Composition and Formation of the Saccharomyces cerevisiae Centromeric Nucleosome

The kinetochore is a complex, multi-protein structure required for proper chromosome segregation in all eukaryotes. The Saccharomyces cerevisiae kinetochore consists of over 65 known proteins which work in concert to facilitate equal distribution of the replicated genome. The S. cerevisiae CenH3 histone variant Cse4 is an evolutionarily conserved histone H3-like inner kinetochore protein that is essential for kinetochore function. Through immunopurification of Cse4 interacting proteins we have identified the previously uncharacterized protein Scm3. Here we report the characterization of S. cerevisiae Scm3, an essential protein with putative orthologs in fungi which possess either point or regional centromeres. We find that Scm3 localizes to all budding yeast centromeres. Construction of a conditional allele of SCM3 has allowed us to characterize the phenotype of cells lacking Scm3. Scm3 depleted cells fail to properly localize the components of the inner kinetochore, including Cse4 and Ndc10, and arrest in metaphase with duplicated spindle poles, short spindles, and unequal DNA distribution in the daughter cells. Our data suggest that Scm3 is not an actual component of the centromeric nucleosome, but rather intimately associates with it. Additional in vivo and in vitro analysis of Cse4 reveals a single centromeric nucleosome that contains an octamer of Cse4, H2A, H2B, and H4. Based on these findings, we hypothesize that Scm3 is a novel yeast inner kinetochore protein that functions in the formation and maintenance of a segregation competent kinetochore through recruitment of the Cse4 octameric nucleosome

Principios de marketing estratégico

Departament d'Administració d'Empreses i Màrqueting. Codis assignatura: AE1018, FC1018, EC101

A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface

HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation

CAL1 is the Drosophila CENP-A assembly factor

Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1. Here, we show that targeting CAL1 to noncentromeric DNA in Drosophila cells is sufficient to heritably recruit CENP-A, kinetochore proteins, and microtubule attachments. CAL1 selectively interacts with CENP-A and is sufficient to assemble CENP-A nucleosomes that display properties consistent with left-handed octamers. The CENP-A assembly activity of CAL1 resides within an N-terminal domain, whereas the C terminus mediates centromere recognition through an interaction with CENP-C. Collectively, this work identifies the “missing” CENP-A chaperone in flies, revealing fundamental conservation between insect and vertebrate centromere-specification mechanisms

The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites

The impact of customer engagement on retailer's brand equity components

Strong brand equity is important for any business. Although the concept of brand equity has been studied in various fields, its analysis has not been as extensive in the retail sector. On the other hand, the analysis of engagement is gaining more importance in recent times. Customer engagement is an increasingly relevant and researched topic. However, studies that relate this concept to retail trade are not common. The present work aims to analyze the effect of engagement on the different components of retail brand equity. The a priori model considers the previous research and the proposed hypotheses. A Confirmatory Factor Analysis is performed, based on the data obtained through a structured questionnaire with closed questions and a 5-point Likert-type response scale. The study sample consists of 623 respondents. This study involved a conceptual model that includes the brand equity dimensions (awareness, perceived quality, image, perceived value, and loyalty) to gain the research goal. The hypothesized causal model relates the variables that make up brand equity and the engagement influence on them. The empirical analysis results showed that customer engagement positively affects all the components of the brand equity retailer (except its image), mainly concerning retailer awareness, loyalty, and perceived quality. The authors concluded that retailer awareness, loyalty towards the retailer, and retailer perceived quality are influenced by engagement. Consequently, it would be necessary for the retailer manager to pay special attention to creating actions that contribute to customers' engagement in the different areas of interaction with them, both online and at the physical point of sale. For future studies, the geographic space should be expanded, considering different regions or even countries and observing possible differences in the behavior of the interviewees

Detrimental incorporation of excess Cenp-A/Cid and Cenp-C into Drosophila centromeres is prevented by limiting amounts of the bridging factor Cal1

