Is PML a tumor suppressor?

The role of the promyelocytic leukemia (PML) protein has been widely tested in many different contexts, as attested by the hundreds of papers present in the literature. In most of these studies, PML is regarded as a tumor suppressor, a notion on the whole accepted by the scientific community. In this review, we examine how the concept of tumor-suppressor gene has evolved until now and then systematically assess whether this assumption for PML is supported by unambiguous experimental evidence

A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry

Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (\u3b3H2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of \u3b3H2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance

P66SHC deletion improves fertility and progeric phenotype of late-generation TERC-deficient mice but not their short lifespan

Oxidative stress and telomere attrition are considered the driving factors of aging. As oxidative damage to telomeric DNA favors the erosion of chromosome ends and, in turn, telomere shortening increases the sensitivity to pro-oxidants, these two factors may trigger a detrimental vicious cycle. To check whether limiting oxidative stress slows down telomere shortening and related progeria, we have investigated the effect of p66SHC deletion, which has been shown to reduce oxidative stress and mitochondrial apoptosis, on late-generation TERC (telomerase RNA component)-deficient mice having short telomeres and reduced lifespan. Double mutant (TERC-/- p66SHC-/-) mice were generated, and their telomere length, fertility, and lifespan investigated in different generations. Results revealed that p66SHC deletion partially rescues sterility and weight loss, as well as organ atrophy, of TERC-deficient mice, but not their short lifespan and telomere erosion. Therefore, our data suggest that p66SHC-mediated oxidative stress and telomere shortening synergize in some tissues (including testes) to accelerate aging; however, early mortality of late-generation mice seems to be independent of any link between p66SHC-mediated oxidative stress and telomere attrition

The emerging role of p53 in stem cells

Among the hundreds of oncogenes and tumor suppressors that have been identified in the past 50 years, p53 is probably the best characterized; nevertheless, new functions are constantly being discovered. As a tumor suppressor, p53 regulates cellular responses to different stress stimuli by inducing reversible cell cycle arrest and DNA repair, or triggering senescence or apoptosis. Recent findings on the regulation of stem cell (SC) division and reprogramming suggest the intriguing possibility that p53 also carries out its tumor suppression function by regulating SC homeostasis. Specifically, p53 activation may counteract SC expansion by several emerging mechanisms including restriction of self-renewing divisions, inhibition of symmetric division and block of reprogramming of somatic/progenitor cells into SCs

Peak shape clustering reveals biological insights

Background: ChIP-seq experiments are widely used to detect and study DNA-protein interactions, such as transcription factor binding and chromatin modifications. However, downstream analysis of ChIP-seq data is currently restricted to the evaluation of signal intensity and the detection of enriched regions (peaks) in the genome. Other features of peak shape are almost always neglected, despite the remarkable differences shown by ChIP-seq for different proteins, as well as by distinct regions in a single experiment. Results: We hypothesize that statistically significant differences in peak shape might have a functional role and a biological meaning. Thus, we design five indices able to summarize peak shapes and we employ multivariate clustering techniques to divide peaks into groups according to both their complexity and the intensity of their coverage function. In addition, our novel analysis pipeline employs a range of statistical and bioinformatics techniques to relate the obtained peak shapes to several independent genomic datasets, including other genome-wide protein-DNA maps and gene expression experiments. To clarify the meaning of peak shape, we apply our methodology to the study of the erythroid transcription factor GATA-1 in K562 cell line and in megakaryocytes. Conclusions: Our study demonstrates that ChIP-seq profiles include information regarding the binding of other proteins beside the one used for precipitation. In particular, peak shape provides new insights into cooperative transcriptional regulation and is correlated to gene expression

Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20\u2013250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus

Modeling cell proliferation in human acute myeloid leukemia xenografts

Motivation: Acute myeloid leukemia (AML) is one of the most common hematological malignancies, characterized by high relapse and mortality rates. The inherent intra-tumor heterogeneity in AML is thought to play an important role in disease recurrence and resistance to chemotherapy. Although experimental protocols for cell proliferation studies are well established and widespread, they are not easily applicable to in vivo contexts, and the analysis of related time-series data is often complex to achieve. To overcome these limitations, model-driven approaches can be exploited to investigate different aspects of cell population dynamics. Results: In this work, we present ProCell, a novel modeling and simulation framework to investigate cell proliferation dynamics that, differently from other approaches, takes into account the inherent stochasticity of cell division events. We apply ProCell to compare different models of cell proliferation in AML, notably leveraging experimental data derived from human xenografts in mice. ProCell is coupled with Fuzzy Self-Tuning Particle Swarm Optimization, a swarm-intelligence settings-free algorithm used to automatically infer the models parameterizations. Our results provide new insights on the intricate organization of AML cells with highly heterogeneous proliferative potential, highlighting the important role played by quiescent cells and proliferating cells characterized by different rates of division in the progression and evolution of the disease, thus hinting at the necessity to further characterize tumor cell subpopulations. Availability and implementation: The source code of ProCell and the experimental data used in this work are available under the GPL 2.0 license on GITHUB at the following URL: https://github.com/aresio/ProCell

The genomic landscape of 8-oxodG reveals enrichment at specific inherently fragile promoters

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most common marker of oxidative stress and its accumulation within the genome has been associated with major human health issues such as cancer, aging, cardiovascular and neurodegenerative diseases. The characterization of the different genomic sites where 8-oxodG accumulates and the mechanisms underlying its formation are still poorly understood. Using OxiDIP-seq, we recently derived the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A). Here, we identify a subset of human promoters that accumulate 8-oxodG under steady-state condition. 8-oxodG nucleotides co-localize with double strand breaks (DSBs) at bidirectional and CG skewed promoters and their density correlate with RNA Polymerase II co-occupancy and transcription. Furthermore, by performing OxiDIP-seq in quiescent (G0) cells, we found a strong reduction of oxidatively-generated damage in the majority of 8-oxodG-positive promoters in the absence of DNA replication. Overall, our results suggest that the accumulation of 8-oxodG at gene promoters occurs through DNA replication-dependent or -independent mechanisms, with a possible contribution to the formation of cancer-associated translocation events

Hdmx recruitment into the nucleus by Hdm2 is essential for its ability to regulate p53 stability and transactivation

The Hdmx gene product is related to the Hdm2 oncoprotein, both of which interact with and regulate p53 stability and function. Like Hdm2, Hdmx is able to inhibit p53 transactivation; however, at variance with Hdm2, which promotes ubiquitination, nuclear export, and degradation of p53, Hdmx increases p53 stability. We report here (i) that overexpressed Hdmx is cytoplasmic and Hdm2 recruits Hdmx into the nucleus and (ii) that nuclear Hdmx blocks Hdm2-mediated nuclear export of p53 and down-regulates p53-dependent transcription. Furthermore we showed that Hdmx inhibits Hdm2-mediated p53 ubiquitination. It appears, therefore, that a regulatory loop exists in which Hdm2 regulates the intracellular localization of Hdmx, and nuclear Hdmx regulates several functions of Hdm2 (ubiquitin ligase activity and p53 nuclear export)