DNA-protein interaction dynamics at the Lamin B2 replication origin

The regulation of human DNA replication operates via a time-defined program of activation and deactivation of approximately 30,000 replication origins distributed along the genome. Due to the complexity of this process, each step requires a sequence of cascade checkpoints and licensing events, most of which are well conserved from yeasts to humans. A multi-protein complex assembles onto each origin causing the local unwinding of the DNA double helix and the start of two oppositely moving replicative forks. Despite the cis-acting elements necessary for origin firing are almost elucidated, the mechanism that governs the selection of a specific DNA sequence as human (and, more generally, metazoan) origin, in the course of G1 phase of the cell cycle, is still poorly understood. The lack of DNA sequence consensus between replication origins characterized so far, together with the poor binding-specificity displayed by the Origin Recognition Complex, suggest that origin selection might rather be determined by local chromatin structures and/or trans-acting factors. With regard to the latter possibility, it was interesting to find out that a DNA region specifically bound by the AP-1 proteins, is located close to the start site of the human Lamin B2 replication origin. In the study conducted during this Ph.D. program, the possible role of AP-1 transcription factors in origin specification was explored by investigating the involvement the principal moieties of this protein family, c-Fos and c-Jun, within the replicative complexes in living human cells. The data reported in this thesis provides evidence that both c-Fos and c-Jun interact with the LaminB2 origin of DNA replication and indeed participates in origin function. Participation of these proteins to origin binding is consistent with their interaction with both ORC4 and HOXC13, two members of the replicative complex, and is cell cycle defined, occurring before origin firing. Furthermore the observations point to the existence of specific and dynamic structural reorganizations of the complexes assembled at the origin region along with origin activation. In this view, AP-1 proteins could contribute to recruit and stabilize the replicative complexes onto the LaminB2 origin, in presence of specific chromatin and topological configurations

Biomechanical defects and rescue of cardiomyocytes expressing pathologic nuclear lamins

Given the clinical impact of LMNA cardiomyopathies, understanding lamin function will fulfill a clinical need and will lead to advancement in the treatment of heart failure. A multidisciplinary approach combining cell biology, atomic force microscopy (AFM) and molecular modeling was used to analyze the biomechanical properties of human lamin A/C gene (LMNA) mutations (E161K, D192G, N195K) using an in vitro neonatal rat ventricular myocyte (NRVM) model

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

The Cardiomyopathy Lamin A/C D192G Mutation Disrupts Whole-Cell Biomechanics in Cardiomyocytes as Measured by Atomic Force Microscopy Loading-Unloading Curve Analysis

Atomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties

Clinical validation of full HR-HPV genotyping HPV Selfy assay according to the international guidelines for HPV test requirements for cervical cancer screening on clinician-collected and self-collected samples

Background According to international guidelines, Human Papillomavirus (HPV) DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more (CIN2+) lesions. The study aimed to assess whether HPV Selfy (Ulisse BioMed - Trieste, Italy), a full-genotyping HPV DNA test that detects and differentiates 14 high-risk HPV (HR-HPV) types, meets the criteria for primary cervical cancer screening described in the international guidelines, on clinician-collected as well as on self-collected samples. Methods For each participant woman, consecutively referring to Azienda Sanitaria Universitaria Giuliano Isontina (Trieste, Italy) and CRO-National Cancer Institute (Aviano, Italy) for the cervical cancer screening program, the following samples were tested: (a) a clinician-collected cervical specimen, analyzed with the reference test (Hybrid Capture (R) 2 test, HC2) and HPV Selfy; and (b) a self-collected vaginal sample, analyzed with HPV Selfy. Enrolled women were also asked to fulfill a questionnaire about self-sampling acceptability. As required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with its reference test. Results HPV Selfy clinical sensitivity and specificity resulted non-inferior to those of HC2. By analysis of a total of 889 cervical liquid-based cytology samples from a screening population, of which 98 were from women with CIN2+, HPV Selfy showed relative sensitivity and specificity for CIN2+ of 0.98 and 1.00 respectively (non-inferiority score test: P = 0.01747 and P = 0.00414, respectively); the test reached adequate intra- and inter-laboratory reproducibility. Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen (0.92 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population. Conclusions HPV Selfy fulfills all the requirements of the international Meijer's guidelines and has been clinically validated for primary cervical cancer screening purposes. Moreover, HPV Selfy has also been validated for self-sampling according to VALHUDES guidelines. Therefore, at date, HPV Selfy is the only full-genotyping test validated both for screening purposes and for self-sampling. Trial registration ASUGI Trieste n. 16008/2018; CRO Aviano n.17149/201

