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PDF file - 663K,Supplementary Figure S1: Confirmation of the quality of RNA in microarray experiments by denaturing gel electrophoresis in 1% agarose. Supplementary Figure S2: Representative pyrograms for PDK4, SPRR2A, SPRR1A, NR1H4 and PYCARD.Supplementary Figure S3: Identification of individual genes and functional gene categories with significant expression changes between BALB/c Mthfr+/- FD and BALB/c Mthfr+/+ CD mice. Supplementary Figure S4: Effect of diet and Mthfr genotype on expression of eight genes in normal intestine of BALB/c mice.Supplementary Figure S5: Correlation of methylation results between quantitative bisulfite-pyrosequencing and the Infinium methylation array. Supplementary Figure S6: Confirmation of DNA methylation differences in normal colon between control subjects and individuals with CRC. Supplementary Table S1: Description of control subjects.Supplementary Table S2: Description of cancer patients.Supplementary Table S3: Primer pairs for qRT-PCR for the 13 genes in Figs S1 and S2, and the internal control, Gapdh.Supplementary Table S4: Taqman primers and probes for qRT-PCR of human genes.Supplementary Table S5: Oligonucleotides used for pyrosequencing (PCR primers and sequencing primers).Supplementary Table S6: List of probe sets showing significant changes comparing Mthfr+/- FD and Mthfr+/+ CD BALB/c mice.Supplementary Table S7: Relative mRNA levels for four genes related to PPAR activation (Bcmo1, Aldh1a1), oxidative stress sensitivity (Sprr2a) or oxidative response (Ugt2b36). Supplementary Table S8: Relative expression of PPAR responsive genes.

The NuA4 lysine acetyltransferase complex acetylates histone and nonhistone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1−Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1Δ and epl1-CΔ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1Δ epl1-CΔ double mutants resembled the epl1-CΔ single mutants, indicating that Eaf1’s role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionarily conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1−Eaf1 interaction regulated picNuA4 amount and activity.

Cadre de la recherche : Le vieillissement de la population est particulièrement rapide au Québec et s’accompagne d’une diminution du soutien aux personnes âgées (Maltais, 2018). De surcroît, les aînés représentent un groupe social à haut risque d’isolement social avec les conséquences importantes sur leur santé qui s’ensuivent (Holt-Lunstad et al., 2017). Ces problématiques requièrent la mise en place de programmes nationaux pour veiller au bien-être des aînés, tel le programme Municipalités amies des aînés (MADA). Or, le partenariat actuel entre les acteurs municipaux et ceux du RSSS est défaillant et diminue son apport à la démarche.Objectifs : Exposer les écueils qui entravent le partenariat intersectoriel dans le programme MADA entre la municipalité et le réseau de la santé et des services sociaux (RSSS), selon le modèle de la gouvernance collaborative d’Ansell et Gash (2007). Dégager des pistes de réflexion fondées sur des expériences réalisées au Québec. L’analyse de celles-ci pourrait servir à améliorer le partenariat intersectoriel et, ultimement, à mieux répondre aux besoins de la population vieillissante.Méthodologie : Pour illustrer l’état actuel du partenariat entre les acteurs municipaux et ceux du RSSS, nous avons eu recours à une méthode qualitative suivant trois dispositifs : 1) une recension sommaire des écrits; 2) les résultats de recherche de l’équipe de recherche MADA-Québec; 3) des entrevues avec les acteurs du milieu (aux niveaux stratégique, gestionnaire et opérationnel).Résultats : Les entrevues ont permis de faire ressortir les écueils présents dans le partenariat intersectoriel au niveau des conditions préalables, selon le modèle de gouvernance collaborative d’Ansell et Gash. Elles ont également aidé à identifier des expériences réalisées au Québec qui peuvent servir de pistes de réflexion pour rétablir le partenariat intersectoriel.Conclusion : Le partenariat entre acteurs municipaux et RSSS doit être optimisé afin d’assurer une réponse adéquate aux besoins des aînés dans la démarche MADA.Contribution : L’analyse selon le modèle d’Ansell et Gash démontre une faiblesse du partenariat intersectoriel dans la démarche MADA. Les expériences présentées pourront inspirer de nouvelles pratiques prometteuses à cet égard., Research Framework : The population of Quebec is ageing particularly fast, yet people over the age of 65 tend to receive less support than other age groups (Maltais, 2018). Older people form a group at higher risk of being socially isolated, which can lead to serious health problems (Holt-Lunstad et al. , 2017). These problems require the creation of national programs to ensure their well-being such as the Age-Friendly Municipality Initiative (AFMI). However, the partnership between actors from municipalities and the Health and Social Services Network (HSSN) is flawed and undermines the initiative’s success. Objectives : Present obstacles that harm the intersectoral partnership in the AFMI between actors from municipalities and the HSSN, using the Ansell and Gash’s (2007) collaborative governance model, then reveal avenues of intervention based on certain experiments. Their analysis may help in the enhancement of intersectoral partnership and eventually help to meet the needs of seniors. Methodology : To illustrate the partnership between actors from municipalities and the HSSN, a qualitative methodology was used via three methods: 1) a brief literature review; 2) research results from the Quebec’s Age-Friendly Cities Research Team; 3) interviews conducted with actors from the field (strategic, administrative and operational level). Results : The interviews allowed the authors to identify obstacles in the intersectoral partnership in terms of starting conditions, based on the Ansell and Gash’s (2007) collaborative governance model. They also helped to identify certain experiments to guide new avenues of intervention and restore the intersectoral partnership. Conclusion : The partnership between actors from municipalities and the HSSN must be enhanced to allow an appropriate response to older people’s needs in the AFM process. Contribution : The analysis based on Ansell and Gash’s model identifies a weakness in the intersectoral partnership in the AFM process. The experiments proposed may inspire new promising practices on this subject.

