DNA demethylase activity maintains intestinal cells in an undifferentiated state following loss of APC.

Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state that was released upon knockdown of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpß and are indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpß. Apc mutants lack retinoic acid as a result of the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1, and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wild-type APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.

A two-step model for colon adenoma initiation and progression caused by APC loss.

Aberrant Wnt/beta-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, we show that homozygous loss of APC causes failed intestinal cell differentiation but that this occurs in the absence of nuclear beta-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing beta-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor C-terminal binding protein-1 (CtBP1), whereas proliferation defects and nuclear accumulation of beta-catenin require the additional activation of KRAS. These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, whereas KRAS activation and beta-catenin nuclear localization promote adenoma progression to carcinomas as a second step. Consistent with this model, human FAP adenomas showed robust upregulation of CtBP1 in the absence of detectable nuclear beta-catenin, whereas nuclear beta-catenin was detected in carcinomas.

A Bridge too Far? Comparison of Transition Metal Complexes of Dibenzyltetraazamacrocycles with and without Ethylene Cross-Bridges: X-ray Crystal Structures, Kinetic Stability, and Electronic Properties.

Tetraazamacrocycles, cyclic molecules with four nitrogen atoms, have long been known to produce highly stable transition metal complexes. Cross-bridging such molecules with two-carbon chains has been shown to enhance the stability of these complexes even further. This provides enough stability to use the resulting compounds in applications as diverse and demanding as aqueous, green oxidation catalysis all the way to drug molecules injected into humans. Although the stability of these compounds is believed to result from the increased rigidity and topological complexity imparted by the cross-bridge, there is insufficient experimental data to exclude other causes. In this study, standard organic and inorganic synthetic methods were used to produce unbridged dibenzyl tetraazamacrocycle complexes of Co, Ni, Cu, and Zn that are analogues of known cross-bridged tetraazamacrocycles and their transition metal complexes to allow direct comparison of molecules that are identical except for the cross-bridge. The syntheses of the known tetraazamacrocycles and the new transition metal complexes were successful with high yields and purity. Initial chemical characterization of the complexes was conducted by UV-Visible spectroscopy, while cyclic voltammetry showed more marked differences in electronic properties from bridged versions. Direct comparison studies of the unbridged and bridged compounds' kinetic stabilities, as demonstrated by decomposition using high acid concentration and elevated temperature, showed that the cyclen-based complex stability did not benefit from cross-bridging. This is likely due to poor complementarity with the Cu2+ ion while cyclam-based complexes benefited greatly. We conclude that ligand-metal complementarity must be maintained in order for the topological and rigidity constraints imparted by the cross-bridge to contribute significantly to complex robustness.

Transcription factor Oct1 is a somatic and cancer stem cell determinant.

Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1(HI) cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24(LO)CD44(HI) cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDH(HI) and dye efflux(HI) cells, and increasing Oct1 increases the proportion of ALDH(HI) cells. Normal ALDH(HI) cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.

Dnmt3 and G9a cooperate for tissue-specific development in zebrafish.

Although DNA methylation is critical for proper embryonic and tissue-specific development, how different DNA methyltransferases affect tissue-specific development and their targets remains unknown. We address this issue in zebrafish through antisense-based morpholino knockdown of Dnmt3 and Dnmt1. Our data reveal that Dnmt3 is required for proper neurogenesis, and its absence results in profound defects in brain and retina. Interestingly, other organs such as intestine remain unaffected suggesting tissue-specific requirements of Dnmt3. Further, comparison of Dnmt1 knockdown phenotypes with those of Dnmt3 suggested that these two families have distinct functions. Consistent with this idea, Dnmt1 failed to complement Dnmt3 deficiency, and Dnmt3 failed to complement Dnmt1 deficiency. Downstream of Dnmt3 we identify a neurogenesis regulator, lef1, as a Dnmt3-specific target gene that is demethylated and up-regulated in dnmt3 morphants. Knockdown of lef1 rescued neurogenesis defects resulting from Dnmt3 absence. Mechanistically, we show cooperation between Dnmt3 and an H3K9 methyltransferase G9a in regulating lef1. Further, like Dnmt1-Suv39h1 cooperativity, Dnmt3 and G9a seemed to function together for tissue-specific development. G9a knockdown, but not Suv39h1 loss, phenocopied dnmt3 morphants and G9a overexpression provided a striking rescue of dnmt3 morphant phenotypes, whereas Suv39h1 overexpression failed, supporting the notion of specific DNMT-histone methyltransferase networks. Consistent with this model, H3K9me3 levels on the lef1 promoter were reduced in both dnmt3 and g9a morphants, and its knockdown rescued neurogenesis defects in g9a morphants. We propose a model wherein specific DNMT-histone methyltransferase networks are utilized to silence critical regulators of cell fate in a tissue-specific manner.

