CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier.

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.

High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers.

Recent advances in high-throughput single-molecule magnetic tweezers have paved the way for obtaining information on individual molecules as well as ensemble-averaged behavior in a single assay. Here we describe how to design robust high-throughput magnetic tweezers assays that specifically require application of high forces (>20pN) for prolonged periods of time (>1000s). We elaborate on the strengths and limitations of the typical construct types that can be used and provide a step-by-step guide towards a high tether yield assay based on two examples. Firstly, we discuss a DNA hairpin assay where force-induced strand separation triggers a tight interaction between DNA-binding protein Tus and its binding site Ter, where forces up to 90pN for hundreds of seconds were required to dissociate Tus from Ter. Secondly, we show how the LTag helicase of Simian virus 40 unwinds dsDNA, where a load of 36pN optimizes the assay readout. The approaches detailed here provide guidelines for the high-throughput, quantitative study of a wide range of DNA-protein interactions.

Invincible DNA tethers: covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments.

Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy.

Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.

INTRODUCTION: Increased expression of αv integrins is frequently associated with tumor cell adhesion, migration, invasion and metastasis, and correlates with poor prognosis in breast cancer. However, the mechanism by which αv integrins can enhance breast cancer progression is still largely unclear. The effects of therapeutic targeting of αv integrins in breast cancer also have yet to be investigated. METHODS: We knocked down αv integrin in MDA-MB-231 and MCF10A-M4 breast cancer cells, or treated these cells with the αv antagonist GLPG0187. The effects of αv integrin depletion on mesenchymal markers, transforming growth factor-ß (TGF-ß)/Smad signaling and TGF-ß-induced target gene expression were analyzed in MDA-MB-231 cells by RNA analysis or Western blotting. The function of αv integrin on breast cancer cell migration was investigated by transwell assay in vitro, and its effect on breast cancer progression was assessed by both zebrafish and mouse xenografts in vivo. In the mouse model, GLPG0187 was administered separately, or in combination with the standard-of-care anti-resorptive agent zoledronate and the chemotherapeutic drug paclitaxel, to study the effects of combinational treatments on breast cancer metastasis. RESULTS: Genetic interference and pharmacological targeting of αv integrin with GLPG0187 in different breast cancer cell lines inhibited invasion and metastasis in the zebrafish or mouse xenograft model. Depletion of αv integrin in MDA-MB-231 cells inhibited the expression of mesenchymal markers and the TGF-ß/Smad response. TGF-ß induced αv integrin mRNA expression and αv integrin was required for TGF-ß-induced breast cancer cell migration. Moreover, treatment of MDA-MB-231 cells with non-peptide RGD antagonist GLPG0187 decreased TGF-ß signaling. In the mouse xenografts GLPG0187 inhibited the progression of bone metastasis. Maximum efficacy of inhibition of bone metastasis was achieved when GLPG0187 was combined with the standard-of-care metastatic breast cancer treatments. CONCLUSION: These findings show that αv integrin is required for efficient TGF-ß/Smad signaling and TGF-ß-induced breast cancer cell migration, and for maintaining a mesenchymal phenotype of the breast cancer cells. Our results also provide evidence that targeting αv integrin could be an effective therapeutic approach for treatment of breast cancer tumors and/or metastases that overexpress αv integrin.

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy.

Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.

The APP intracellular domain (AICD) inhibits Wnt signalling and promotes neurite outgrowth.

ß- and γ-secretase cleave the amyloid precursor protein (APP) to release the amyloidogenic ß-amyloid peptides (Aß) and the APP intracellular domain (AICD). Aß has been widely believed to initiate pathogenic cascades culminating in Alzheimer's disease (AD). However, the physiological functions of the AICD remain elusive. In this study, we found the AICD to strongly inhibit Wnt-induced transcriptional reporter activity, and to counteract Wnt-induced c-Myc expression. Loss of the AICD resulted in an increased responsiveness to Wnt/ß-catenin-mediated transcription. Mechanically, the AICD was found to interact with glycogen synthase kinase 3 beta (GSK3ß) and promote its kinase activity. The subsequent AICD-strengthened Axin-GSK3ß complex potentiates ß-catenin poly-ubiquitination. Functional studies in N(2)a mouse neuroblastoma cells, rat pheochromocytoma PC12 cells and primary neurons showed that the AICD facilitated neurite outgrowth. And AICD antagonised Wnt3a-suppressed growth arrest and neurite outgrowth in N2a and PC12 cells. Taken together, our results identify the AICD as a novel inhibitory factor of the canonical Wnt signalling pathway and suggest its regulatory role in neuronal cell proliferation and differentiation.

