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This study proposes a Fuzzy Inference based Intelligent Fish Feeding Model (FIIFF Inference Model) based on fish activity status, especially Convict grouper activities. The proposed FIIFF Inference Model estimated the feeding amount according to the 3 kinds of fish activities (bottom swimming, slow swimming, jump), the weight of fishes, the temperature of water, and the amount of oxygen in the aqua farm. The advantages of the FIIFF Inference Model intelligently estimated the fish feeding amount according to the fish hungry status in real time.

Hydroxyurea (HU), the first of two drugs approved by the US Food and Drug Administration for treating patients with sickle cell disease (SCD), produces anti-sickling effect by re-activating fetal γ-globin gene to enhance production of fetal hemoglobin. However, approximately 30% of the patients do not respond to HU therapy. The molecular basis of non-responsiveness to HU is not clearly understood. To address this question, we examined HU-induced changes in the RNA and protein levels of transcription factors NF-Y, GATA-1, -2, BCL11A, TR4, MYB and NF-E4 that assemble the γ-globin promoter complex and regulate transcription of γ-globin gene. In erythroblasts cultured from peripheral blood CD34+ cells of patients with SCD, we found that HU-induced changes in the protein but not the RNA levels of activator GATA-2 and repressors GATA-1, BCL11A and TR4 correlated with HU-induced changes in fetal hemoglobin (HbF) levels in the peripheral blood of HU high and low responders. However, HU did not significantly induce changes in the protein or RNA levels of activators NF-Y and NF-E4. Based on HU-induced changes in the protein levels of GATA-2, -1 and BCL11A, we calculated an Index of Hydroxyurea Responsiveness (IndexHU-3). Compared to the HU-induced fold changes in the individual transcription factor protein levels, the numerical values of IndexHU-3 statistically correlated best with the HU-induced peripheral blood HbF levels of the patients. Thus, IndexHU-3 can serve as an appropriate indicator for inherent HU responsiveness of patients with SCD.

Motivation Chromatin accessibility plays a key role in epigenetic regulation of gene activation and silencing. Open chromatin regions allow regulatory elements such as transcription factors and polymerases to bind for gene expression while closed chromatin regions prevent the activity of transcriptional machinery. Recently, Methyltransferase Accessibility Protocol for individual templates-Bisulfite Genome Sequencing (MAPit-BGS) and nucleosome occupancy and methylome sequencing (NOMe-seq) have been developed for simultaneously profiling chromatin accessibility and DNA methylation on single molecules. Therefore, there is a great demand in developing computational methods to identify chromatin accessibility from MAPit-BGS and NOMe-seq. Results In this article, we present CAME (Chromatin Accessibility and Methylation), a seed-extension based approach that identifies chromatin accessibility from NOMe-seq. The efficiency and effectiveness of CAME were demonstrated through comparisons with other existing techniques on both simulated and real data, and the results show that our method not only can precisely identify chromatin accessibility but also outperforms other methods. Availability and Implementation CAME is implemented in java and the program is freely available online at http://sourceforge.net/projects/came/ Supplementary information Supplementary data are available at Bioinformatics online.

High-fat diet consumption and sedentary lifestyle elevates risk for obesity, non-alcoholic fatty liver disease, and cancer. Exercise training conveys health benefits in populations with or without these chronic conditions. Diet and exercise regulate gene expression by mediating epigenetic mechanisms in many tissues; however, such effects are poorly documented in the liver, a central metabolic organ. To dissect the consequences of diet and exercise on the liver epigenome, we measured DNA methylation, using reduced representation bisulfite sequencing, and transcription, using RNA-seq, in mice maintained on a fast food diet with sedentary lifestyle or exercise, compared with control diet with and without exercise. Our analyses reveal that genome-wide differential DNA methylation and expression of gene clusters are induced by diet and/or exercise. A combination of fast food and exercise triggers extensive gene alterations, with enrichment of carbohydrate/lipid metabolic pathways and muscle developmental processes. Through evaluation of putative protective effects of exercise on diet-induced DNA methylation, we show that hypermethylation is effectively prevented, especially at promoters and enhancers, whereas hypomethylation is only partially attenuated. We assessed diet-induced DNA methylation changes associated with liver cancer-related epigenetic modifications and identified significant increases at liver-specific enhancers in fast food groups, suggesting partial loss of liver cell identity. Hypermethylation at a subset of gene promoters was associated with inhibition of tissue development and promotion of carcinogenic processes. Our study demonstrates extensive reprogramming of the epigenome by diet and exercise, emphasizing the functional relevance of epigenetic mechanisms as an interface between lifestyle modifications and phenotypic alterations.

