Functions In Adult Tissues Research Articles

Functional Profiling of Human MeCP2 by Automated Data Comparison Analysis and Computerized Expression Pathway Modeling.

ObjectivesMethyl-CpG binding protein 2 (MeCP2) is a ubiquitous epigenetic factor that represses gene expression by modifying chromatin. Mutations in the MeCP2 gene cause Rett syndrome, a progressive neurodevelopmental disorder. Recent studies also have shown that MeCP2 plays a role in carcinogenesis. Specifically, functional ablation of MeCP2 suppresses cell growth and leads to the proliferation of cancer cells. However, MeCP2's function in adult tissues remains poorly understood. We utilized a weight matrix-based comparison software to identify transcription factor binding site (TFBS) of MeCP2-regulated genes, which were recognized by cDNA microarray analysis.MethodsMeCP2 expression was silenced using annealed siRNA in HEK293 cells, and then a cDNA microarray analysis was performed. Functional analysis was carried out, and transcriptional levels in target genes regulated by MeCP2 were investigated. TFBS analysis was done within genes selected by the cDNA microarray analysis, using a weight matrix-based program and the TRANSFAC 6.0 database.ResultsAmong the differentially expressed genes with a change in expression greater than two-fold, 189 genes were up-regulated and 91 genes were down-regulated. Genes related to apoptosis and cell proliferation (JUN, FOSL2, CYR61, SKIL, ATF3, BMABI, BMPR2, RERE, and FALZ) were highly up-regulated. Genes with anti-apoptotic and anti-proliferative functions (HNRPA0, HIS1, and FOXC1) were down-regulated. Using TFBS analysis within putative promoters of novel candidate target genes of MeCP2, disease-related transcription factors were identified.ConclusionsThe present results provide insights into the new target genes regulated by MeCP2 under epigenetic control. This information will be valuable for further studies aimed at clarifying the pathogenesis of Rett syndrome and neoplastic diseases.

Abstract C130: The oncogenic transcription factors TBX2 and TBX3 as attractive targets for anticancer drug development

Abstract TBX2 and TBX3 are transcription factors that play critical roles in embryonic development but have no known function in adult tissue, and their overexpression has been associated with a growing list of cancers including melanoma, sarcomas, breast, pancreatic, liver and bladder cancers (1). Previous studies in our laboratory have shown that TBX2 and TBX3 play central but different roles in oncogenesis, where TBX2 functions as a potent growth promoting factor and TBX3 contributes to tumour formation, metastasis and invasion (2-4). Additionally, our data have demonstrated that TBX2 confers resistance to cisplatin by promoting DNA repair and may also promote oncogenesis through inappropriate survival of cells with damaged DNA (5). Recent work from our group has provided strong in vitro and in vivo biological evidence that TBX2 and TBX3 are novel targets for development of anti-cancer drugs for cancers where they are overexpressed. Knocking down TBX2 by shRNA induces senescence in advanced melanoma cells, and it results in profound inhibition of proliferation of several metastatic breast cancer cell lines (2,6). Furthermore, depleting TBX2 in metastatic melanoma and cisplatin-resistant breast cancer cells leads to their sensitization to this widely used chemotherapeutic drug, suggesting that targeting TBX2 in combination with currently used therapies could improve their efficacy (5). On the other hand, when TBX3 is depleted by shRNA in some breast cancers and melanomas, cells display reduced anchorage independence, cell migration, colony forming ability and a less aggressive phenotype (2). We are in the process of exploring ways of identifying potential inhibitors of TBX2/TBX3 oncogenic activity by combining a structure-based approach that focuses on molecular recognition and interaction of TBX2/TBX3 with their target genes, and a medium throughput approach involving screening of small molecule libraries.

DNA hydroxymethylation age of human blood determined by capillary hydrophilic-interaction liquid chromatography/mass spectrometry.

