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We analyzed 8.55 million LongSAGE tags generated from 72 libraries. Each LongSAGE library was prepared from a different mouse tissue. Analysis of the data revealed extensive overlap with existing gene data sets and evidence for the existence of [approximately equal to] 24,000 previously undescribed genomic loci. The visual cortex, pancreas, mammary gland, preimplantation embryo, and placenta contain the largest number of differentially expressed transcripts, 25% of which are previously undescribed loci. alternative transcripts | development | serial analysis of gene expression

SHIP is a (SH2 protein containing inositol-5-phospate) that is found in hemopoietic cells and it plays an important role in cytokine signaling. Targeted disruption of SHIP results in only 40% of mice thriving past the 14th week. Infiltration from myeloid cells leads to extensive consolidation of the legs. The other changes and the impact of SHIP deficiency are discussed.

Age-related senescence of T-cell mediated responses is well recognized. This study was desgined to determine how aging affects the T-cell mediated Interleukin 2 (IL2) response to influenza vaccination. A group of healthy elderly individuals were compared to a control group of healthy young adults for their response to the 1990 influenza vaccine. Cultures of peripheral blood mononuclear cells (PBMC) were prepared from venous blood samples taken prevaccination (pre) and 8 and 12 weeks post-vaccination (post). PBMC cultures stimulated with inactivated A/Shanghai/16/89 (contained in the 1990 vaccine) and A/Philippine/2/82 (not contained in the vaccine) were assayed for peak IL2 activity. We find that after influenza vaccination, there was an insignificant increase in IL2 activity when PBMC from the young control group were stimulated with A/Shanghai/16/89 (pre, 5.14 U/mL/[10.sup.6] PBMC; post, 6.64 U/mL/[10.sup.6] PBMC but there was a significant increase in IL2 activity when stimulated with A/Philippine/2/82 (pre, 1.5 U/mL/[10.sup.6] PBMC; post 8.3 U/m/[10.sup.6] PBMC). In similar cultures of PBMC from the elderly group, there was a significant increase in IL2 response to both A/Shanghai/16/89 (pre, 1.6 U/mL/[10.sup.6] PBMC; post, 3.5 U/mL/[10.sup.6] PBMC) and A/Philippine/2/82 (pre, 0.86 U/mL/[10.sup.6] PBMC; post, 8.3 U/mL/[10.sup.6] PBMC). Measurements of [CD4.sup.+]/[CD8.sup.8+] populations were not affected by vaccination and were not significantly different in two groups. Subgroup analysis of the elderly group revealed that previous influenza vaccination in 1989 did not significantly affect IL2 levels measured in the present study. This study shows that in healthy elderly, influenza vaccination effectively restores IL2 activity to normal. There appears to be an age-related decrease in the duration of T-cell memory. The IL2 response to influenza vaccination in healthy elderly suggests that age-related changes in the immune response to influenza viral infection may be reversed by yearly influenza vaccination.

Abstract Early prostate cancer (PCa) is generally treatable and associated with good prognosis. After a variable time, PCa evolves into a highly metastatic and treatment-refractory disease: castration-resistant PCa (CRPC). Currently, few prognostic factors are available to predict the emergence of CRPC, and no curative option is available. Epigenetic gene regulation has been shown to trigger PCa metastasis and androgen-independence. Most epigenetic studies have focused on DNA and histone methyltransferases. While DNA methylation leads to gene silencing, histone methylation can trigger gene activation or inactivation, depending on the target amino acid residues and the extent of methylation (me1, me2, or me3). Interestingly, some histone modifiers are essential for PCa tumor-initiating cell (TIC) self-renewal. TICs are considered the seeds responsible for metastatic spreading and androgen-independence. Histone Lysine Demethylases (KDMs) are a novel class of epigenetic enzymes which can remove both repressive and activating histone marks. KDMs are currently grouped into 7 major classes, each one targeting a specific methylation site. Since their discovery, KDM expression has been found to be deregulated in several neoplasms. In PCa, KDMs may act as either tumor suppressors or oncogenes, depending on their gene regulatory function. For example, KDM1A and KDM4C are essential for PCa androgen-dependent proliferation, while PHF8 is involved in PCa migration and invasion. Interestingly, the possibility of pharmacologically targeting KDMs has been demonstrated. In the present paper, we summarize the emerging role of KDMs in regulating the metastatic potential and androgen-dependence of PCa. In addition, we speculate on the possible interaction between KDMs and other epigenetic effectors relevant for PCa TICs. Finally, we explore the role of KDMs as novel prognostic factors and therapeutic targets. We believe that studies on histone demethylation may add a novel perspective in our efforts to prevent and cure advanced PCa.