Snail and Slug, key regulators of TGF-ß-induced EMT, are sufficient for the induction of single-cell invasion.

TGF-ß plays a dual role in cancer; in early stages it inhibits tumor growth, whereas later it promotes invasion and metastasis. TGF-ß is thought to be pro-invasive by inducing epithelial-to-mesenchymal transition (EMT) via induction of transcriptional repressors, including Slug and Snail. In this study, we investigated the role of Snail and Slug in TGF-ß-induced invasion in an in vitro invasion assay and in an embryonic zebrafish xenograft model. Ectopic expression of Slug or Snail promoted invasion of single, rounded amoeboid cells in vitro. In an embryonic zebrafish xenograft model, forced expression of Slug and Snail promoted single cell invasion and metastasis. Slug and Snail are sufficient for the induction of single-cell invasion in an in vitro invasion assay and in an embryonic zebrafish xenograft model.

A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint.

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation.

The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies. Conversely, single-molecule studies of CMG motion have thus far relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents. Here, we report the activation of CMG fully reconstituted from purified yeast proteins and the quantification of its motion at the single-molecule level. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG independently of DNA melting. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication.

Characterizing single-molecule dynamics of viral RNA-dependent RNA polymerases with multiplexed magnetic tweezers.

Multiplexed single-molecule magnetic tweezers (MT) have recently been employed to probe the RNA synthesis dynamics of RNA-dependent RNA polymerases (RdRp). Here, we present a protocol for simultaneously probing the RNA synthesis dynamics of hundreds of single polymerases with MT. We describe the preparation of a dsRNA construct for probing single RdRp kinetics. We then detail the measurement of RdRp RNA synthesis kinetics using MT. The protocol is suitable for high-throughput probing of RdRp-targeting antiviral compounds for mechanistic function and efficacy. For complete details on the use and execution of this protocol, please refer to Janissen et al. (2021).

SMC complexes can traverse physical roadblocks bigger than their ring size.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

VEGF and inhibitors of TGFbeta type-I receptor kinase synergistically promote blood-vessel formation by inducing alpha5-integrin expression.

Vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGFbeta) are potent regulators of angiogenesis. How VEGF and TGFbeta signaling pathways crosstalk is not well understood. Therefore, we analyzed the effects of the TGFbeta type-I-receptor inhibitors (SB-431542 and LY-2157299) and VEGF on endothelial cell (EC) function and angiogenesis. We show that SB-431542 dramatically enhances VEGF-induced formation of EC sheets from fetal mouse metatarsals. Sub-optimal doses of VEGF and SB-431542 synergistically induced EC migration and sprouting of EC spheroids, whereas overexpression of a constitutively active form of TGFbeta type-I receptor had opposite effects. Using quantitative PCR, we demonstrated that VEGF and SB-431542 synergistically upregulated the mRNA expression of genes involved in angiogenesis, including the integrins alpha5 and beta3. Specific downregulation of alpha5-integrin expression or functional blocking of alpha5 integrin with a specific neutralizing antibody inhibited the cooperative effect of VEGF and SB-431542 on EC sprouting. In vivo, LY-2157299 induced angiogenesis and enhanced VEGF- and basic-fibroblast-growth-factor-induced angiogenesis in a Matrigel-plug assay, whereas adding an alpha5-integrin-neutralizing antibody to the Matrigel selectively inhibited this enhanced response. Thus, induction of alpha5-integrin expression is a key determinant by which inhibitors of TGFbeta type-I receptor kinase and VEGF synergistically promote angiogenesis.

Wnt/ß-catenin signal pathway stabilizes APP intracellular domain (AICD) and promotes its transcriptional activity.

Amyloid precursor protein (APP), a key protein in pathogenesis of Alzheimer's disease (AD), is a type I transmembrane protein which can be cleaved by ß- and γ-secretase to release the amyloidogenic ß-amyloid peptides (Aß) and the APP intracellular domain (AICD). While Aß has been widely believed to initiate pathogenic cascades culminating AD, the physiological functions and regulations of AICD remain elusive. In present study, endogenous AICD was demonstrated to be increased by canonical Wnt signal. Instead of due to γ-secretase activity, enhanced AICD expression was found due to the increased protein stability by Wnt/ß-catenin. ß-Catenin was demonstrated to be an associating partner of AICD, capable of promoting AICD mediated transcriptional activity. Investigation by AICD mutants proved that Fe65, a previously identified AICD binding partner, is not involved in this regulation. Taken together, our results suggest that AICD is stabilized and the AICD mediated transcriptional activity is promoted by canonical Wnt/ß-catenin signaling independent of Fe65.