BackgroundAging is a complex phenomenon and characterized by a progressive decline in physiology and function of adult tissues. However, it hasn’t been well established of the correlation between aging and global DNA methylation and hydroxymethylation that regulate the growth and development of higher organisms.ResultsWe developed an on-line trapping/capillary hydrophilic-interaction liquid chromatography/electrospray ionization-mass spectrometry method for ultra-sensitive and simultaneous quantification of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in genomic DNA from human blood. Limits of detection for 5-mC and 5-hmC were 0.04 and 0.13 fmol, respectively. The imprecision and recovery of the method were determined with the relative standard deviations (RSDs) and relative errors being <11.2 and 14.0 %, respectively. We analyzed the contents of 5-mC and 5-hmC in genomic DNA of blood from 238 healthy people aged from 1 to 82 years. The results showed that 5-hmC content was significantly decreased and highly correlated with aging process, while 5-mC only showed slight correlation with age. We then established a DNA hydroxymethylation age model according to 5-hmC content with a mean absolute deviation (MAD) of approximate 8.9 years. We also calculated the mean relative error (MRE) using the predicted ages based on the age model and the chronological ages. The results showed that the MRE was 18.3 % for samples with ages from 20 to 82 years (95 % confidence interval, N = 190).ConclusionsThe global DNA hydroxymethylation represents a strong and reproducible mark of chronological age, which could be potentially applied in health assessment and prevention of diseases. The identification of biological or environmental factors that influence DNA hydroxymethylation aging rate may permit quantitative assessments of their impacts on health.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0109-x) contains supplementary material, which is available to authorized users.

Abstract A51: Novel Hedgehog co-receptors in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. The Hedgehog (Hh) signaling pathway is upregulated in PDAC. 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is active in early PanIN lesions and persists as the cancer progresses. Interestingly, Hh ligands are secreted by the tumor cells and function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor growth. PDAC is characterized by hypovascularity and a highly desmoplastic stroma. The downstream effects of Hh activation in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdon), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Gas1, Boc, and Cdon are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. During cancer progression, expression of all three co-receptors is prevalent throughout the desmoplastic stroma. Functional studies on Gas1-/-;Boc-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wildtype MEFs, indicating that these co-receptors are important for Hh signal transduction. Despite a reduced Hh-response, we found that Gas1-/-;Boc-/- MEFs promoted the growth of significantly larger, more vascularized tumors than their wildtype counterparts. A more physiologically relevant experiment with pancreatic fibroblasts and primary pancreatic tumor cells yielded the same counterintuitive result: removal of Gas1 and Boc from fibroblasts decreases Hh-response, and yet tumor-promoting potential was significantly increased. Moreover, chick chorioallantoic membrane (CAM) assays with tumor cells and fibroblasts revealed that Gas1-/-;Boc-/- MEFs stimulated blood vessel growth. We repeated these experiments with MEFs in which all three co-receptors were removed. Gas1-/-;Boc-/-;Cdon-/- MEFs displayed a more significantly abrogated Hh-response than both their Gas1-/-;Boc-/- and wildtype counterparts. However subcutaneous co-injection and CAM experiments revealed that these MEFs did not promote tumor growth, behaving similarly to Smo-/- MEFs in previously published findings. These results indicate the importance of Hh dosage in pancreatic cancer, which has important clinical implications. In particular, we found that intermediate levels of Hh signaling, such as that resulting from pharmaceutical inhibition, resulted in a marked angiogenesis response. This finding may partially account for the recent failure of Hh inhibition in a pancreatic cancer clinical trial. Citation Format: Esha Mathew, Yaqing Zhang, Alexander M. Holtz, Kevin T. Kane, Jane Song, Benjamin L. Allen, Marina Pasca di Magliano. Novel Hedgehog co-receptors in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A51.

Not miR‐ly micromanagers: the functions and regulatory networks of microRNAs in mammalian skin

The microRNA (miRNA) pathway is a widespread mechanism of post-transcriptional gene regulation in eukaryotic cells. In animals, each miRNA species can regulate hundreds of protein-coding genes, resulting in pervasive functions for miRNAs in numerous cellular processes. Since the identification of the first mammalian miRNA, the function of miRNAs in mammals has been a topic of great interest, both because of the versatile roles of miRNAs in biological systems, as well as the clinical potential of these regulatory RNAs. With well-defined cell lineages and the availability of versatile tools for both in vivo and in vitro studies, mammalian skin has emerged as an important system in which to examine miRNAs' functions in adult tissues. In this review, we discuss recent insights into the functions and regulatory networks of miRNAs in mammals, with a specific focus on murine skin development as a model system. We first introduce functional analyses of the miRNA biogenesis pathway in the skin, then highlight the functions of individual miRNAs in skin development, followed by an examination of miRNA roles in skin stress responses. We finish with a discussion of miRNA regulatory networks and emphasize future challenges and emerging technologies that permit the genome-wide study of miRNA functions and regulatory mechanisms in mammalian skin.

Palliative Care for Salivary Gland Dysfunction Highlights the Need for Regenerative Therapies: A Review on Radiation and Salivary Gland Stem Cells.

Radiotherapy remains the major course of treatment for Head and Neck cancer patients. A common consequence of radiation treatment is dysfunction of the salivary glands, which leads to a number of oral complications including xerostomia and dysphagia, for which there is no existent cure. Here, we briefly describe the current palliative treatments available for patients undergoing these conditions, such as oral lubricants, saliva substitutes, and saliva stimulants. None of these options achieves restoration of normal quality of life due to their limited effectiveness, and in some cases, adverse side effects of their own. Other therapies under development, such as acupuncture and electrostimulation have also yielded mixed results in clinical trials. Due to the ineffectiveness of palliative care to restore quality of life, it is reasonable to aim for the development of regenerative therapies that allow restoration of function of the salivary epithelium following radiation treatment. Adult stem cells are a necessary component of wound healing, and play important roles in preserving normal function of adult tissues. Thus, the present review mainly focuses on the effects of radiation on adult stem cells in a variety of tissues, which may be at play in the response of salivary glands to radiation treatment. This is of clinical importance because progenitor cells of the salivary glands have shown partial regenerative potential in mouse transplantation assays. Therefore, understanding how these progenitor cells are affected by radiation offers potential for development of new therapies for patients with xerostomia.

Abstract PR10: Novel hedgehog co-receptors in pancreatic cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. Currently there is inadequate understanding of the molecular mechanisms behind PDAC initiation and progression from pancreatic intraepithelial neoplasia (PanINs), the most common precursor lesions, to full blown carcinoma. Thus, there is a lack of effective treatment modalities. The Hedgehog (Hh) signaling pathway plays an important role in PDAC; 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is found in early PanIN lesions and promotes tumor growth, and persists as the cancer progresses. Interestingly, Hh ligands do not act on the tumor cells themselves, but instead function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor progression. PDAC is characterized by a highly desmoplastic stroma, however the mechanisms that activate Hh signaling in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdo), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Boc and Gas1 are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. We did not detect Cdo expression. Interestingly, during cancer progression, Gas1 and Boc expression is significantly increased throughout the cancer stroma. This finding is notable as expression of Gli2, a vital transcription factor in the Hh pathway, is also significantly increased in the stroma during tumorigenesis. Functional studies on Boc-/-;Gas1-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wild-type MEFs, indicating that these co-receptors are important for Hh signal transduction. To further functionally characterize these receptors, we performed co-injection experiments with wild-type pancreatic fibroblasts and tumor cells or Boc-/-;Gas1-/- fibroblasts and tumor cells to determine the effect of co-receptor loss on tumor growth. Strikingly, tumors co-injected with Boc-/-;Gas1-/- fibroblasts formed significantly larger tumors. Histological analysis revealed that these tumors had a higher degree of vascularity. Future studies include dissecting the manner in which mutant fibroblasts support increased vascularity and whether this increase renders these tumors more susceptible to chemotherapy. This abstract is also presented as poster B37. Citation Format: Esha Mathew, Benjamin L. Allen, Marina Pasca di Magliano. Novel hedgehog co-receptors in pancreatic cancer progression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR10.