Centromere protein-A (CENP-A), a histone-H3 variant, plays an essential role in cell division by ensuring proper formation and function of centromeres and kinetochores. Elevated CENP-A expression has been associated with cancer development. This study aimed to establish whether elevated CENP-A expression can be used as a prognostic and predictive cancer biomarker. Molecular profiling of CENP-A in human cancers was investigated using genomic, transcriptomic and patient information from databases, including COSMIC, Oncomine, Kaplan-Meier plotter and cBioPortal. A network of CENP-A co-expressed genes was derived from cBioPortal and analyzed using Ingenuity Pathway Analysis (IPA) and Oncomine protocols to explore the function of CENP-A and its predictive potential. Transcriptional and post-transcriptional regulation of CENP-A expression was analyzed in silico. It was found that CENP-A expression was elevated in 20 types of solid cancer compared with normal counterparts. Elevated CENP-A expression highly correlated with cancer progression and poor patient outcome. Genomic analysis indicated that the elevated CENP-A expression was not due to alterations in the sequence or copy number of the CENP-A gene. Furthermore, CENP-A can be regulated by key oncogenic proteins and tumor-suppressive microRNAs. CENP-A co-expression network analysis indicated that CENP-A function is associated with cell cycle progression. Oncomine analysis showed a strong correlation between elevated CENP-A expression and oncolytic response of breast cancer patients to taxane-based chemotherapy. In conclusion, elevated CENP-A expression is coupled to malignant progression of numerous types of cancer. It may be useful as a biomarker of poor patient prognosis and as a predictive biomarker for taxane-based chemotherapy. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs) can orchestrate oncogenic or tumor-suppressive functions in cancer biology. Accordingly, PCGEM1 and PRNCR1 were implicated in progression of prostate cancer (PCa) as transcriptional co-regulators of the androgen receptor (AR). However, these findings were recently refuted asserting that neither gene physically binds to the AR. Despite evidence for differing AR transcriptional programs in vivo and in vitro, studies investigating AR-regulation of these genes hitherto have only been conducted in vitro. Here, we further examine the relevance of PCGEM1 and PRNCR1 in PCa, and their relationship with AR signaling, using patient-derived xenograft models. Findings: RNA sequencing of two distinct androgen-dependent models shows PCGEM1 to be considerably expressed, while PRNCR1 showed scant basal expression. PCGEM1 was sharply down-regulated following castration and up-regulated upon AR activation in vivo. However, we found no parallel evidence following AR stimulation in vitro. A PCGEM1-associated gene expression signature (PES) was significantly repressed in response to androgen ablation therapy and in hormone-refractory versus hormone-naïve PCa patients. Furthermore, we found PCGEM1 was uniformly distributed in PCa cell nucleus and cytoplasm which remained unaltered upon AR transcriptional activation. PCGEM1 was up-regulated in primary PCa but not in metastasized PCa. Accordingly, the PES was significantly down-regulated in advanced and higher grade PCa patients from multiple independent studies. Conclusion: Our results demonstrate PCGEM1 as an in vivo androgen-regulated transcript with potential nuclear and/or cytoplasmic function(s). Importantly, the clinical expression profile of PCGEM1 implicates it in the early stages of PCa warranting further research in this direction. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have emerged as key regulators of numerous biological processes, and increasing evidence suggests that circulating miRNAs may be useful biomarkers of clinical disease. In this study, we sought to identify plasma miRNAs that differentiate patients with metastatic castration resistant prostate cancer (mCRPC) from those with localized prostate cancer (PCa). Pooled plasma samples from patients with localized PCa or mCRPC (25 per group) were assayed using the Exiqon miRNA qPCR panel, and the differential expression of selected candidates was validated using qRT-PCR. We identified 63 miRNAs upregulated in mCRPC versus localized PCa, while only four were downregulated. Pearson's correlation analysis revealed two highly correlated groups: one consisting of miR-141, miR375 and miR-200c and the other including miR151-3p, miR423-3p, miR-126, miR152 and miR-21. A third group, containing miR-16 and miR-205, showed less correlation. One miRNA from each group (miR-141, miR151-3p and miR-16) was used for logistic regression analysis and proved to increase the sensitivity of the prostate-specific antigen (PSA) test alone. While no miRNA alone differentiated localized PCa and mCRPC, combinations had greater sensitivity and specificity. The expression of these 10 candidates was assayed for association with clinical parameters of disease progression through the cBio portal. Our results demonstrate that plasma levels of selected miRNAs are potential biomarkers to differentiate localized PCa and mCRPC. [ABSTRACT FROM AUTHOR]