Propagation of centromere identity during cell cycle progression in higher eukaryotes depends critically on the faithful incorporation of a centromere-specific histone H3 variant encoded by CENPA in humans and cid in Drosophila. Cenp-A/Cid is required for the recruitment of Cenp-C, another conserved centromere protein. With yeast three-hybrid experiments, we demonstrate that the essential Drosophila centromere protein Cal1 can link Cenp-A/Cid and Cenp-C. Cenp-A/Cid and Cenp-C interact with the N- and C-terminal domains of Cal1, respectively. These Cal1 domains are sufficient for centromere localization and function, but only when linked together. Using quantitative in vivo imaging to determine protein copy numbers at centromeres and kinetochores, we demonstrate that centromeric Cal1 levels are far lower than those of Cenp-A/Cid, Cenp-C and other conserved kinetochore components, which scale well with the number of kinetochore microtubules when comparing Drosophila with budding yeast. Rather than providing a stoichiometric link within the mitotic kinetochore, Cal1 limits centromeric deposition of Cenp-A/Cid and Cenp-C during exit from mitosis. We demonstrate that the low amount of endogenous Cal1 prevents centromere expansion and mitotic kinetochore failure when Cenp-A/Cid and Cenp-C are present in excess

Influence of the Internet on Retailer's perceived quality in the generation of retailer's brand equity

This study analyses the relationship between the different components of the retailer's brand equity, as well as the influence that the use of internet has on the formation of retailer’s brand equity. As some authors point out, there is a need for a measure of the retailer's brand equity (Boo et al, 2008; Lee and Back, 2010). The model proposed to analyse retailer’s brand equity is based on the one used by Boo et al. (2008) who, in turn, start from the brand equity proposal of the authors Aaker (1991) and Keller (1993). A theoretical a priori model was specified according to the results in previous literature and our hypotheses. Prior to testing the model, the dimensionality of the scales was stablished with Confirmatory Factor Analyses (CFAs). Once the dimensionality was stablished, full structural equations model (SEM) was tested. This study provides evidences on the effect of the use of internet on retailer´s perceived quality. In turn, the influence that this variable has on the retailer's image and on its perceived value is shown. These relationships have an influence on consumer loyalty to the retailer, which, in turn, generates brand equity for the retailer

Poesia en guitarra

El present treball tracta sobre els diferents tipus de relacions que poden establir-se entre poesia i música, a partir de l’estudi d’obres del repertori guitarrístic. Les obres elegides tenen la particularitat d’haver estat creades a partir de uns textos poètics concrets, els quals són motiu d’inspiració pel compositor. Cadascuna de les obres objecte d’estudi és contextualitzada i analitzada segons diferents paràmetres, segons com aquests es relacionen amb els textos. L’objectiu final és poder utilitzar les conclusions derivades del treball com a eina a l’hora de crear i defensar la interpretació d’obres vinculades a textos poètics.El siguiente trabajo trata sobre las diferentes clases de relaciones que pueden establecerse entre poesía y música, a partir del estudio de obras pertenecientes al repertorio guitarrístico. Las obras elegidas tienen la particularidad de haver sido creadas a partir de determinados textos poéticos, los cuáles son motivo de inspiración para el compositor. Cada una de las obras objeto de estudio se contextualiza y se analiza según diferentes parámetros, según como estos se relacionan con los textos. El objetivo final es poder utilizar las conclusiones derivadas del trabajo como herramienta para crear y defensar la interpretación de obras vinculadas a textos poéticos.This project talks about different types of relationships that can be stablished beween potry and music, out of the study of works from the guitar repertory. The chosen works share the peculiarity of having been created out of concrete poetical texts, which have become compositor’s inspiration cause. Each of every studied work is analised in its context through different parameters, depending on how their relatioship with the corresponding texts is. The final objective is to be able to use the project conclusions as a tool to create and defend the interpretation of works linked to poetical texts

The quantitative architecture of centromeric chromatin

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation. DOI: http://dx.doi.org/10.7554/eLife.02137.00