Vassilis Roukos

Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis.

Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re‐replication. We show here that the N‐terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S–G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1‐Cul4‐mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF‐Skp2, which recognizes the Cy‐motif‐mediated Cyclin E/A‐cyclin‐dependent kinase‐phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S–G2 phases. The Cul4‐containing E3 is active during ongoing replication, while SCF‐Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N‐terminal amino acids. PCNA is essential for Cul4‐ but not Skp2‐directed degradation during DNA replication and following ultraviolet‐irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re‐replication.

Spatial dynamics of chromosome translocations in living cells.

Chromosome Choreography Chromosome translocations arise through the illegitimate pairing of broken chromosome ends and are commonly found in many cancers. Roukos et al. (p. 660) used ultrahigh-throughput time-lapse imaging on human tissue culture cells containing marked chromosomes to capture very rare translocation events. Double-strand breaks in the DNA underwent an initial “partner search,” with a fraction of the ends moving into spatial proximity to each other, which resulted in persistent pairing and the merging of DNA repair foci. Most paired ends arose from breaks in close proximity, but occasionally translocations formed from more distantly positioned breaks. Proteins of the DNA repair machinery could influence the pairing and/or translocation process. An experimental system allows the visualization of human cell chromosome translocations in real time. Chromosome translocations are a hallmark of cancer cells. We have developed an experimental system to visualize the formation of translocations in living cells and apply it to characterize the spatial and dynamic properties of translocation formation. We demonstrate that translocations form within hours of the occurrence of double-strand breaks (DSBs) and that their formation is cell cycle–independent. Translocations form preferentially between prepositioned genome elements, and perturbation of key factors of the DNA repair machinery uncouples DSB pairing from translocation formation. These observations generate a spatiotemporal framework for the formation of translocations in living cells.

The biogenesis of chromosome translocations

Chromosome translocations are catastrophic genomic events and often play key roles in tumorigenesis. Yet the biogenesis of chromosome translocations is remarkably poorly understood. Recent work has delineated several distinct mechanistic steps in the formation of translocations, and it has become apparent that non-random spatial genome organization, DNA repair pathways and chromatin features, including histone marks and the dynamic motion of broken chromatin, are critical for determining translocation frequency and partner selection.

Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B.

Cell cycle staging of individual cells by fluorescence microscopy

Progression through the cell cycle is one of the most fundamental features of cells. Studies of the cell cycle have traditionally relied on the analysis of populations, and they often require specific markers or the use of genetically modified systems, making it difficult to determine the cell cycle stage of individual, unperturbed cells. We describe a protocol, suitable for use in high-resolution imaging approaches, for determining cell cycle staging of individual cells by measuring their DNA content by fluorescence microscopy. The approach is based on the accurate quantification by image analysis of the integrated nuclear intensity of cells stained with a DNA dye, and it can be used in combination with several histochemical methods. We describe and provide the algorithms for two automated image analysis pipelines and the derivation of cell cycle profiles with both commercial and open-source software. This 1–2-d protocol is applicable to adherent cells, and it is adaptable for use with several DNA dyes.

Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage

For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1Cdt2 ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21Cip1 also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21Cip1 exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation.

Geminin cleavage during apoptosis by caspase-3 alters its binding ability to the SWI/SNF subunit Brahma.

Geminin has been proposed to coordinate cell cycle and differentiation events through balanced interactions with the cell cycle regulator Cdt1 and with homeobox transcription factors and chromatin remodeling activities implicated in cell fate decisions. Here we show that Geminin is cleaved in primary cells and cancer cell lines induced to undergo apoptosis by a variety of stimuli. Geminin targeting is mediated by caspase-3 both in vivo and in vitro. Two sites at the carboxyl terminus of Geminin (named C1 and C2) are cleaved by the caspase, producing truncated forms of Geminin. We provide evidence that Geminin cleavage is regulated by phosphorylation. Casein kinase II alters Geminin cleavage at site C1 in vitro, whereas mutating phosphorylation competent Ser/Thr residues proximal to site C1 affects Geminin cleavage in vivo. We show that truncated Geminin produced by cleavage at C1 can promote apoptosis. In contrast, Geminin cleaved at site C2 has lost the ability to interact with Brahma (Brm), a catalytic subunit of the SWI/SNF chromatin remodeling complex, while binding efficiently to Cdt1, indicating that targeting of Geminin during apoptosis differentially affects interactions with its binding partners.

Systematic identification of pathological lamin A interactors

As essential components of the cell nucleus, lamins play key roles in organizing genomes and as protein–protein interaction platforms. Mutations in lamin A cause a diverse set of human diseases. This work describes the identification of lamin A partners and assesses how interactions are affected by a comprehensive set of lamin A disease mutations.

Interconnected contribution of tissue morphogenesis and the nuclear protein NuMA to the DNA damage response.

Epithelial tissue morphogenesis is accompanied by the formation of a polarity axis – a feature of tissue architecture that is initiated by the binding of integrins to the basement membrane. Polarity plays a crucial role in tissue homeostasis, preserving differentiation, cell survival and resistance to chemotherapeutic drugs among others. An important aspect in the maintenance of tissue homeostasis is genome integrity. As normal tissues frequently experience DNA double-strand breaks (DSBs), we asked how tissue architecture might participate in the DNA damage response. Using 3D culture models that mimic mammary glandular morphogenesis and tumor formation, we show that DSB repair activity is higher in basally polarized tissues, regardless of the malignant status of cells, and is controlled by hemidesmosomal integrin signaling. In the absence of glandular morphogenesis, in 2D flat monolayer cultures, basal polarity does not affect DNA repair activity but enhances H2AX phosphorylation, an early chromatin response to DNA damage. The nuclear mitotic apparatus protein 1 (NuMA), which controls breast glandular morphogenesis by acting on the organization of chromatin, displays a polarity-dependent pattern and redistributes in the cell nucleus of basally polarized cells upon the induction of DSBs. This is shown using high-content analysis of nuclear morphometric descriptors. Furthermore, silencing NuMA impairs H2AX phosphorylation – thus, tissue polarity and NuMA cooperate to maintain genome integrity.

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Background: MCM2–7 loading onto chromatin licenses origins for replication. Results: MCMs exhibit transient interactions with chromatin in late mitosis, stable binding in G1 phase and increased loading in late G1 phase. Conclusion: Multilevel regulation of MCM2–7 loading to chromatin occurs during mitosis and preceding the G1/S phase transition. Significance: The dynamics of the DNA licensing system within live human cells reveal multiple control points. Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2–7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.