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors. [ABSTRACT FROM AUTHOR]

Background: Serial Analysis of Gene Expression (SAGE) is a DNA sequencing-based method for large-scale gene expression profiling that provides an alternative to microarray analysis. Most analyses of SAGE data aimed at identifying co-expressed genes have been accomplished using various versions of clustering approaches that often result in a number of false positives. Principal Findings: Here we explore the use of seriation, a statistical approach for ordering sets of objects based on their similarity, for large-scale expression pattern discovery in SAGE data. For this specific task we implement a seriation heuristic we term 'progressive construction of contigs' that constructs local chains of related elements by sequentially rearranging margins of the correlation matrix. We apply the heuristic to the analysis of simulated and experimental SAGE data and compare our results to those obtained with a clustering algorithm developed specifically for SAGE data. We show using simulations that the performance of seriation compares favorably to that of the clustering algorithm on noisy SAGE data. Conclusions: We explore the use of a seriation approach for visualization-based pattern discovery in SAGE data. Using both simulations and experimental data, we demonstrate that seriation is able to identify groups of co-expressed genes more accurately than a clustering algorithm developed specifically for SAGE data. Our results suggest that seriation is a useful method for the analysis of gene expression data whose applicability should be further pursued. [ABSTRACT FROM AUTHOR]

Background: The full-length mammalian homologs of groucho, Tle1, 2, 3, and 4, act as transcriptional corepressors and are recruited by transcription factors containing an eh1 or WRPW/Y domain. Many transcription factors critical to pancreas development contain a Gro/TLE interaction domain and several have been shown to require Gro/TLE interactions for proper function during neuronal development. However, a detailed analysis of the expression patterns of the Gro/TLE proteins in pancreas development has not been performed. Moreover, little is known about the ability of Gro/TLE proteins to interact with transcription factors in the pancreas. Results: We describe the expression of Gro/TLE family members, and of 34 different transcription factors that contain a Gro/TLE interaction motif, in the pancreas utilizing nine SAGE libraries created from the developing and adult pancreas, as well as the GenePaint database. Next, we show the dynamic expression of Tle1, 2, 3, 4, 5 and 6 during pancreas development by qRT-PCR. To further define the cell-type specificity of the expression of these proteins we use immunofluorescence to co-localize them with Pdx1 at embryonic day 12.5 (E12.5), Ngn3 at E14.5, Pdx1, Nkx2-2, Insulin, Glucagon, Pancreatic polypeptide and Somatostatin at E18.5, as well as Insulin and Glucagon in the adult. We then show that Tle2 can interact with Nkx2-2, Hes1, Arx, and Nkx6-1 which are all critical factors in pancreas development. Finally, we demonstrate that Tle2 modulates the repressive abilities of Arx in a β-cell line. Conclusion: Although Tle1, 2, 3, and 4 show overlapping expression in pancreatic progenitors and in the adult islet, the expression of these factors is restricted to different cell types during endocrine cell maturation. Of note, Tle2 and Tle3 are co-expressed with Gro/TLE interaction domain containing transcription factors that are essential for endocrine pancreas development. We further demonstrate that Tle2 can interact with several of these factors and that Tle2 modulate Arx's repressive activity. Taken together our studies suggest that Gro/TLE proteins play a role in the repression of target genes during endocrine cell specification. [ABSTRACT FROM AUTHOR]

