction reaction involves step-like events of 200 nm (600 bp) size and is strongly suppressed by forces above 1 pN, consistent with a loop-capture mechanism for initial binding and compaction. The compaction reactions are largely insensitive to DNA torsional stress. Our results suggest a physiological role for oligomerized condensin in driving gradual chromatin compaction by step-like and slow "creeping" dynamics consistent with a loop-extrusion mechanism.

Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis.

Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.

Centrosome-Dependent Bypass of the DNA Damage Checkpoint by the Polo Kinase Cdc5.

Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage.