Andrea Ballabeni

Loss of Geminin induces rereplication in the presence of functional p53

Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of γH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.

Human Geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre‐replicative complex (pre‐RC) formation in Xenopus egg extracts. Here we describe a novel function of Geminin. We demonstrate that human Geminin protects CDT1 from proteasome‐mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre‐RC formation and DNA replication during the following cell cycle. Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre‐RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that Geminin is both a negative and positive regulator of pre‐RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2–M, and preventing relicensing of origins in S–G2.

Cell cycle adaptations of embryonic stem cells

ES cells proliferate with very short gap phases yet maintain their capacity to differentiate. It had been thought that the levels of cyclins and other substrates of ubiquitin ligase APC/C remain nearly constant and Cdk activity remains constitutively high in mouse ES cells. Here we demonstrate that APC/C (anaphase-promoting complex/cyclosome) enzyme is active in ES cells but attenuated by high levels of the Emi1 (early mitotic inhibitor-1) protein. Despite the presence of high Cdk activity during the G1 phase, chromatin can be effectively licensed for DNA replication and fast entry into the S phase can still occur. High Cdk activity during S-G2-M phases produces high levels of the DNA replication factor Cdt1, and this leads to efficient Mcm proteins loading on chromatin after mitotic exit. Although disturbing the usual balance between Cdk activity and APC/C activity found in somatic cells, a few key adaptations allow normal progression of a very rapid cell cycle.

Gap 1 phase length and mouse embryonic stem cell self-renewal

In somatic cells, the length of the G1 phase of the cell cycle is tightly linked to differentiation, and its elongation can drive differentiation in many cases. Although it has been suggested that the situation is very similar in embryonic stem cells (ESCs), where a rapid cell cycle and a short G1 phase maintain the pluripotent state, evidence has been contradictory. Here we show that, in murine ESCs, elongation of the cell cycle and elongation of G1 are compatible with their pluripotent state. Multiple methods that lengthen the cell cycle and that target cyclin-dependent kinase, retinoblastoma protein, and E2F activity all fail to induce differentiation on their own or even to facilitate differentiation. The resistance of murine ESCs to differentiation induced by lengthening G1 and/or the cell cycle could allow for separate control of these events and provide new opportunities for investigation and application.

An Advanced Pre-Processing Pipeline to Improve Automated Photogrammetric Reconstructions of Architectural Scenes

Automated image-based 3D reconstruction methods are more and more flooding our 3D modeling applications. Fully automated solutions give the impression that from a sample of randomly acquired images we can derive quite impressive visual 3D models. Although the level of automation is reaching very high standards, image quality is a fundamental pre-requisite to produce successful and photo-realistic 3D products, in particular when dealing with large datasets of images. This article presents an efficient pipeline based on color enhancement, image denoising, color-to-gray conversion and image content enrichment. The pipeline stems from an analysis of various state-of-the-art algorithms and aims to adjust the most promising methods, giving solutions to typical failure causes. The assessment evaluation proves how an effective image pre-processing, which considers the entire image dataset, can improve the automated orientation procedure and dense 3D point cloud reconstruction, even in the case of poor texture scenarios.

A nontranscriptional role for Oct4 in the regulation of mitotic entry

Significance Embryonic stem cells and induced pluripotent stem cells have abbreviated cell cycles. To achieve this rapid proliferation, several molecular safeguards that normally distinguish healthy from transformed cells are altered. Understanding how these pluripotent stem cells balance the demands of their unique cell cycles against the need to maintain a stable genome is critical to unlocking their great promise for regenerative medicine. Here, we demonstrate that Oct4 (octamer-binding transcription factor 4), a transcription factor required to maintain pluripotency, inhibits the activation of cyclin-dependent kinase (Cdk) 1, the master regulator of mitosis, and delays mitotic entry in a nontranscriptional manner. To our knowledge, our study is the first demonstration of a nontranscriptional function of the pluripotency regulator Oct4. Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry.