Cet article pose la thèse que les pratiques réflexives engagées avec une pluralité d’acteurs, incluant les individus dits « vulnérables », participent à des changements sur les plan individuels et interpersonnels. Pour ce faire, des espaces de rencontre, qu’ils soient concrets ou symboliques, doivent être créés. Les pratiques qui se développent dans ces espaces favorisent la conscientisation ainsi que la réflexion collective et viennent enrichir et transformer les pratiques d’action sociale. Par ailleurs, les réflexions qui en émergent rendent possible le développement de processus de subjectivation individuelle et collective, et sont garantes de bénéfices pour tous. This article underlines the thesis that reflective practices, initiated with multiple actors including the so-called "vulnerable" individuals, contribute to change both at individual and interpersonal levels. In order to achieve this, one must create physical or figurative meeting spaces. The practices that then develop promote awareness and collective thinking, as well as enriching and transforming social action. In turn, the collective thoughts that emerge permit individual and collective subjectivation processes, and are guarantees of benefits for all. Reflective practices, awareness, subjectivation, space, collectivisation

Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5' to the upstream translational start site or in another region 3' to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.

Background: Cancer cells maintain high rates of glycolysis. Pyruvate dehydrogenase kinases (PDK) contribute to this phenomenon, which favours apoptosis resistance and cellular transformation. We previously reported upregulation of PDK4 in normal mucosa of colorectal cancer (CRC) patients compared with controls and in preneoplastic intestine of our mouse model. Decreased methylation of four consecutive PDK4 CpGs was observed in normal mucosa of patients. Although other members of the PDK family have been investigated for transformation potential, PDK4 has not been extensively studied. Methods: PDK4 methylation in blood of CRC patients and controls was evaluated by pyrosequencing. PDK4 expression in human colon carcinoma cells was down-regulated by RNAi. Cellular migration and invasion, apoptosis and qRT-PCR of key genes were assessed. Results: Pyrosequencing revealed decreased methylation of the same four consecutive CpGs in the blood of patients compared with controls. Cellular migration and invasion were reduced and apoptosis was increased following transient or stable inhibition of PDK4. Expression of vimentin, HIF-1 and VEGFA was reduced. Conclusions: These studies demonstrate the involvement of PDK4 in transformation. Methylation assessment of PDK4 in the blood may be useful for non-invasive CRC detection. PDK4 should be considered as a target for development of anticancer strategies and therapies

The common R653Q variant (∼20% homozygosity in Caucasians) in the synthetase domain of the folate-metabolizing enzyme MTHFD1 reduces purine synthesis. Although this variant does not appear to affect risk for colorectal cancer, we questioned whether it would affect growth of colorectal tumors. We induced tumor formation in a mouse model for MTHFD1-synthetase deficiency (Mthfd1S