DNA replication origins retain mobile licensing proteins.

DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.

Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB.

Metastasis is a major factor in the malignancy of cancers, and is often responsible for the failure of cancer treatment. Anoikis (apoptosis resulting from loss of cell-matrix interactions) has been suggested to act as a physiological barrier to metastasis; resistance to anoikis may allow survival of cancer cells during systemic circulation, thereby facilitating secondary tumour formation in distant organs. In an attempt to identify metastasis-associated oncogenes, we designed an unbiased, genome-wide functional screen solely on the basis of anoikis suppression. Here, we report the identification of TrkB, a neurotrophic tyrosine kinase receptor, as a potent and specific suppressor of caspase-associated anoikis of non-malignant epithelial cells. By activating the phosphatidylinositol-3-OH kinase/protein kinase B pathway, TrkB induced the formation of large cellular aggregates that survive and proliferate in suspension. In mice, these cells formed rapidly growing tumours that infiltrated lymphatics and blood vessels to colonize distant organs. Consistent with the ability of TrkB to suppress anoikis, metastases--whether small vessel infiltrates or large tumour nodules--contained very few apoptotic cells. These observations demonstrate the potent oncogenic effects of TrkB and uncover a specific pro-survival function that may contribute to its metastatic capacity, providing a possible explanation for the aggressive nature of human tumours that overexpress TrkB.

Expression of BNIP3 in invasive breast cancer: correlations with the hypoxic response and clinicopathological features.

BACKGROUND: Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a pro-apoptotic member of the Bcl-2 family induced under hypoxia. Low or absent expression has recently been described in human tumors, including gastrointestinal tumors, resulting in poor prognosis. Little is known about BNIP3 expression in invasive breast cancer. The aim of the present study was to investigate the expression of BNIP3 in invasive breast cancer at the mRNA and protein level in correlation with the hypoxic response and clinicopathological features. METHODS: In 40 cases of invasive breast cancer, BNIP3 mRNA in situ hybridization was performed on frozen sections with a digoxigenin labeled anti-BNIP3 probe. Paraffin embedded sections of the same specimens were used to determine protein expression of BNIP3, Hypoxia Inducible Factor 1 alpha (HIF-1alpha) and its downstream targets Glucose Transporter 1 (Glut-1) and Carbonic Anhydrase (CAIX) by immunohistochemistry. RESULTS: BNIP3 mRNA was expressed in 16/40 (40%) of the cases and correlated with BNIP3 protein expression (p = 0.0218). Neither BNIP3 protein nor mRNA expression correlated with expression of HIF-1alpha expression or its downstream targets. Tumors which showed loss of expression of BNIP3 had significantly more often lymph node metastases (82% vs 39%, p = 0.010) and showed a higher mitotic activity index (p = 0.027). BNIP3 protein expression was often nuclear in normal breast, but cytoplasmic in tumor cells. CONCLUSION: BNIP3 expression is lost in a significant portion of invasive breast cancers, which is correlated with poor prognostic features such as positive lymph node status and high proliferation, but not with the hypoxic response.

Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers.

RNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events. Here, we use a high-throughput magnetic tweezers platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRp-RNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides.

Mutations of the PU.1 Ets domain are specifically associated with murine radiation-induced, but not human therapy-related, acute myeloid leukaemia.

Murine radiation-induced acute myeloid leukaemia (AML) is characterized by loss of one copy of chromosome 2. Previously, we positioned the critical haematopoietic-specific transcription factor PU.1 within a minimally deleted region. We now report a high frequency (>65%) of missense mutation at codon 235 in the DNA-binding Ets domain of PU.1 in murine AML. Earlier studies, outside the context of malignancy, determined that conversion of arginine 235 (R235) to any other amino-acid residue leads to ablation of DNA-binding function and loss of expression of downstream targets. We show that mutation of R235 does not lead to protein loss, and occurs specifically in those AMLs showing loss of one copy of PU.1 (P=0.001, Fisher's exact test). PU.1 mutations were not found in the coding region, UTRs or promoter of human therapy-related AMLs. Potentially regulatory elements upstream of PU.1 were located but no mutations found. In conclusion, we have identified the cause of murine radiation-induced AML and have shown that loss of one copy of PU.1, as a consequence of flanking radiation-sensitive fragile domains on chromosome 2, and subsequent R235 conversion are highly specific to this mouse model. Such a mechanism does not operate, or is extremely rare, in human AML.