Human CDT1 Associates with CDC7 and Recruits CDC45 to Chromatin during S Phase

The initiation of DNA replication is a tightly controlled process that involves the formation of distinct complexes at origins of DNA replication at specific periods of the cell cycle. Pre-replicative complexes are formed during telophase and early G1. They rearrange at the start of S phase to form pre-initiation complexes, which are a prerequisite for DNA replication. The CDT1 protein is required for the formation of the pre-replicative complexes. Here we show that human CDT1 associates with the CDC7 kinase and recruits CDC45 to chromatin. Moreover, we show that the amount of CDT1 bound to chromatin is regulated by CDC7. We propose a model in which chromatin-bound CDT1 is first stabilized and subsequently displaced by CDC7 activity, thereby ensuring the timely execution of DNA replication.

Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

Significance The master cell-cycle processes governing DNA replication and mitosis in eukaryotic cells are regulated by cyclin/cyclin dependent kinase 1 and the anaphase-promoting complex, with checkpoint activity on these regulators. It is not these regulators but rather intermediaries that communicate to the processes. Here we show that the protein Geminin acts centrally in controlling DNA replication by ensuring that DNA is replicated during S phase and only once. This paper describes the Geminin “sub-master” regulatory circuit and the central role of Geminin in controlling events of the cell cycle. Cdc10-dependent transcript 1 (Cdt1) is an essential DNA replication protein whose accumulation at the end of the cell cycle promotes the formation of pre-replicative complexes and replication in the next cell cycle. Geminin is thought to be involved in licensing replication by promoting the accumulation of Cdt1 in mitosis, because decreasing the Geminin levels prevents Cdt1 accumulation and impairs DNA replication. Geminin is known to inhibit Cdt1 function; its depletion during G2 leads to DNA rereplication and checkpoint activation. Here we show that, despite rapid Cdt1 protein turnover in G2 phase, Geminin promotes Cdt1 accumulation by increasing its RNA and protein levels in the unperturbed cell cycle. Therefore, Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents its rereplication.

Basic Research and Knowledge Production Modes A View from the Harvard Medical School

A robust body of literature analyzes the shift of academic science toward more business-oriented models. This paper presents the findings of an empirical study investigating basic scientists’ attitudes toward publicly funded basic research at the Harvard Medical School and affiliated institutions. The study finds that scientists at the Harvard Medical School construe publicly funded basic research as inquiries that, whether use oriented or not, must be governed by the cognitive and social norms of the traditional mode of knowledge production (mode 1 paradigm). They recognize that academic science is vulnerable to access by external capital but maintain that it remains distinct from research done in the private sector. Overall, the study demonstrates that important segments of academia have preserved a traditional approach to knowledge production, which is yet to be transformed by the entrepreneurial turn.

Countries’ Biomedical Publications and Attraction Scores. A PubMed-based assessment

Studying publication volumes at the country level is key to understanding and improving a country’s research system. PubMed is a public search engine of publications in all life sciences areas. Here, we show how this search engine can be used to assess the outputs of life science-related research by country. We have measured the numbers of publications during different time periods based on the country of affiliation of the first authors. Moreover, we have designed scores, which we have named Attraction Scores, to appraise the relative focus either toward particular types of studies, such as clinical trials or reviews, or toward specific research areas, such as public health and pharmacogenomics, or toward specific topics, for instance embryonic stem cells; we have also investigated a possible use of these Attraction Scores in connection with regulatory policies. We have weighed the statistics against general indicators such as country populations and gross domestic products (GDP). During the 5-year period 2008-2012, the United States was the country with the highest number of publications and Denmark the one with the highest number of publications per capita. Among the 40 countries with the highest GDPs, Israel had the highest publications-to-GDP ratio. Among the 20 countries with the most publications, Japan had the highest Attraction Score for induced pluripotent stem (iPS) cells and Italy the highest proportion of review publications. More than 50% of publications in English were from countries in which English is not the primary language. We show an assorted and extensive collection of rankings and charts that will inform scholars and policymakers in studying and improving the research systems both at the national and international level.