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_ at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types. [ABSTRACT FROM AUTHOR]

Groucho (Gro)/TLE transcriptional corepressors are involved in a variety of developmental mechanisms, including neuronal differentiation. They contain a conserved C-terminal WD40 repeat domain that mediates interactions with several DNA-binding proteins. In particular, Gro/TLE1 interacts with forkhead transcription factor brain factor 1 (BF-1: also termed FoxG1). BF-1 is an essential regulator of neuronal differentiation during cerebral cortex development and represses transcription together with Gro/TLE1. Gro/TLE-related gene product 6 (Grg6) shares with Gro/TLEs a conserved WD40 repeat domain but is more distantly related at its N-terminal half. We demonstrate that Grg6 is expressed in cortical neural progenitor cells and interacts with BF-1. In contrast to Gro/TLE1, however, Grg6 does not promote, but rather suppresses, BF-1-mediated transcriptional repression. Consistent with these observations, Grg6 interferes with the binding of Gro/TLE1 to BF-1 and does not repress transcription when targeted to DNA. Moreover, coexpression of Grg6 and BF-1 in cortical progenitor cells leads to a decrease in the number of proliferating cells and increased neuronal differentiation. Conversely, Grg6 knockdown by RNA interference causes decreased neurogenesis. These results identify a new role for Grg6 in cortical neuron development and establish a functional link between Grg6 and BF-1. [ABSTRACT FROM AUTHOR]

The hematopoietic-restricted protein Src homology 2?containing inositol-5-phosphatase (SHIP) blunts phosphatidylinositol-3-kinase-initiated signaling by dephosphorylating its major substrate, phosphatidylinositol-3,4,5-trisphosphate. As SHIP-/- mice contain increased numbers of osteoclast precursors, that is, macrophages, we examined bones from these animals and found that osteoclast number is increased two-fold. This increased number is due to the prolonged life span of these cells and to hypersensitivity of precursors to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Similar to pagetic osteoclasts, SHIP-/- osteoclasts are enlarged, containing upwards of 100 nuclei, and exhibit enhanced resorptive activity. Moreover, as in Paget disease, serum levels of interleukin-6 are markedly increased in SHIP-/- mice. Consistent with accelerated resorptive activity, 3D trabecular volume fraction, trabecular thickness, number and connectivity density of SHIP-/- long bones are reduced, resulting in a 22% loss of bone-mineral density and a 49% decrease in fracture energy. Thus, SHIP negatively regulates osteoclast formation and function and the absence of this enzyme results in severe osteoporosis. [ABSTRACT FROM AUTHOR]

: ObjectiveThe Hox gene family of transcription factors is thought to be involved in the regulation of primitive hematopoietic cells, including stem cells and early committed progenitors, and has also been directly implicated in leukemia. To gain further insight into Hox gene–mediated regulation of hematopoiesis, we investigated the expression pattern of representative Hox genes and two of their cofactors, Pbx1 and Meis1, at different stages of murine hematopoiesis: MethodsFunctionally distinct subpopulations of murine bone marrow (BM) and fetal liver day 14.5 (FL) cells were isolated by flow cytometry, and gene expression of various homeobox-containing genes was assessed by global cDNA amplification technique.: ResultsHox genes were found preferentially expressed in hematopoietic stem cell (HSC)–enriched subpopulations and downregulated following differentiation and maturation. This profile of expression was observed at both adult and fetal stages of hematopoiesis. The Pbx1 and Meis1 genes had important differences in their expression pattern but were both detected in Hox expressing subpopulations. In particular, Meis1 consistently showed an expression profile closely resembling that of Hox genes. Finally, using the in vitro embryonic stem (ES) cell differentiation model to mimic embryonic hematopoiesis, we found coexpression of Hox genes and their cofactors coincided with the appearance of hematopoietic progenitor cells.: ConclusionTogether, these results further support the notion that Hox genes are involved in the regulation of early hematopoietic cells and provide strong evidence that they are involved in the regulation of hematopoiesis throughout ontogeny. [Copyright &y& Elsevier]