MYCN amplification drives the development of neuronal cancers in children and adults. Given the challenge in therapeutically targeting MYCN directly, we searched for MYCN-activated metabolic pathways as potential drug targets. Here we report that neuroblastoma cells with MYCN amplification show increased transcriptional activation of the serine-glycine-one-carbon (SGOC) biosynthetic pathway and an increased dependence on this pathway for supplying glucose-derived carbon for serine and glycine synthesis. Small molecule inhibitors that block this metabolic pathway exhibit selective cytotoxicity to MYCN-amplified cell lines and xenografts by inducing metabolic stress and autophagy. Transcriptional activation of the SGOC pathway in MYCN-amplified cells requires both MYCN and ATF4, which form a positive feedback loop, with MYCN activation of ATF4 mRNA expression and ATF4 stabilization of MYCN protein by antagonizing FBXW7-mediated MYCN ubiquitination. Collectively, these findings suggest a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited as a strategy for selective cancer therapy. Significance: This study identifies a MYCN-dependent metabolic vulnerability and suggests a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited for cancer therapy. See related commentary by Rodriguez Garcia and Arsenian-Henriksson, p. 3818

Crustacean amphipods are important trophic links between primary producers and higher consumers. Although most amphipods occur in or around aquatic environments, the family Talitridae is the only family found in terrestrial and semi-terrestrial habitats. The sand-hopper Trinorchestia longiramus is a talitrid species often found in the sandy beaches of South Korea. In this study, we present the first draft genome assembly and annotation of this species. We generated ~380.3 Gb of sequencing data assembled in a 0.89 Gb draft genome. Annotation analysis estimated 26,080 protein-coding genes, with 89.9% genome completeness. Comparison with other amphipods showed that T. longiramus has 327 unique orthologous gene clusters, many of which are expanded gene families responsible for cellular transport of toxic substances, homeostatic processes, and ionic and osmotic stress tolerance. This first talitrid genome will be useful for further understanding the mechanisms of adaptation in terrestrial environments, the effects of heavy metal toxicity, as well as for studies of comparative genomic variation across amphipods., Measurement(s)DNA • RNA • sequence_assembly • sequence feature annotationTechnology Type(s)DNA sequencing • RNA sequencing • sequence assembly process • sequence annotationSample Characteristic - OrganismTrinorchestia longiramus Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11897430

Background While aberrant DNA methylation is a characteristic feature of tumor cells, our knowledge of how these DNA methylation patterns are established and maintained is limited. DNA methyltransferases and ten-eleven translocation methylcytosine dioxygenases (TETs) function has been found altered in a variety of cancer types. Results Here, we report that in T cell acute lymphoblastic leukemia (T-ALL) the MYC oncogene controls the expression of TET1 and TET2 to maintain 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) patterns, which is associated with tumor cell-specific gene expression. We found that cellular senescence and tumor regression upon MYC inactivation in T-ALL was associated with genome-wide changes in 5mC and 5hmC patterns. Correlating with the changes in DNA (hydroxy)methylation, we found that T-ALL overexpress TET1, while suppressing TET2 in a MYC-dependent fashion. Consequently, MYC inactivation led to an inverse expression pattern, decreasing TET1, while increasing TET2 levels. Knockdown of TET1 or ectopic expression of TET2 in T-ALL was associated with genome-wide changes in 5mC and 5hmC enrichment and decreased cell proliferation, suggesting a tumor promoting function of TET1, and a tumor suppressing role for TET2. Among the genes and pathways controlled by TET1, we found ribosomal biogenesis and translational control of protein synthesis highly enriched. Conclusions Our finding that MYC directly deregulates the expression of TET1 and TET2 in T-ALL provides novel evidence that MYC controls DNA (hydroxy)methylation in a genome-wide fashion. It reveals a coordinated interplay between the components of the DNA (de)methylating machinery that contribute to MYC-driven tumor maintenance, highlighting the potential of specific TET enzymes for therapeutic strategies. Electronic supplementary material The online version of this article (10.1186/s13072-019-0278-5) contains supplementary material, which is available to authorized users.

