The Polo-like kinases as recipients and enablers of epigenetic modifications in tumourigenesis

Many highly conserved proteins have evolved specific niches in the cell cycle. For the Polo-like kinases (PLKs), these roles include centrosome duplication and maturation, the interaction with key DNA damage response proteins, cytokinesis, and chromosome separation. The PLKs deliver their effects via phosphorylation of their substrates at serine and threonine residues. Due to their importance in the cell cycle, expression of the PLKs is strictly governed. PLK deregulation is ubiquitously associated with malignancy. Elucidating how these proteins are regulated is key to understanding how their proper function can be restored in tumourigenesis. In recent years, the study of epigenetics as an additional mechanism controlling gene expression has come to the forefront. Epigenetic mechanisms include the addition or removal of methyl groups at the DNA and histone levels. My studies describe the regulation of PLK expression at the DNA level through the epigenetic mechanism of DNA methylation, the microenvironmental alteration of PLK epigenetic marks, and how these modifications translate in vivo in the context of carcinogenesis. Furthermore, I describe a novel interaction between PLK4, the most structurally divergent of the PLKs, and PRMT5, another evolutionarily conserved protein that is responsible for the methylation of arginine residues. Here I propose that the DNA hypermethylation of PLK4 promoter, and its subsequent reduction at the protein level, contributes to a tumourigenic state. As a signalling protein, the significant decrease in PLK4 creates a domino effect destabilizing global epigenetic marks, inhibiting the expression of the guardian of the genome, p53, and potentially contributing to the upregulation of the pro-mitotic protein, PLK1, the original member of the PLKs

Gene expression patterns in heterozygous Plk4 murine embryonic fibroblasts

<p>Abstract</p> <p>Background</p> <p>The polo-like kinases (Plks) are a group of serine/threonine kinases which have roles in many aspects of cellular function including the regulation of mitotic activity and cellular stress responses. This study focuses on Plk4, the most divergent member of the Plk family, which is necessary for proper cellular proliferation. More specifically, alterations in Plk4 levels cause significantly adverse mitotic defects including abnormal centrosome duplication and aberrant mitotic spindle formation. We sought to clarify the effect of reduced Plk4 levels on the cell by examining transcript profiles of <it>Plk4 </it>wild-type and heterozygous mouse embryonic fibroblasts (MEFs). Subsequently, the levels of several key proteins involved in the DNA damage response were examined.</p> <p>Results</p> <p>143 genes were found to be significantly up-regulated in the heterozygous MEFs compared to their wild-type counterparts, while conversely, 9 genes were down-regulated. Numerous genes with increased transcript levels in heterozygous MEFs were identified to be involved in p53-dependent pathways. Furthermore, examination of the promoter regions of all up- and down-regulated genes revealed that the majority contained putative p53 responsive elements.</p> <p>An analysis of transcript levels in MEFs after exposure to either ionizing or ultraviolet radiation revealed a significant change between wild type and heterozygous MEFS for Plk4 transcript levels upon only UV exposure. Furthermore, changes in protein levels of several important cell check-point and apoptosis regulators were examined, including p53, Chk1, Chk2, Cdc25C and p21. In heterozygous MEFs, p53, p21 and Chk2 protein levels were at significantly higher levels. Furthermore, p53 activity was increased 5 fold in the <it>Plk4 </it>heterozygous MEFs.</p> <p>Conclusion</p> <p>Global transcript profiles and levels of key proteins involved in cellular proliferation and DNA damage pathways were examined in wild-type and <it>Plk4 </it>heterozygous MEFs. It was determined that Plk4 haploinsufficiency leads to changes in the levels of RNA accumulation for a number of key cellular genes as well as changes in protein levels for several important cell cycle/DNA damage proteins. We propose a model in which reduced Plk4 levels invoke an increase in p53 levels that leads to the aforementioned changes in global transcription profiles.</p

The Last Cordilleran Ice Sheet in Southern Yukon Territory

The Cordilleran Ice Sheet in Yukon radiated from ice-divides in the Selwyn, PeIIy1 Cassiar, and eastern Coast Mountains and was contiguous with a piedmond glacier complex from the St. Elias Mountains. Expansion of glaciers in divide areas could have been underway by 29 ka BP but these did not merge to form the ice sheet until after 24 ka BP. The firn line fell to approximately 1500 m at the climax of McConnell Glaciation. Flow within the ice sheet was more analogous to a complex of merged valley glaciers than to that of extant ice sheets: topographic relief was typically equal to or exceeded ice thickness, and strongly influenced ice flow. Surface gradients on the ice sheet were fractions of a degree. Steeper ice-surface gradients occurred locally along the digitate ice margin. Retreat from the terminal moraine was initially gradual as indicated by recessional moraines within a few tens of kilometres of the terminal moraine. Small magnitude readvances occurred locally. The ice sheet eventually disappeared through regional stagnation and downwasting in response to a rise in the firn line to above the surface of the ice sheet. Regional déglaciation was complete prior to approximately 10 ka BP.L'Inlandsis de la Cordillère a progressé dans le Yukon à partir des lignes de partage des glaces des monts Selwyn, PeIIy et Cassiar et de l'est de la chaîne Côtière; il était contigu à un glacier de piémont complexe en provenance des monts St. Elias. Les glaciers ont pu se développer dans les régions de partage des glaces à partir de 29 ka BP, mais ils ne se sont fusionnés pour former un inlandsis qu'à partir de 24 ka BP. À l'optimum de la Glaciation de McConnell, la ligne de névé s'est abaissée à environ 1500 m. L'écoulement à l'intérieur de l'inlandsis ressemblait davantage à celui d'un complexe de glaciers de vallées coalescents qu'à celui des inlandsis actuels: le relief rejoignait l'épaisseur de glace ou la dépassait et influençait grandement l'écoulement glaciaire. Sur l'inlandsis, les gradients du profil topographique se mesuraient en fractions de degrés, mais le long de la marge digitée, les gradients étaient par endroits plus prononcés. Le retrait à partir de la moraine frontale a au départ été graduel comme l'indiquent les moraines de retrait à quelques dizaines de kilomètres de la moraine frontale. Il y eut localement quelques récurrences mineures. L'inlandsis disparut avec le temps par stagnation à une échelle régionale et fonte en réponse à une hausse de la ligne de névé au-dessus de la surface de l'inlandsis. À l'échelle régionale, la déglaciation était terminée avant 10kaBP environ.Die Kordilleren-Eisdecke in Yukon breitete sich strahlenfôrmig von den Eisscheiden der Seywyn-, Pelley- und Cassiar-Berge und den ôstlichen Kùstenbergen aus und grenzte an einen Vorlandgletscher von den St. Elias-Bergen. Die Ausdehnung der Gletscher in den Eisscheiden-Gebieten kônnte schon um 29 ka v.u.Z. eingesetzt haben; doch sind sie erst nach 24 ka v.u.Z. zu der Eisdecke verschmolzen. Die Firn-Linie sank auf etwa 1500m wâhrend des Hôhepunkts der McConnell-Vereisung. Das Fliessen innerhalb der Eisdecke entsprach mehr einer Einheit verschmolzener Talgletscher als dem der noch vorhandenen Eisdecke: das topographische relief stimmt in beispielhafter Weise mit der Dicke des Eises ùberein, oder ùbertraf dièse und beeinflusste sehr stark die Eisstrômung. Auf der Oberflàche der Eisdecke betrug das Gefàlle nur Bruchteile eines Grads. Steilere Eisoberflàchengefàlle gab es ôrtlich entlang des fingerfôrmigen Eisrands. Der Rùckzug von der Endmoràne geschah anfangs graduell, was aus den einige 10 km von der Endmoràne entfernten Rùckzugsmorànen hervogeht. Ôrtlich gab es einige kleinere Rùckvorstôsse. Die Eisdecke verschwand schliesslich durch régionale Stagnation und Abzehrung infolge eines Anhubs der Firnlinie ùber die Oberflàche der Eisdecke hinaus. Die régionale Enteisung war vor etwa 10 ka v.u.Z. vollendet

Aberrant methylation of Polo-like kinase CpG islands in Plk4 heterozygous mice

<p>Abstract</p> <p>Background</p> <p>Hepatocellular carcinoma (HCC), one of the most common cancers world-wide occurs twice as often in men compared to women. Predisposing conditions such as alcoholism, chronic viral hepatitis, aflatoxin B1 ingestion, and cirrhosis all contribute to the development of HCC.</p> <p>Methods</p> <p>We used a combination of methylation specific PCR and bisulfite sequencing, qReal-Time PCR (qPCR), and Western blot analysis to examine epigenetic changes for the <it>Polo-like kinases </it>(<it>Plks</it>) during the development of hepatocellular carcinoma (HCC) in <it>Plk4 </it>heterozygous mice and murine embryonic fibroblasts (MEFs).</p> <p>Results</p> <p>Here we report that the promoter methylation of <it>Plk4 </it>CpG islands increases with age, was more prevalent in males and that <it>Plk4 </it>epigenetic modification and subsequent downregulation of expression was associated with the development of HCC in <it>Plk4 </it>mutant mice. Interestingly, the opposite occurs with another Plk family member, <it>Plk1 </it>which was typically hypermethylated in normal liver tissue but became hypomethylated and upregulated in liver tumours. Furthermore, upon alcohol exposure murine embryonic fibroblasts exhibited increased <it>Plk4 </it>hypermethylation and downregulation along with increased centrosome numbers and multinucleation.</p> <p>Conclusions</p> <p>These results suggest that aberrant <it>Plk </it>methylation is correlated with the development of HCC in mice.</p