Trachea-Derived Dpp Controls Adult Midgut Homeostasis in Drosophila

Homeostasis in adult tissues is maintained by resident stem cells and their progeny. Little is known about the regulation of tissue homeostasis by organ-organ interaction. Here we demonstrate that trachea-derived Decapentaplegic (Dpp), the main bone morphogenetic protein ligand in Drosophila, is essential for adult midgut homeostasis. We show that Dpp signaling is primarily activated in enterocytes (ECs). Depletion of Dpp signaling in ECs results in excess amounts of intestinal stem-cell-like cells and their progeny. Importantly, we find that Dpp is expressed specifically in tracheal cells that reach the intestinal cells through the visceral muscles. Depletion of dpp expression in tracheal cells phenocopies the Dpp loss-of-function defects in ECs. Our data demonstrate that the Drosophila trachea not only exchanges air for bodily needs but also produces a Dpp morphogen essential for neighboring tissue homeostasis. This work will provide important insights into the mechanisms of tissue homeostasis control by interorgan communication.

SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Abstract 2315SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures:No relevant conflicts of interest to declare.

Glucocorticoid overexposure in neonatal life alters pancreatic beta-cell function in newborn foals1

Studies in humans and animals have linked abnormal programming of adult tissue function to excess glucocorticoids during perinatal development. The current study investigated the hypothesis that physiological variations in glucocorticoid concentrations during early neonatal life of the foal alter the secretory responses of the pancreatic β cells 2 and 12 wk after treatment. Spontaneously delivered foals received either saline or long-acting ACTH for 5 d from 1 d after birth to maintain an endogenous rise in cortisol concentrations. Starting at d 10, pancreatic β cell function was studied using an intravenous (i.v.) glucose tolerance test, an i.v. arginine challenge, and an i.v. tolbutamide challenge. The maximum increment in plasma insulin achieved in response to exogenous glucose was less in ACTH-treated foals at both 2 and 12 wk of age (P<0.05). By 12 wk of age, developmental changes also occurred in the magnitude and biphasic pattern of glucose-stimulated insulin release. The area under the insulin curve during the early phase of insulin secretion (0 to 30 min) was not different between the 2- and 12-wk-old animals but was significantly greater during the later phase (30 to 120 min) at 12 wk than at 2 wk (P<0.05). Arginine infusion induced a brief 5 to 15 min increase in plasma concentrations of insulin that was not different in saline- and ACTH-treated foals. The β-cell response to tolbutamide infusion was rapid and monophasic, and there was no difference (P>0.05) in the area under the insulin curve with treatment at 2 or at 12 wk. However, after tolbutamide, plasma insulin concentrations remained increased for a longer period in the ACTH-treated than in the saline-treated foals at 12 wk of age (P<0.05). Hence, this is the first study to show altered pancreatic β-cell function after ACTH-induced glucocorticoid overexposure during early postnatal life in foals.

Autocrine Platelet-derived Growth Factor-Vascular Endothelial Growth Factor Receptor-related (Pvr) Pathway Activity Controls Intestinal Stem Cell Proliferation in the Adult Drosophila Midgut