An understanding of early genetic/epigenetic changes in colorectal cancer would aid in diagnosis and prognosis. To identify these changes in human preneoplastic tissue, we first studied our mouse model in which Mthfr+/− BALB/c mice fed folate-deficient diets develop intestinal tumors in contrast to Mthfr+/+ BALB/c mice fed control diets. Transcriptome profiling was performed in normal intestine from mice with low or high tumor susceptibility. We identified 12 upregulated and 51 downregulated genes in tumor-prone mice. Affected pathways included retinoid acid synthesis, lipid and glucose metabolism, apoptosis and inflammation. We compared murine candidates from this microarray analysis, and murine candidates from an earlier strain-based comparison, with a set of human genes that we had identified in previous methylome profiling of normal human colonic mucosa, from colorectal cancer patients and controls. From the extensive list of human methylome candidates, our approach uncovered five orthologous genes that had shown changes in murine expression profiles (PDK4, SPRR1A, SPRR2A, NR1H4, and PYCARD). The human orthologs were assayed by bisulfite-pyrosequencing for methylation at 14 CpGs. All CpGs exhibited significant methylation differences in normal mucosa between colorectal cancer patients and controls; expression differences for these genes were also observed. PYCARD and NR1H4 methylation differences showed promise as markers for presence of polyps in controls. We conclude that common pathways are disturbed in preneoplastic intestine in our animal model and morphologically normal mucosa of patients with colorectal cancer, and present an initial version of a DNA methylation-based signature for human preneoplastic colon. Cancer Prev Res; 6(11); 1171–81. ©2013 AACR.

Background: b-Carotene 15,15#-monooxygenase 1 (BCMO1) converts b-carotene to retinaldehyde. Increased b-carotene consumption is linked to antitumor effects. Retinoic acid reduces the invasiveness in cancer, through inhibition of matrix metalloproteinases (MMPs). In our studies of a mouse model that develops intestinal tumors after low dietary folate, we found reduced BCMO1 expression in normal preneoplastic intestine of folate-deficient tumor-prone mice. Objective: Our goal was to determine whether BCMO1 expression could influence transformation potential in human colorectal carcinoma cells, by examining the effect of BCMO1 modulation on cellular migration and invasion, and on expression of MMPs. Design: LoVo colon carcinoma cells were transfected with BCMO1 small interfering RNA (siRNA) or scrambled siRNA. Migration and invasion were measured, and the expression of BCMO1, MMP7, and MMP28 was assessed by quantitative reverse-transcriptase polymerase chain reaction. These variables were also measured after treatment of cells with retinoic acid, 5-aza-2#-deoxycytidine, folate-depleted/ high-methionine medium, and b-carotene. Results: Retinoic acid decreased the migration, invasion, and expression of MMP28 mRNA. Transfection of cells with BCMO1 siRNA inhibited BCMO1 expression, enhanced migration and invasion, and increased expression of MMP7 and MMP28. 5-Aza2#-deoxycytidine decreased, whereas folate-depleted/high-methionine medium increased invasiveness. b-Carotene increased BCMO1 expression and reduced invasiveness with a decrease in expression of MMP7 and MMP28. Conclusions: Inhibition of BCMO1 expression is associated with increased invasiveness of colon cancer cells and increased expression of MMP7 and MMP28. b-Carotene can upregulate BCMO1 and reverse these effects. These novel associations suggest a critical role for BCMO1 in cancer and provide a mechanism for the proposed antitumor effects of b-carotene. Am J Clin Nutr doi: 10. 3945/ajcn.113.060996.

Chromatin structure is important for the compaction of eukaryotic genomes, thus chromatin modifications play a fundamental role in regulating many cellular processes. The coordinated activities of various chromatin-remodelling and -modifying complexes are crucial in maintaining distinct chromatin neighbourhoods, which in turn ensure appropriate gene expression, as well as DNA replication, repair, and recombination. SWR1-C is an ATP-dependent histone deposition complex for the histone variant H2A.Z, whereas NuA4 is a histone acetyltransferase for histones H4, H2A, and H2A.Z. Together the NuA4 and SWR1-C chromatin-modifying complexes alter the chromatin structure through 3 distinct modifications in yeast: post-translational addition of chemical groups, ATP-dependent chromatin remodelling, and histone variant incorporation. These 2 multi-protein complexes share 4 subunits and function together to regulate the circuitry of H2A.Z biology. The components and functions of both multi-protein complexes are evolutionarily conserved and play important roles in multi-cellular development and cellular differentiation in higher eukaryotes. This review will summarize recent findings about NuA4 and SWR1-C and will focus on the connection between these complexes by investigating their physical and functional interactions through eukaryotic evolution.

Background: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure. Results: We cloned and analyzed the complete telomerase RNA loci ( TLC1 ) from all known Saccharomyces species belonging to the "sensu stricto" group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core. Conclusions: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.