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative (XLP) disease. Although the exact role and mechanism of action of SAP are not known, it has the capacity to interact with the cytoplasmic region of SLAM and other related immune cell receptors. As SAP is composed almost exclusively of a Src homology 2 (SH2) domain, it has been proposed that it functions as a natural blocker of SH2 domain?mediated interactions. We report here that the SLAM receptor is capable of triggering a protein tyrosine phosphorylation signal in T cells via a mechanism that is strictly dependent on SAP expression. This signal involves the SH2 domain?containing inositol phosphatase (SHIP); the adaptor molecules Dok2, Dok1 and Shc; and Ras GTPase?activating protein RasGAP. SAP is essential for this pathway because it facilitates the selective recruitment and activation of the Src-related protein tyrosine kinase FynT. We also show that signaling via the SLAM-SAP pathway in an established T cell line can alter the profile of cytokine production during T cell activation. These findings identify a mechanism by which a putative adaptor molecule is required for receptor-mediated signaling events in the immune system. They also provide insights into the pathophysiology of a severe human lymphoproliferative disease. [ABSTRACT FROM AUTHOR]

Reports that targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis. Inactivation of the spgp gene in mice; Analysis of spgp[sup -/-] mice; Secretion of bile acids; Tetra-hydroxylated bile acids; Lipid composition of bile in spgp[sup -/-] mice.

Objective: We previously demonstrated that c-kit expression decreases during murine embryonic stem cell (ESC) differentiation induced by leukemia inhibitory factor removal. In this study, we addressed the possibility that c-kit is a marker of undifferentiated murine ESC and, moreover, that it plays a role in maintaining the undifferentiated state of these cells. Materials and Methods: c-kit expression was analyzed under various differentiation conditions by flow cytometry and quantitative reverse transcription polymerase chain reaction. ESC were then sorted on the basis of c-kit expression and functionality was investigated using embryoid body and colony-forming cell assays. Imatinib (Gleevec) and ACK2 were used to block, and stem cell factor was used to stimulate, c-kit activity. Results: c-kit expression decreased in two murine ESC lines under various differentiation conditions. Sorting of ESC populations on the basis of c-kit expression revealed significant differences in the functional capacities and gene expression profiles of the sorted populations. The inhibition studies revealed an important role for tyrosine kinase activity in maintaining ESC viability and differentiation capacity, at least in part by preventing apoptosis and enhancing cell cycle progression. However, activation of c-kit alone is not sufficient for maintaining undifferentiated ESC. Conclusion: The results suggest that c-kit may represent a useful marker for monitoring ESC functionality. Moreover, tyrosine kinase signaling plays an important role in maintaining undifferentiated ESC. This work provides valuable insights into the complex signaling pathways that synergize to maintain the undifferentiated state of murine ESC. [Copyright &y& Elsevier]