The Role of Extracellular Vesicles in ß-Cell Function and Viability: A Scoping Review.

Extracellular vesicles (EVs) released by cells throughout the body have been implicated in diabetes pathogenesis. Understanding the role of EVs in regulation of ß-cell function and viability may provide insights into diabetes etiology and may lead to the development of more effective screening and diagnostic tools to detect diabetes earlier and prevent disease progression. This review was conducted to determine what is known from the literature about the effect of EV crosstalk on pancreatic ß-cell function and viability in the pathogenesis of diabetes mellitus, to perform a gap analysis for future research directions, and to discuss implications of available evidence for diabetes care. The literature search yielded 380 studies from which 31 studies were determined to meet eligibility criteria. The majority of studies had the disease context of autoimmunity in T1DM. The most commonly studied EV crosstalk dynamics involved localized EV-mediated communication between ß-cells and other islet cells, or between ß-cells and immune cells. Other organs and tissues secreting EVs that affect ß-cells include skeletal muscle, hepatocytes, adipocytes, immune cells, bone marrow, vascular endothelium, and mesenchymal stem cells. Characterization of EV cargo molecules with regulatory effects in ß-cells was conducted in 24 studies, with primary focus on microRNA cargo. Gaps identified included scarcity of evidence for the effect on ß-cell function and viability of EVs from major metabolic organs/tissues such as muscle, liver, and adipose depots. Future research should address these gaps as well as characterize a broader range of EV cargo molecules and their activity in ß-cells.

Zebra fish Dnmt1 and Suv39h1 regulate organ-specific terminal differentiation during development.

DNA methylation and histone methylation are two key epigenetic modifications that help govern heterochromatin dynamics. The roles for these chromatin-modifying activities in directing tissue-specific development remain largely unknown. To address this issue, we examined the roles of DNA methyltransferase 1 (Dnmt1) and the H3K9 histone methyltransferase Suv39h1 in zebra fish development. Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the intestine, exocrine pancreas, and retina. Interestingly, not all tissues required Dnmt1, as differentiation of the liver and endocrine pancreas appeared normal. Proper differentiation depended on Dnmt1 catalytic activity, as Dnmt1 morphants could be rescued by active zebra fish or human DNMT1 but not by catalytically inactive derivatives. Dnmt1 morphants exhibited dramatic reductions of both genomic cytosine methylation and genome-wide H3K9 trimethyl levels, leading us to investigate the overlap of in vivo functions of Dnmt1 and Suv39h1. Embryos lacking Suv39h1 had organ-specific terminal differentiation defects that produced largely phenocopies of Dnmt1 morphants but retained wild-type levels of DNA methylation. Remarkably, suv39h1 overexpression rescued markers of terminal differentiation in Dnmt1 morphants. Our results suggest that Dnmt1 activity helps direct histone methylation by Suv39h1 and that, together, Dnmt1 and Suv39h1 help guide the terminal differentiation of particular tissues.