A dynamic pool of undifferentiated somatic stem cells proliferate and differentiate to replace dead or dying mature cell types and maintain the integrity and function of adult tissues. Intestinal stem cells (ISCs) in the Drosophila posterior midgut are a well established model to study the complex genetic circuitry that governs stem cell homeostasis. Exposure of the intestinal epithelium to environmental toxins results in the expression of cytokines and growth factors that drive the rapid proliferation and differentiation of ISCs. In the absence of stress signals, ISC homeostasis is maintained through intrinsic pathways. In this study, we uncovered the PDGF- and VEGF-receptor related (Pvr) pathway as an essential regulator of ISC homeostasis under unstressed conditions in the posterior midgut. We found that Pvr is coexpressed with its ligand Pvf2 in ISCs and that hyperactivation of the Pvr pathway distorts the ISC developmental program and drives intestinal dysplasia. In contrast, we show that mutant ISCs in the Pvf/Pvr pathway are defective in homeostatic proliferation and differentiation, resulting in a failure to generate mature cell types. Additionally, we determined that extrinsic stress signals generated by enteropathogenic infection are epistatic to the hypoplasia generated in Pvf/Pvr mutants, making the Pvr pathway unique among all previously studied intrinsic pathways. Our findings illuminate an evolutionarily conserved signal transduction pathway with essential roles in metazoan embryonic development and direct involvement in numerous disease states.

CECR2 Is Involved in Spermatogenesis and Forms a Complex with SNF2H in the Testis

The regulation of nucleosome positioning and composition by ATP-dependent chromatin remodeling enzymes and their associated binding partners plays important biological roles in mammals. CECR2 is a binding partner to the ISWI (imitation switch) ATPase SNF2L/SMARCA1 and is involved in neural tube closure and inner ear development; however, its functions in adult tissues have not been examined. Here, we report that CECR2 contributes to spermatogenesis and forms a complex that includes the other ISWI ATPase SNF2H/SMARCA5 in the testis. Cecr2 mutant males non-penetrant for neural tube defects sired smaller litters than wild-type males. Strikingly, while we found that Cecr2 mutants have normal seminiferous epithelium morphology, sperm count, motility, and morphology, the mutant spermatozoa were compromised in their ability to fertilize oocytes. Investigation of CECR2/ISWI complexes in the testis showed that SNF2H interacted with CECR2, and this interaction was also observed in embryonic stem cells, suggesting that CECR2 may interact with SNF2H or SNF2L depending on the cell type. Finally, we found that Cecr2 mutants exhibit misregulation of the homeobox transcription factor Dlx5 in the testis, suggesting that CECR2 complexes may regulate gene expression during spermatogenesis. Taken together, our results demonstrate a novel role of CECR2-containing complexes in spermatogenesis and show that CECR2 interacts predominantly with SNF2H instead of SNF2L in the testis.

The Oncoprotein EVI1 and the DNA Methyltransferase Dnmt3 Co-Operate in Binding and De Novo Methylation of Target DNA

EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation.

Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor

C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.

The cell cycle regulator Cdh1 controls the pool sizes of hematopoietic stem cells and mature lineage progenitors by protecting from genotoxic stress

Various key cell cycle components, especially G0/G1 regulators, have effects not only on cell proliferation but also on cell differentiation. Cdh1, one of the co-activators that maintain anaphase-promoting complex/cyclosome activity, plays a crucial role in the mitotic phase, but has recently been identified as a G0/G1 regulator, suggesting that the role of Cdh1 in cell differentiation. Here, we generated Cdh1 conditional gene-trap mice to examine Cdh1 functions in adult tissues by overcoming the embryonic lethality of Cdh1 homozygous gene-trap mice. We focused on the hematopoietic system and found that Cdh1-deficient mice exhibited a general decrease in mature lineage progenitor cells and a significant increase in short-term hematopoietic stem cells. This phenomenon became conspicuous by irradiation shortly after Cdh1 downregulation, suggesting that Cdh1 regulates the pool sizes of the hematopoietic stem cells and mature lineage progenitor cells by protecting cells from genotoxic stress. We also found that the irradiation-induced G2/M checkpoint was defective in Cdh1-deficient BM cells, causing the loss of stem/progenitor cells. This is the first report revealing Cdh1 function in adult hematopoiesis and showing a role of Cdh1 in a G2/M checkpoint regulation in vivo.