SummaryThe histone lysine demethylase KDM4C is often overexpressed in cancers primarily through gene amplification. The molecular mechanisms of KDM4C action in tumorigenesis are not well defined. Here, we report that KDM4C transcriptionally activates amino acid biosynthesis and transport, leading to a significant increase in intracellular amino acid levels. Examination of the serine-glycine synthesis pathway reveals that KDM4C epigenetically activates the pathway genes under steady-state and serine deprivation conditions by removing the repressive histone modification H3 lysine 9 (H3K9) trimethylation. This action of KDM4C requires ATF4, a transcriptional master regulator of amino acid metabolism and stress responses. KDM4C activates ATF4 transcription and interacts with ATF4 to target serine pathway genes for transcriptional activation. We further present evidence for KDM4C in transcriptional coordination of amino acid metabolism and cell proliferation. These findings suggest a molecular mechanism linking KDM4C-mediated H3K9 demethylation and ATF4-mediated transactivation in reprogramming amino acid metabolism for cancer cell proliferation.

The complete mitochondrial genome of sand-hopper Trinorchestia longiramus was analyzed in this study, which is the first for the genus within the family Talitridae. The mitogenome sequence is 15,40...

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER−, not ER+, cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor–based immunotherapy. Cancer Immunol Res; 5(4); 330–44. ©2017 AACR.

Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the Δ exoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression.

// Emily L. Putiri 1 , Rochelle L. Tiedemann 1,2 , Chunsheng Liu 1 , Jeong-Hyeon Choi 2 and Keith D. Robertson 1 1 Department of Molecular Pharmacology and Experimental Therapeutics and Center for Individualized Medicine, Mayo Clinic, Rochester, MN 2 Cancer Center, Georgia Regents University, Augusta, GA Correspondence: Keith D. Robertson, email: // Keywords : DNA methylation, epigenetics, cancer, histone modification, histone methylation Received : May 05, 2014 Accepted : July 13, 2014 Published : July 14, 2014 Abstract The correlation between DNA methylation and a subset of histone post-translational modifications (positive and negative) has hinted at an underlying regulatory crosstalk between histone marks and DNA methylation in patterning the human DNA methylome, an idea further supported by corresponding alterations to both histone marks and DNA methylation during malignant transformation. This study investigated the framework by which histone marks influence DNA methylation at a genome-wide level. Using RNAi in a pluripotent human embryonic carcinoma cell line we depleted essential components of the MLL/COMPASS, polycomb repressive complex 2 (PRC2), and PRC1 histone modifying complexes that establish, respectively, the post-translational modifications H3K4me3, H3K27me3, and H2AK119ub, and assayed the impact of the subsequent depletion of these marks on the DNA methylome. Absence of H2AK119ub resulted predominantly in hypomethylation across the genome. Depletion of H3K4me3 and, surprisingly, H3K27me3 caused CpG island hypermethylation at a subset of loci. Intriguingly, many promoters were co-regulated by all three histone marks, becoming hypermethylated with loss of H3K4me3 or H3K27me3 and hypomethylated with depletion of H2AK119ub, and many of these co-regulated loci were among those commonly targeted for aberrant hypermethylation in cancer. Taken together, our results elucidate novel roles for polycomb and MLL/COMPASS in regulating DNA methylation and define targets of this regulation.

The genetic basis is unknown for ∼60% of normosmic hypogonadotropic hypogonadism (nHH)/Kallmann syndrome (KS). DNAs from (17 male and 31 female) nHH/KS patients were analyzed by targeted next generation sequencing (NGS) of 261 genes involved in hypothalamic, pituitary, and/or olfactory pathways, or suggested by chromosome rearrangements. Selected variants were subjected to Sanger DNA sequencing, the gold standard. The frequency of Sanger-confirmed variants was determined using the ExAC database. Variants were classified as likely pathogenic (frameshift, nonsense, and splice site) or predicted pathogenic (nonsynonymous missense). Two novel FGFR1 mutations were identified, as were 18 new candidate genes including: AMN1, CCKBR, CRY1, CXCR4, FGF13, GAP43, GLI3, JAG1, NOS1, MASTL, NOTCH1, NRP2, PALM2, PDE3A, PLEKHA5, RD3, and TRAPPC9, and TSPAN11. Digenic and trigenic variants were found in 8/48 (16.7%) and 1/48 (2.1%) patients, respectively. NGS with confirmation by Sanger sequencing resulted in the identification of new causative FGFR1 gene mutations and suggested 18 new candidate genes in nHH/KS.

LTR retrotransposons are repetitive DNA elements comprising ∼10% of the human genome. However, LTR sequences are disproportionately present in human long, non-coding RNAs (lncRNAs). Whether and how the LTR lncRNAs serve biological functions are largely unknown. Here we show that in primary human erythroblasts, lncRNAs transcribed from the LTR retrotransposons of ERV-9 human endogenous retrovirus activated transcription of key erythroid genes and modulated ex vivo erythropoiesis. To dissect the functional mechanism of ERV-9 lncRNAs, we performed genome-wide RNA and ChIRP analyses before and after global knockdown or locus-specific deletion of ERV-9 lncRNAs in human erythroblasts carrying ∼4000 copies of the ERV-9 LTRs and in transgenic mouse erythroblasts carrying a single copy of the primate-specific ERV-9 LTR in the 100 kb human β-globin gene locus. We found that ERV-9 lncRNAs acted in cis to stabilize assembly of the ERV-9 LTR enhancer complex and facilitate long-range LTR enhancer function in activating transcription of downstream, cis-linked globin genes. Our findings suggested that LTR lncRNAs transcribed from many of the 4000 copies of ERV-9 LTR retrotransposons acted by a similar cis mechanism to modulate LTR enhancer function in activating transcription of downstream genes critical to cellular processes including erythropoiesis.

Splicing factor SF3B1 is frequently mutated in chronic lymphocytic leukemia (CLL) patients and has been suggested as a potential therapeutic target. In this study, we demonstrated that SF3B1 modulator sudemycin D6 (SD6) effectively inhibits cell growth and induces apoptosis in CLL cells. RNA sequencing analysis revealed significant increases in global intron retention in SD6-treated CLL cells. Pathway analysis of the genes associated with increased intron retention suggested that B-cell receptor (BCR) and PI3K signaling pathways were among the most important pathways being affected by SD6. The increases in intron retention were inversely correlated with decreases in mRNA and protein levels of the affected BCR/PI3K pathway molecules including BLNK, BTK, AKT, PLCγ2, and PI3Kδ. SD6 treatment also induced a time-dependent exon-skipping event in MCL-1 mRNA and resulted in significant down-regulation of another anti-apoptotic gene TRAF1, thus collectively contributing to the SD6-induced apoptosis. Furthermore, SD6 treatment can overcome the pro-survival and pro-growth signals and synergize with established CLL therapies ibrutinib, idelalisib, and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Finally, in vivo treatment with SD6 at 10mg/kg/day for 7 days significantly inhibited the growth of xenograft tumors that were established by subcutaneous inoculation of 5×106 MEC1 CLL cells into NOD mice. Collectively, these results provide a strong rationale for the future clinical development of spliceosome modulators and potential combination therapies for the treatment of CLL. Citation Format: Qimei Han, Jianbo Wang, Austin Y. Shull, Fang Shi, Libin Deng, Jeong-Hyeon Choi, Eun-Jeong Park, Shuo Tu, Lirong Pei, Farrukh T. Awan, Roni Bollag, Locke J. Bryan, Hong-bo Xin, Chandraiah Lagisetti, Thomas R. Webb, Victor Jin, Huidong Shi. Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5230.