Objective: Dendritic cells (DC) play a critical role in establishment and maintenance of central and peripheral tolerance. Despite intensive research, our knowledge of the molecular mechanisms regulating DC development and function is limited, thus hindering our ability to generate appropriate DC populations for manipulating immune tolerance. We utilized mice deficient in the SH2-containing inositol-5-phosphatase (SHIP) to examine the role of cytokine signaling in DC development and function. Methods: We analyzed the phenotype of both primary and bone marrow (BM)-derived DC (BMDC) using flow cytometry. In addition, cytokine production was measured using cytometric bead arrays and the ability of DC to induce allogeneic T-cell proliferation was assessed using thymidine incorporation assays. Results: We demonstrated that spleen DC isolated from SHIP-deficient mice are increased in number and have an altered phenotype. In vitro analyses revealed that SHIP-deficient BM cells give rise to a higher frequency of myeloid, but not plasmacytoid, DC due to both an increased progenitor frequency and enhanced cytokine sensitivity. The BMDC exhibit an altered phenotype that correlates with a reduced capacity to induce allogeneic T-cell proliferation. Addition of interleukin-6 to WT BM cultures during DC differentiation partially induces a KO phenotype. Conclusion: These studies suggest that myeloid and plasmacytoid DC progenitors are differentially sensitive to signaling pathways in which SHIP is involved. Moreover, they suggest that interleukin-6 may have an important role in regulating the phenotype and function of myeloid DC. [Copyright &y& Elsevier]

Abstract: To identify genes and pathways not previously implicated in the mesenchymal–epithelial (M/E) interactions that are critical for normal mouse prostate development, we constructed six serial analysis of gene expression (SAGE) libraries. Bioinformatic analyses revealed expression of various members of numerous signalling pathways and the differential expression of several members of the wingless-related MMTV integration site (Wnt) signalling pathway. This pathway has not been previously implicated in prostate development thus expression of selected Wnt pathway members in the developing prostate was confirmed by RT-qPCR. Of particular interest, an antagonist of the Wnt pathway, secreted frizzled related protein 2 (Sfrp2), was highly expressed in the early prostate libraries and down regulated at later developmental stages. The expression levels of four Wnt ligands reported to interact with Sfrp2 were, therefore, examined by RT-qPCR. We found that only Wnt4 transcripts were detectable in the developing prostate. Expression of Sfrp2 was validated using RT-qPCR and localization of Sfrp2 transcripts and protein was carried out using in situ hybridization and immunofluorescence, respectively. These studies provide the first evidence that Wnt pathway members are expressed in the developing prostate. Functional analyses are now required to establish the biological significance of this observation. [Copyright &y& Elsevier]

The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet- and liver-specific gene expression. [ABSTRACT FROM AUTHOR]

Background: While localized prostate cancer (PCa) can be effectively cured, metastatic disease inevitably progresses to a lethal state called castration-resistant prostate cancer (CRPC). Emerging evidence suggests that aberrant epigenetic repression by the polycomb group (PcG) complexes fuels PCa progression, providing novel therapeutic opportunities., Results: In the search for potential epigenetic drivers of CRPC, we analyzed the molecular profile of PcG members in patient-derived xenografts and clinical samples. Overall, our results identify the PcG protein and methyl-lysine reader CBX2 as a potential therapeutic target in advanced PCa. We report that CBX2 was recurrently up-regulated in metastatic CRPC and that elevated CBX2 expression was correlated with poor clinical outcome in PCa cohorts. Furthermore, CBX2 depletion abrogated cell viability and induced caspase 3-mediated apoptosis in metastatic PCa cell lines. Mechanistically explaining this phenotype, microarray analysis in CBX2-depleted cells revealed that CBX2 controls the expression of many key regulators of cell proliferation and metastasis., Conclusions: Taken together, this study provides the first evidence that CBX2 inhibition induces cancer cell death, positioning CBX2 as an attractive drug target in lethal CRPC.

Biomarkers are molecules or features which can provide clinically-relevant information about a particular disease state, thus providing useful tools for oncologists. Recently, a number of studies have demonstrated that DNA methylation holds great promise as a novel source of cancer biomarkers. Although promoter regions have been the focus of most investigations thus far, mounting evidence demonstrates that enhancer sequences also undergo extensive differential methylation in cancer cells. Moreover, enhancer methylation correlates with target gene expression better than promoter methylation, providing unexplored strategies for biomarker development. Here, we review important considerations associated with the clinical analysis of DNA methylation at distal regulatory regions. Notably, we highlight emerging literature addressing the methylation status of enhancers in development and cancer, and subsequently discuss how enhancer methylation can be exploited to guide disease management. While acknowledging current limitations, we propose that the methylation state of enhancer regions has the potential to headline the next generation of epigenetic biomarkers.