Up-regulation of CYP26A1 in adenomatous polyposis coli-deficient vertebrates via a WNT-dependent mechanism: implications for intestinal cell differentiation and colon tumor development.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene seem to underlie the initiation of many colorectal carcinomas. Loss of APC function results in accumulation of beta-catenin and activation of beta-catenin/TCF-dependent transcription. Recent studies have implicated APC in controlling retinoic acid biosynthesis during normal intestinal development through a WNT-independent mechanism. Paradoxically, however, previous studies found that dietary supplementation of Apc(MIN) mice with retinoic acid failed to abrogate adenoma formation. While investigating the above finding, we found that expression of CYP26A1, a major retinoic acid catabolic enzyme, was up-regulated in Apc(MIN) mouse adenomas, human FAP adenomas, human sporadic colon carcinomas, and in the intestine of apc(mcr) mutant zebrafish embryos. Mechanistically, cyp26a1 induction following apc mutation is dependent on WNT signaling as antisense morpholino knockdown of tcf4 or injection of a dnLEF construct into apc(mcr) mutant zebrafish suppressed expression of cyp26a1 along with known WNT target genes. In addition, injection of stabilized beta-catenin or dnGSK3beta into wild-type embryos induced cyp26a1 expression. Genetic knockdown or pharmacologic inhibition of cyp26a1 in apc(mcr) mutant zebrafish embryos rescued gut differentiation defects such as expression of intestinal fatty acid-binding protein and pancreatic trypsin. These findings support a novel role for APC in balancing retinoic acid biosynthesis and catabolism through WNT-independent and WNT-dependent mechanisms.

Dnmt2 functions in the cytoplasm to promote liver, brain, and retina development in zebrafish.

The roles of DNA methyltransferase-2 (DNMT2) enzymes are controversial; whether DNMT2 functions primarily as a nuclear DNA methyltransferase or as a cytoplasmic tRNA methyltransferase, and whether DNMT2 activity impacts development, as dnmt2 mutant mice or Drosophila lack phenotypes. Here we show that morpholino knockdown of Dnmt2 protein in zebrafish embryos confers differentiation defects in particular organs, including the retina, liver, and brain. Importantly, proper organ differentiation required Dnmt2 activity in the cytoplasm, not in the nucleus. Furthermore, zebrafish Dnmt2 methylates an RNA species of approximately 80 bases, consistent with tRNA methylation. Thus, Dnmt2 promotes zebrafish development, likely through cytoplasmic RNA methylation.

The adenomatous polyposis coli tumor suppressor gene regulates expression of cyclooxygenase-2 by a mechanism that involves retinoic acid.

Mutations in the adenomatous polyposis coli (APC) gene result in uncontrolled proliferation of intestinal epithelial cells and are associated with the earliest stages of colorectal carcinogenesis. Cyclooxygenase-2 (COX-2) is elevated in human colorectal cancers and plays an important role in colorectal tumorigenesis; however, the mechanisms by which APC mutations result in increased COX-2 expression remain unclear. We utilized APC mutant zebrafish and human cancer cells to investigate how APC modulates COX-2 expression. We report that COX-2 is up-regulated in APC mutant zebrafish because of a deficiency in retinoic acid biosynthesis. Treatment of both APC mutant zebrafish and human carcinoma cell lines with retinoic acid significantly reduces COX-2 expression. Retinoic acid regulates COX-2 levels by a mechanism that involves participation of the transcription factor C/EBP-beta. APC mutant zebrafish express higher levels of C/EBP-beta than wild-type animals, and retinoic acid supplementation reduces C/EBP-beta expression to basal levels. Both morpholino knockdown of C/EBP-beta in APC mutant zebrafish and silencing of C/EBP-beta using small interfering RNA in HT29 colon cancer cells robustly decrease COX-2 expression. Our findings support a sequence of events in which mutations in APC result in impaired retinoic acid biosynthesis, elevated levels of C/EBP-beta, up-regulation of COX-2, increased prostaglandin E(2) accumulation, and activation of Wnt target genes.

Adenomatous polyposis coli control of C-terminal binding protein-1 stability regulates expression of intestinal retinol dehydrogenases.

Mutations in the human adenomatous polyposis coli (APC) gene are thought to initiate colorectal tumorigenesis. The tumor suppressor function of APC is attributed primarily to its ability to regulate the WNT pathway by targeting the destruction of beta-catenin. We report here a novel role for APC in regulating degradation of the transcriptional co-repressor C-terminal-binding protein-1 (CtBP1) through a proteasome-dependent process. Further, CtBP1 suppresses the expression of intestinal retinol dehydrogenases, which are required for retinoic acid production and intestinal differentiation. In support of a role for CtBP1 in initiation of colorectal cancer, adenomas taken from individuals with familial adenomatous polyposis contain high levels of CtBP1 protein in comparison with matched, uninvolved tissue. The relationship between APC and CtBP1 is conserved between humans and zebrafish and provides a mechanistic model explaining APC control of intestinal retinoic acid biosynthesis.