The calcium-binding protein S100P in normal and malignant human tissues

<p>Abstract</p> <p>Background</p> <p>S100P is a Ca²⁺binding protein overexpressed in a variety of cancers, and thus, has been considered a potential tumor biomarker. Very little has been studied about its normal expression and functions.</p> <p>Methods</p> <p>We examined S100P expression in normal human tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. S100P protein expression was also studied in a series of tumors, consisting of 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas using a novel anti-S100P monoclonal antibody.</p> <p>Results</p> <p>Among the normal tissues, the highest S100P mRNA levels were observed in the placenta and esophagus. Moderate signals were also detected in the stomach, duodenum, large intestine, prostate and leukocytes. At the protein level, the highest reactions for S100P were seen in the placenta and stomach. Immunostaining of tumor specimens showed that S100P protein is expressed in all the tumor categories included in the study, being most prevalent in gastric tumors.</p> <p>Conclusion</p> <p>Based on our observations, S100P is widely expressed in both normal and malignant tissues. The high expression in some tumors suggests that it may represent a potential target molecule for future diagnostic and therapeutic applications.</p

Molecular and Behavioral Differentiation among Brazilian Populations of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae)

Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. There is strong evidence that L. longipalpis is a species complex, but there is still no consensus regarding the number of species occurring in Brazil. We combined molecular and behavioral analyses of a number of L. longipalpis populations in order to help clarify this question. This approach has allowed us to identify two main groups of populations in Brazil. One group probably represents a single species distributed mainly throughout the coastal regions of North and Northeast Brazil and whose males produce the same type of copulation song and pheromone. The second group is more heterogeneous, probably represented by a number of incipient species with different levels of genetic divergence among the siblings that produce different combinations of copulation songs and pheromones. The high level of complexity observed raises important questions concerning the epidemiological consequences of this incipient speciation process

Modified penetrance of coding variants by cis-regulatory variation contributes to disease risk

Coding variants represent many of the strongest associations between genotype and phenotype; however, they exhibit interindividual differences in effect, termed 'variable penetrance'. Here, we study how cis-regulatory variation modifies the penetrance of coding variants. Using functional genomic and genetic data from the Genotype-Tissue Expression Project (GTEx), we observed that in the general population, purifying selection has depleted haplotype combinations predicted to increase pathogenic coding variant penetrance. Conversely, in cancer and autism patients, we observed an enrichment of penetrance increasing haplotype configurations for pathogenic variants in disease-implicated genes, providing evidence that regulatory haplotype configuration of coding variants affects disease risk. Finally, we experimentally validated this model by editing a Mendelian single-nucleotide polymorphism (SNP) using CRISPR/Cas9 on distinct expression haplotypes with the transcriptome as a phenotypic readout. Our results demonstrate that joint regulatory and coding variant effects are an important part of the genetic architecture of human traits and contribute to modified penetrance of disease-causing variants.Peer reviewe

Monitoring the Morphology of M87* in 2009–2017 with the Event Horizon Telescope

The Event Horizon Telescope (EHT) has recently delivered the first resolved images of M87*, the supermassive black hole in the center of the M87 galaxy. These images were produced using 230 GHz observations performed in 2017 April. Additional observations are required to investigate the persistence of the primary image feature—a ring with azimuthal brightness asymmetry—and to quantify the image variability on event horizon scales. To address this need, we analyze M87* data collected with prototype EHT arrays in 2009, 2011, 2012, and 2013. While these observations do not contain enough information to produce images, they are sufficient to constrain simple geometric models. We develop a modeling approach based on the framework utilized for the 2017 EHT data analysis and validate our procedures using synthetic data. Applying the same approach to the observational data sets, we find the M87* morphology in 2009–2017 to be consistent with a persistent asymmetric ring of ~40 μas diameter. The position angle of the peak intensity varies in time. In particular, we find a significant difference between the position angle measured in 2013 and 2017. These variations are in broad agreement with predictions of a subset of general relativistic magnetohydrodynamic simulations. We show that quantifying the variability across multiple observational epochs has the potential to constrain the physical properties of the source, such as the accretion state or the black hole spin