Murine MTHFD1-synthetase deficiency, a model for the human MTHFD1 R653Q polymorphism, decreases growth of colorectal tumors.

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+/- ) using combined administration of azoxymethane (AOM) and dextran sodium sulfate (DSS) in male and female wild-type and Mthfd1S+/- mice. Tumor size was significantly smaller in MthfdS+/- mice, particularly in males. A reduction in the proliferation of MthfdS+/- mouse embryonic fibroblast cell lines, compared with wild-type lines, was also observed. Tumor number was not influenced by genotype. The amount of inflammation observed within tumors from male Mthfd1S+/- mice was lower than that in wild-type mice. Gene expression analysis in tumor adjacent normal (pre-neoplastic) tissue identified several genes involved in proliferation (Fosb, Fos, Ptk6, Esr2, Atf3) and inflammation (Atf3, Saa1, TNF-α) that were downregulated in MthfdS+/- males. In females, MthfdS+/- genotype was not associated with these gene expression changes, or with differences in tumor inflammation. These findings suggest that the mechanisms directing tumor growth differ significantly between males and females. We suggest that restriction of purine synthesis, reduced expression of genes involved in proliferation, and/or reduced inflammation lead to slower tumor growth in MTHFD1-synthetase deficiency. These findings may have implications for CRC tumor growth and prognosis in individuals with the R653Q variant. © 2016 Wiley Periodicals, Inc.

Murine diet/tissue and human brain tumorigenesis alter Mthfr/MTHFR 5'-end methylation.

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.

Integrated Place-Based Primary Interventions: Levers and Tensions Related to Multilevel Governance for Community Integrated Pathways, A Multiple Case Study.

Integrated Place-Based Primary Interventions (IPPIs) are considered an innovative response to the challenges and complex issues faced in disadvantaged areas where traditional institutional services have difficulty reaching people in vulnerable situations. IPPIs are an innovative approach to the delivery of in services, conceived as an original community-based local care and service pathways. However, these intervention practices require adaptive modes of governance. In this article, we explore how and to what extent the mode of governance of IPPIs influences the performance of community-integrated pathways. To this end, using a qualitative exploratory multiple-case study design (observation and semi-structured interviews), we describe 4 IPPIs in 3 territories in Quebec. This includes an examination of the levers of action and tensions related to their governance and the performance levels of the community-integrated pathways. We conclude that collaborative and shared multilevel governance, despite its demanding nature, appears to contribute to the longevity of the actions and benefits of IPPIs and could prevent their relevance from being questioned.

A balancing act: interactions within NuA4/TIP60 regulate picNuA4 function in Saccharomyces cerevisiae and humans.

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.

Loss of H3 K79 trimethylation leads to suppression of Rtt107-dependent DNA damage sensitivity through the translesion synthesis pathway.

Genomic integrity is maintained by the coordinated interaction of many DNA damage response pathways, including checkpoints, DNA repair processes, and cell cycle restart. In Saccharomyces cerevisiae, the BRCA1 C-terminal domain-containing protein Rtt107/Esc4 is required for restart of DNA replication after successful repair of DNA damage and for cellular resistance to DNA-damaging agents. Rtt107 and its interaction partner Slx4 are phosphorylated during the initial phase of DNA damage response by the checkpoint kinases Mec1 and Tel1. Because the natural chromatin template plays an important role during the DNA damage response, we tested whether chromatin modifications affected the requirement for Rtt107 and Slx4 during DNA damage repair. Here, we report that the sensitivity to DNA-damaging agents of rtt107Δ and slx4Δ mutants was rescued by inactivation of the chromatin regulatory pathway leading to H3 K79 trimethylation. Further analysis revealed that lack of Dot1, the H3 K79 methyltransferase, led to activation of the translesion synthesis pathway, thereby allowing the survival in the presence of DNA damage. The DNA damage-induced phosphorylation of Rtt107 and Slx4, which was mutually dependent, was not restored in the absence of Dot1. The antagonistic relationship between Rtt107 and Dot1 was specific for DNA damage-induced phenotypes, whereas the genomic instability caused by loss of Rtt107 was not rescued. These data revealed a multifaceted functional relationship between Rtt107 and Dot1 in the DNA damage response and maintenance of genome integrity.

NuA4 and SWR1-C: two chromatin-modifying complexes with overlapping functions and components.

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.

A phylogenetically based secondary structure for the yeast telomerase RNA.

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.

High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice.

BACKGROUND: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions. OBJECTIVE: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism. DESIGN: Folic acid-supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr(+/+) and Mthfr(+/-) mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined. RESULTS: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr(+/-) mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr(+/-) livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr(+/-) mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile. CONCLUSIONS: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.

ß,ß-carotene 15,15'-monooxygenase and its substrate ß-carotene modulate migration and invasion in colorectal carcinoma cells.

BACKGROUND: ß,ß-Carotene 15,15'-monooxygenase (BCMO1) converts ß-carotene to retinaldehyde. Increased ß-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 ß-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-Aza-2'-deoxycytidine decreased, whereas folate-depleted/high-methionine medium increased invasiveness. ß-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. ß-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 ß-carotene.

Genes with aberrant expression in murine preneoplastic intestine show epigenetic and expression changes in normal mucosa of colon cancer patients.

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.