Dual roles for adenomatous polyposis coli in regulating retinoic acid biosynthesis and Wnt during ocular development.

Congenital hypertrophy/hyperplasia of the retinal pigmented epithelium is an ocular lesion found in patients harboring mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. We report that Apc-deficient zebrafish display developmental abnormalities of both the lens and retina. Injection of dominant-negative Lef reduced Wnt signaling in the lens but did not rescue retinal differentiation defects. In contrast, treatment of apc mutants with all-trans retinoic acid rescued retinal differentiation defects but had no apparent effect on the lens. We identified Rdh5 as a retina-specific retinol dehydrogenase controlled by APC. Morpholino knockdown of Rdh5 phenocopied the apc mutant retinal differentiation defects and was rescued by treatment with exogenous all-trans retinoic acid. Microarray analyses of apc mutants and Rdh5 morphants revealed a profound overlap in the transcriptional profile of these embryos. These findings support a model wherein Apc serves a dual role in regulating Wnt and retinoic acid signaling within the eye and suggest retinoic acid deficiency as an explanation for APC mutation-associated retinal defects such as congenital hypertrophy/hyperplasia of the retinal pigmented epithelium.

The zebrafish retinol dehydrogenase, rdh1l, is essential for intestinal development and is regulated by the tumor suppressor adenomatous polyposis coli.

Retinoic acid (RA) is a potent signaling molecule that plays important roles in multiple and diverse developmental processes. The contribution of retinoic acid to promoting the development and differentiation of the vertebrate intestine and the factors that regulate RA production in the gut remain poorly defined. Herein, we report that the novel retinol dehydrogenase, rdh1l, is required for proper gut development and differentiation. rdh1l is expressed ubiquitously during early development but becomes restricted to the gut by 3 days postfertilization. Knockdown of rdh1l results in a robust RA-deficient phenotype including lack of intestinal differentiation, which can be rescued by the addition of exogenous retinoic acid. We report that adenomatous polyposis coli (APC) mutant zebrafish harbor an RA-deficient phenotype including aberrant intestinal differentiation and that these mutants can be rescued by treatment with retinoic acid or injection of rdh1l mRNA. Further, we have found that although APC mutants are deficient in rdh1l expression, they harbor increased expression of raldh2 suggesting the control of RA production by APC is via retinol dehydrogenase activity. These results provide genetic evidence that retinoic acid is required for vertebrate gut development and that the tumor suppressor APC controls the production of RA in the gut by regulating the expression of the retinol dehydrogenase, rdh1l.

Adenomatous polyposis coli control of retinoic acid biosynthesis is critical for zebrafish intestinal development and differentiation.

Mutations in the APC (adenomatous polyposis coli) tumor suppressor gene cause uncontrolled proliferation and impaired differentiation of intestinal epithelial cells. Recent studies indicate that human colon adenomas and carcinomas lack retinol dehydrogenases (RDHs) and that APC regulates the expression of human RDHL. These data suggest a model wherein APC controls enterocyte differentiation by controlling retinoic acid production. However, the importance of APC and retinoic acid in mediating control of normal enterocyte development and differentiation remains unclear. To examine the relationship between APC and retinoic acid biosynthesis in normal enterocytes, we have identified two novel zebrafish retinol dehydrogenases, termed zRDHA and zRDHB, that show strong expression within the gut of developing zebrafish embryos. Morpholino knockdown of either APC or zRDHB in zebrafish embryos resulted in defects in structures known to require retinoic acid. These defects included cardiac abnormalities, pericardial edema, failed jaw and pectoral fin development, and the absence of differentiated endocrine and exocrine pancreas. In addition, APC or zRDHB morphant fish developed intestines that lacked columnar epithelial cells and failed to express the differentiation marker intestinal fatty acid-binding protein. Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes. Downstream of retinoic acid production, we identified hoxc8 as a retinoic acid-induced gene that, when ectopically expressed, rescued phenotypes of APC- and zRDHB-deficient zebrafish. Our data establish a genetic link supporting a critical role for retinoic acid downstream of APC and confirm the importance of retinoic acid in enterocyte differentiation.