Aim: Imatinib is a tyrosine kinase inhibitor that has revolutionized the treatment of chronic myeloid leukemia (CML). Despite its efficacy, about a third of patients discontinue the treatment due to therapy failure or intolerance. The rational identification of patients less likely to respond to imatinib would be of paramount clinical relevance. We have shown that transmembrane transporter hOCT1 genotyping predicts imatinib activity. In parallel, Polycomb group genes (PcGs) are epigenetic repressors implicated in CML progression and in therapy resistance., Patients & Methods: We measured the expression of eight PcGs in paired pre- and post-imatinib bone marrow samples from 30 CML patients., Results: BMI1, PHC3, CBX6 and CBX7 expression was significantly increased during imatinib treatment. Post-treatment levels of CBX6 and CBX7 predicted 3-month response rate. Measurement of post-treatment BMI1 levels improved the predictive power of hOCT1 genotyping., Conclusion: These results suggest that the expression levels of PcGs might be useful for a more accurate risk stratification of CML patients.

Metastatic prostate cancer (PCa) is still an incurable disease. Long non-coding RNAs (lncRNAs) may be an overlooked source of cancer biomarkers and therapeutic targets. We therefore performed RNA sequencing on paired metastatic/non-metastatic PCa xenografts derived from clinical specimens. The most highly up-regulated transcript was LOC728606, a lncRNA now designated PCAT18. PCAT18 is specifically expressed in the prostate compared to 11 other normal tissues (p<0.05) and up-regulated in PCa compared to 15 other neoplasms (p<0.001). Cancer-specific up-regulation of PCAT18 was confirmed on an independent dataset of PCa and benign prostatic hyperplasia samples (p<0.001). PCAT18 was detectable in plasma samples and increased incrementally from healthy individuals to those with localized and metastatic PCa (p<0.01). We identified a PCAT18-associated expression signature (PES), which is highly PCa-specific and activated in metastatic vs. primary PCa samples (p<1E-4, odds ratio>2). The PES was significantly associated with androgen receptor (AR) signalling. Accordingly, AR activation dramatically up-regulated PCAT18 expression in vitro and in vivo. PCAT18 silencing significantly (p<0.001) inhibited PCa cell proliferation and triggered caspase 3/7 activation, with no effect on non-neoplastic cells. PCAT18 silencing also inhibited PCa cell migration (p<0.01) and invasion (p<0.01). These results position PCAT18 as a potential therapeutic target and biomarker for metastatic PCa.

Prostate cancer (PCa) is a very common neoplasm, which is generally treated by chemo-, radio-, and/or hormonal-therapy. After a variable time, PCa becomes resistant to conventional treatment, leading to patient death. Prostate tumor-initiating cells (TICs) and cancer repopulating cells (CRCs) are stem-like populations, driving respectively cancer initiation and progression. Histone modifiers (HMs) control gene expression in normal and cancer cells, thereby orchestrating key physiological and pathological processes. In particular, Polycomb group genes (PcGs) are a set of HMs crucial for lineage-specific gene silencing and stem cell self renewal. PcG products are organized into two main Polycomb Repressive Complexes (PRCs). At specific loci, PRC2 catalyzes histone H3 Lys27 trimethylation, which triggers gene silencing by recruiting PRC1, histone deacetylases and DNA methyl transferases. PRC1 catalyzes addition of the repressive mark histone H2A ubiquitination. Recently, the catalytic component of PRC1 (BMI1) was shown to play critical roles in prostate CRC self-renewal and resistance to chemotherapy, resulting in poorer prognosis. Similarly, pharmacological disruption of PRC2 by a small molecule inhibitor reduced the tumorigenicity and metastatic potential of prostate CRCs. Along with PcGs, some histone lysine demethylases (KDMs) are emerging as critical regulators of TIC/CRC biology. KDMs may be inhibited by specific small molecules, some of which display antitumor activity in PCa cells at micromolar concentrations. Since epigenetic gene regulation is crucial for stem cell biology, exploring the role of HMs in prostate cancer is a promising path that may lead to novel treatments.