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations

Atomic force microscopy and lamins: a review study towards future, combined investigations

In the last decades, atomic force microscopy (AFM) underwent a rapid and stunning development, especially for studying mechanical properties of biological samples. The numerous discoveries relying to this approach, have increased the credit of AFM as a versatile tool, and potentially eligible as a diagnostic equipment. Meanwhile, it has become strikingly evident that lamins are involved on the onset and development of certain diseases, including cancer, Hutchinson-Gilford progeria syndrome, cardiovascular pathologies, and muscular dystrophy. A new category of pathologies has been defined, the laminopathies, which are caused by mutations in the gene encoding for A-type lamins. As the majority of medical issues, lamins, and all their related aspects can be considered as a quite complex problem. Indeed, there are many facets to explore, and this definitely requires a multidisciplinary approach. One of the most intriguing aspects concerning lamins is their remarkable contribute to cells mechanics. Over the years, this has led to the speculation of the so-called \u201cstructural hypothesis\u201d, which attempts to elucidate the etiology and some features of the laminopathies. Among the various techniques tried to figure out the role of lamins in the cells mechanics, the AFM has been already successfully applied, proving its versatility. Therefore, the present work aims both to highlight the qualities of AFM and to review the most relevant knowledge about lamins, in order to promote the study of the latter, taking advantage from the forme

DNA-protein interaction dynamics at the Lamin B2 replication origin

To date, a complete understanding of the molecular events leading to DNA replication origin activation in mammalian cells still remains elusive. In this work, we report the results of a high resolution chromatin immunoprecipitation study to detect proteins interacting with the human Lamin B2 replication origin. In addition to the pre-RC component ORC4 and to the transcription factors USF and HOXC13, we found that 2 components of the AP-1 transcription factor, c-Fos and c-Jun, are also associated with the origin DNA during the late G1 phase of the cell cycle and that these factors interact with ORC4. Both DNA replication and AP-1 factor binding to the origin region were perturbed by cell treatment with merbarone, a topoisomerase II inhibitor, suggesting that DNA topology is essential for determining origin function

Knock Down of Plakophillin 2 Dysregulates Adhesion Pathway through Upregulation of miR200b and Alters the Mechanical Properties in Cardiac Cells

Abstract: Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte\u2013myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-\u3b11 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM

Easy fabrication of aligned PLLA nanofibers-based 2D scaffolds suitable for cell contact guidance studies

An easy, low-cost and fast wet processing-based method named ASB-SANS (Auxiliary Solvent-Based Sublimation-Aided NanoStructuring) has been used to fabricate poly(L-lactic acid) (PLLA) highly ordered and hierarchically organized 2D fibrillar patterns,with fiber widths between 40 and 500 nmand lengths exceeding tens of microns. A clear contact guidance effect of these nanofibrillar scaffolds with respect to HeLa and NIH-3T3 cells growth has been observed, on top of an overall good viability. For NIH-3T3 pronounced elongation of the cells was observed, as well as a remarkable ability of the patterns to guide the extension of pseudopodia. Moreover, SEM imaging revealed filopodia stemming from both sides of the pseudopodia and aligned with the secondary PLLA nanofibrous structures created by the ASB-SANS procedure. These results validate ASB-SANS as a technique capable to provide biocompatible 2D nanofibrillar patterns suitable for studying phenomena of contact guidance (and,more in general, the behavior of cells onto nanofibrous scaffolds), at very lowcosts and in an extremely easy way, accessible to virtually any laboratory