Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding.

Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome binding hot-spots exist remain unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that disrupts all exposed histone surfaces to comprehensively assess how proteins recognize nucleosomes. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk interactions, whereas nearly half of the nucleosome disk participates only minimally in protein binding. Our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly engages the acidic patch, and we elucidate a redundant mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub and establishes universal principles of nucleosome recognition.

Ubiquitin chain-elongating enzyme UBE2S activates the RING E3 ligase APC/C for substrate priming.

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.

Mass spectrometry-based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells.

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.

Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence.

To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.

Evidence that the human cell cycle is a series of uncoupled, memoryless phases.

The cell cycle is canonically described as a series of four consecutive phases: G1, S, G2, and M. In single cells, the duration of each phase varies, but the quantitative laws that govern phase durations are not well understood. Using time-lapse microscopy, we found that each phase duration follows an Erlang distribution and is statistically independent from other phases. We challenged this observation by perturbing phase durations through oncogene activation, inhibition of DNA synthesis, reduced temperature, and DNA damage. Despite large changes in durations in cell populations, phase durations remained uncoupled in individual cells. These results suggested that the independence of phase durations may arise from a large number of molecular factors that each exerts a minor influence on the rate of cell cycle progression. We tested this model by experimentally forcing phase coupling through inhibition of cyclin-dependent kinase 2 (CDK2) or overexpression of cyclin D. Our work provides an explanation for the historical observation that phase durations are both inherited and independent and suggests how cell cycle progression may be altered in disease states.

Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI.

Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions.

Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication.

The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.

The Cell Cycle Browser: An Interactive Tool for Visualizing, Simulating, and Perturbing Cell-Cycle Progression.

The cell cycle is driven by precise temporal coordination among many molecular activities. To understand and explore this process, we developed the Cell Cycle Browser (CCB), an interactive web interface based on real-time reporter data collected in proliferating human cells. This tool facilitates visualizing, organizing, simulating, and predicting the outcomes of perturbing cell-cycle parameters. Time-series traces from individual cells can be combined to build a multi-layered timeline of molecular activities. Users can simulate the cell cycle using computational models that capture the dynamics of molecular activities and phase transitions. By adjusting individual expression levels and strengths of molecular relationships, users can predict effects on the cell cycle. Virtual assays, such as growth curves and flow cytometry, provide familiar outputs to compare cell-cycle behaviors for data and simulations. The CCB serves to unify our understanding of cell-cycle dynamics and provides a platform for generating hypotheses through virtual experiments.

Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and key regulator of cell cycle progression. Since APC/C promotes the degradation of mitotic cyclins, it controls cell cycle-dependent oscillations in cyclin-dependent kinase (CDK) activity. Both CDKs and APC/C control a large number of substrates and are regulated by analogous mechanisms, including cofactor-dependent activation. However, whereas substrate dephosphorylation is known to counteract CDK, it remains largely unknown whether deubiquitinating enzymes (DUBs) antagonize APC/C substrate ubiquitination during mitosis. Here, we demonstrate that Cezanne/OTUD7B is a cell cycle-regulated DUB that opposes the ubiquitination of APC/C targets. Cezanne is remarkably specific for K11-linked ubiquitin chains, which are formed by APC/C in mitosis. Accordingly, Cezanne binds established APC/C substrates and reverses their APC/C-mediated ubiquitination. Cezanne depletion accelerates APC/C substrate degradation and causes errors in mitotic progression and formation of micronuclei. These data highlight the importance of tempered APC/C substrate destruction in maintaining chromosome stability. Furthermore, Cezanne is recurrently amplified and overexpressed in numerous malignancies, suggesting a potential role in genome maintenance and cancer cell proliferation.

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none.

The Temporal Regulation of S Phase Proteins During G1.

Successful DNA replication requires intimate coordination with cell-cycle progression. Prior to DNA replication initiation in S phase, a series of essential preparatory events in G1 phase ensures timely, complete, and precise genome duplication. Among the essential molecular processes are regulated transcriptional upregulation of genes that encode replication proteins, appropriate post-transcriptional control of replication factor abundance and activity, and assembly of DNA-loaded protein complexes to license replication origins. In this chapter we describe these critical G1 events necessary for DNA replication and their regulation in the context of both cell-cycle entry and cell-cycle progression.

Sequential replication-coupled destruction at G1/S ensures genome stability.

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4(Cdt2), which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4(Cdt2) targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNA(DNA)) and recruits CRL4(Cdt2). Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNA(DNA). Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability.

Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors.

We identify the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle-regulated genes. FOXM1 is bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle transcription factors (E2F1, E2F4, E2F6, and GABPA) from the Encyclopedia of DNA Elements and ChIP-seq data for the DREAM complex finds that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle-regulated genes in a second cancer-derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle.

Transcription factor binding profiles reveal cyclic expression of human protein-coding genes and non-coding RNAs.

Cell cycle is a complex and highly supervised process that must proceed with regulatory precision to achieve successful cellular division. Despite the wide application, microarray time course experiments have several limitations in identifying cell cycle genes. We thus propose a computational model to predict human cell cycle genes based on transcription factor (TF) binding and regulatory motif information in their promoters. We utilize ENCODE ChIP-seq data and motif information as predictors to discriminate cell cycle against non-cell cycle genes. Our results show that both the trans- TF features and the cis- motif features are predictive of cell cycle genes, and a combination of the two types of features can further improve prediction accuracy. We apply our model to a complete list of GENCODE promoters to predict novel cell cycle driving promoters for both protein-coding genes and non-coding RNAs such as lincRNAs. We find that a similar percentage of lincRNAs are cell cycle regulated as protein-coding genes, suggesting the importance of non-coding RNAs in cell cycle division. The model we propose here provides not only a practical tool for identifying novel cell cycle genes with high accuracy, but also new insights on cell cycle regulation by TFs and cis-regulatory elements.

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters.

Transcription of long noncoding RNAs (lncRNAs) within gene regulatory elements can modulate gene activity in response to external stimuli, but the scope and functions of such activity are not known. Here we use an ultrahigh-density array that tiles the promoters of 56 cell-cycle genes to interrogate 108 samples representing diverse perturbations. We identify 216 transcribed regions that encode putative lncRNAs, many with RT-PCR-validated periodic expression during the cell cycle, show altered expression in human cancers and are regulated in expression by specific oncogenic stimuli, stem cell differentiation or DNA damage. DNA damage induces five lncRNAs from the CDKN1A promoter, and one such lncRNA, named PANDA, is induced in a p53-dependent manner. PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes; PANDA depletion markedly sensitized human fibroblasts to apoptosis by doxorubicin. These findings suggest potentially widespread roles for promoter lncRNAs in cell-growth control.

Common markers of proliferation.

When normal tissue and tumour samples are compared by microarray analysis, the biggest differences most often occur in the expression levels of genes that control cell proliferation. However, this difference is detected whenever mRNA samples that are taken from two cell populations with different proliferation rates are compared. Although the exact genes that comprise this 'proliferation signature' often differ, they are almost always genes that are involved in the fundamental process of cell proliferation. Can the proliferation signature be used to improve our understanding of the cell cycle and cancer pathogenesis, as well as being used as a biomarker for cancer diagnosis and prognosis?