Wnt signalling and cancer stem cells

Intracellular signalling mediated by secreted Wnt proteins is essential for the establishment of cell fates and proper tissue patterning during embryo development and for the regulation of tissue homeostasis and stem cell function in adult tissues. Aberrant activation of Wnt signalling pathways has been directly linked to the genesis of different tumours. Here, the components and molecular mechanisms implicated in the transduction of Wnt signal, along with important results supporting a central role for this signalling pathway in stem cell function regulation and carcinogenesis will be briefl y reviewed.

Construction and characterization of a doxycycline‐inducible transgenic system in Msx2 expressing cells

Homeobox gene Msx2 is widely expressed during both embryogenesis and postnatal development and plays important roles during organogenesis. We developed an Msx2-rtTA BAC transgenic line which can activate TetO-Cre expression in Msx2-expressing cells upon doxycycline (Dox) treatment. Using the Rosa26-LacZ (R26R) reporter line, we show that rtTA is activated in Msx2-expressing organs including the limb, heart, external genitalia, urogenital system, hair follicles and craniofacial regions. Moreover, we show that in body appendages, the transgene can be activated in different domains depending on the timing of Dox treatment. In addition, the transgene can also be effectively activated in adult tissues such as the hair follicle and the urogenital system. Taken together, this Msx2-rtTA;TetO-Cre system is a valuable tool for studying gene function in the development of the aforementioned organs in a temporal and spatially-restricted manner, as well as for tissue lineage tracing of Msx2-expressing cells. When induced postnatally, this system can also be used to study gene function in adult tissues without compromising normal development and patterning.

Dicer Is Required for Maintaining Adult Pancreas

Dicer1, an essential component of RNA interference and the microRNA pathway, has many important roles in the morphogenesis of developing tissues. Dicer1 null mice have been reported to die at E7.5; therefore it is impossible to study its function in adult tissues. We previously reported that Dicer1-hypomorphic mice, whose Dicer1 expression was reduced to 20% in all tissues, were unexpectedly viable. Here we analyzed these mice to ascertain whether the down-regulation of Dicer1 expression has any influence on adult tissues. Interestingly, all tissues of adult (8–10 week old) Dicer1-hypomorphic mice were histologically normal except for the pancreas, whose development was normal at the fetal and neonatal stages; however, morphologic abnormalities in Dicer1-hypomorphic mice were detected after 4 weeks of age. This suggested that Dicer1 is important for maintaining the adult pancreas.

Small molecule–mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer

SUMMARYThe pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two novel classes of small molecules that disrupt Wnt pathway responses - whereas one class inhibits the activity of Porcupine (Porcn), a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, suppressors of Wnt/β-catenin pathway activity. With these small molecules we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/β-catenin pathway response in vivo, and establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

Neuroprotective effect of sonic hedgehog up‐regulated in Schwann cells following sciatic nerve injury

The physiological roles of sonic hedgehog (Shh) have been intensively characterized in development of various organs. However, their functions in adult tissues have not been fully elucidated. We investigated the expression and the potential function of Shh in crush-injured adult rat sciatic nerves. The Shh expression was up-regulated in Schwann cells adjacent to the injured site. The time-course analyses of various neurotrophic factors revealed the up-regulation of Shh mRNA followed by that of brain-derived neurotrophic factor (BDNF) mRNA. The continuous administration of cyclopamine, a hedgehog signal inhibitor, to the injured site suppressed the increase of BDNF expression and deteriorated the survival of motor neurons in lumbar spinal cord. Treatment of exogenous Shh in cultured Schwann cells enhanced the BDNF expression. The BDNF promoter activity (exon I and II) was increased in IMS32 cells co-transfected with Shh and its receptor Smoothened. These findings imply that the up-regulated expression of Shh in Schwann cells may play an important role in injured motor neurons through the induction of BDNF.