Although several groups have investigated the role of SHIP in macrophage (M) development and function, SHIP's contribution to the generation, maturation, and innate immune activation of dendritic cells (DCs) is poorly understood. We show herein that SHIP negatively regulates the generation of DCs from bone marrow precursors in vitro and in vivo, as illustrated by the enhanced expansion of DCs from SHIP(-/-) GM-CSF cultures, as well as increased numbers of DCs in the spleens of SHIP-deficient mice. Interestingly, however, these SHIP(-/-) DCs display a relatively immature phenotype and secrete substantially lower levels of IL-12 after TLR ligand stimulation than wild type DCs. This, in turn, leads to a dramatically reduced stimulation of Ag-specific T cell proliferation and Th1 cell responses in vitro and in vivo. This immature phenotype of SHIP(-/-) DCs could be reversed with the PI3K inhibitors LY294002 and wortmannin, suggesting that SHIP promotes DC maturation by reducing the levels of the PI3K second messenger phosphatidylinositol-3,4,5-trisphosphate. These results are consistent with SHIP being a negative regulator of GM-CSF-derived DC generation but a positive regulator of GM-CSF-derived DC maturation and function.

Background: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts., Results: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development., Conclusion: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

Groucho (Gro)/TLE transcriptional corepressors are involved in a variety of developmental mechanisms, including neuronal differentiation. They contain a conserved C-terminal WD40 repeat domain that mediates interactions with several DNA-binding proteins. In particular, Gro/TLE1 interacts with forkhead transcription factor brain factor 1 (BF-1; also termed FoxG1). BF-1 is an essential regulator of neuronal differentiation during cerebral cortex development and represses transcription together with Gro/TLE1. Gro/TLE-related gene product 6 (Grg6) shares with Gro/TLEs a conserved WD40 repeat domain but is more distantly related at its N-terminal half. We demonstrate that Grg6 is expressed in cortical neural progenitor cells and interacts with BF-1. In contrast to Gro/TLE1, however, Grg6 does not promote, but rather suppresses, BF-1-mediated transcriptional repression. Consistent with these observations, Grg6 interferes with the binding of Gro/TLE1 to BF-1 and does not repress transcription when targeted to DNA. Moreover, coexpression of Grg6 and BF-1 in cortical progenitor cells leads to a decrease in the number of proliferating cells and increased neuronal differentiation. Conversely, Grg6 knockdown by RNA interference causes decreased neurogenesis. These results identify a new role for Grg6 in cortical neuron development and establish a functional link between Grg6 and BF-1.

Global gene expression profiling was performed on murine embryonic stem cells (ESCs) induced to differentiate by removal of leukemia inhibitory factor (LIF) to identify genes whose change in expression correlates with loss of pluripotency. To identify appropriate time points for the gene expression analysis, the dynamics of loss of pluripotency were investigated using three functional assays: chimeric mouse formation, embryoid body generation, and colony-forming ability. A rapid loss of pluripotency was detected within 24 hours, with very low residual activity in all assays by 72 hours. Gene expression profiles of undifferentiated ESCs and ESCs cultured for 18 and 72 hours in the absence of LIF were determined using the Affymetrix GeneChip U74v2. In total, 473 genes were identified as significantly differentially expressed, with approximately one third having unknown biological function. Among the 275 genes whose expression decreased with ESC differentiation were several factors previously identified as important for, or markers of, ESC pluripotency, including Stat3, Rex1, Sox2, Gbx2, and Bmp4. A significant number of the decreased genes also overlap with previously published mouse and human ESC data. Furthermore, several membrane proteins were among the 48 decreased genes correlating most closely with the functional assays, including the stem cell factor receptor c-Kit. Through identification of genes whose expression closely follows functional properties of ESCs during early differentiation, this study lays the foundation for further elucidating the molecular mechanisms regulating the maintenance of ESC pluripotency and facilitates the identification of more reliable molecular markers of the undifferentiated state.