Induction of differentiation is a therapeutic strategy in neuroblastoma, a common pediatric cancer of the sympathetic nervous system. The homeobox protein HOXC9 is a key regulator of neuroblastoma differentiation. To gain a molecular understanding of the function of HOXC9 in promoting differentiation of neuroblastoma cells, we conducted a genome-wide analysis of the HOXC9-induced differentiation program by microarray gene expression profiling and chromatin immunoprecipitation in combination with massively parallel sequencing (ChIP-seq). Here we describe in detail the experimental system, methods, and quality control for the generation of the microarray and ChIP-seq data associated with our recent publication [1].

Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive cyclic diguanosine monophosphate phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchro- matic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, oftendistant, genes. Sixty-four percent of genes areexpressed detectably in atleastone line, but only 21%are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ''off'' andsurvival/growth pathways''on.''Roughly 50%ofthe genesexpressed by eachlinearenot part ofthe commonset, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most re- markable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ''cell line'' gene expression pattern. (Supplemental material is available for this article. The data from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession nos. GSE15596, GSE16269-GSE16290, GSE16321- GSE16322, GSE16325, and GSE18040. All of the microarray data, RNA-seq data, and expression scores for genes and exons are available from the Data Coordination Center of modENCODE (http://modencode.org), and much of the data are also available from the Drosophila Genomic Resources Center (https://dgrc.cgb.indiana.edu/) and FlyBase (http:// flybase.org/).)

Four percent of female Nasonia vitripennis carry the son-killer bacterium Arsenophonus nasoniae, a microbe with notably different biology from other inherited parasites and symbionts. In this paper, we examine a draft genome sequence of the bacterium for open reading frames (ORFs), structures and pathways involved in interactions with its insect host. The genome data suggest that A. nasoniae carries multiple type III secretion systems, and an array of toxin and virulence genes found in Photorhabdus, Yersinia and other gammaproteobacteria. Of particular note are ORFs similar to those known to affect host innate immune functioning in other bacteria, and four ORFs related to pro-apoptotic exotoxins. The genome sequences for both A. nasoniae and its Nasonia host are useful tools for examining functional genomic interactions of microbial survival in hostile immune environments, and mechanisms of passage through gut epithelia, in a whole organism context.

Background Internal circadian (circa, about; dies, day) clocks enable organisms to maintain adaptive timing of their daily behavioral activities and physiological functions. Eukaryotic clocks consist of core transcription-translation feedback loops that generate a cycle and post-translational modifiers that maintain that cycle at about 24 h. We use the pitcher-plant mosquito, Wyeomyia smithii (subfamily Culicini, tribe Sabethini), to test whether evolutionary divergence of the circadian clock genes in this species, relative to other insects, has involved primarily genes in the core feedback loops or the post-translational modifiers. Heretofore, there is no reference transcriptome or genome sequence for any mosquito in the tribe Sabethini, which includes over 375 mainly circumtropical species. Methods We sequenced, assembled and annotated the transcriptome of W. smithii containing nearly 95 % of conserved single-copy orthologs in animal genomes. We used the translated contigs and singletons to determine the average rates of circadian clock-gene divergence in W. smithii relative to three other mosquito genera, to Drosophila, to the butterfly, Danaus, and to the wasp, Nasonia. Results Over 1.08 million cDNA sequence reads were obtained consisting of 432.5 million nucleotides. Their assembly produced 25,904 contigs and 54,418 singletons of which 62 % and 28 % are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. Discussion The W. smithii transcriptome includes all nine circadian transcription-translation feedback-loop genes and all eight post-translational modifier genes we sought to identify (Fig. 1). After aligning translated W. smithii contigs and singletons from this transcriptome with other insects, we determined that there was no significant difference in the average divergence of W. smithii from the six other taxa between the core feedback-loop genes and post-translational modifiers. Conclusions The characterized transcriptome is sufficiently complete and of sufficient quality to have uncovered all of the insect circadian clock genes we sought to identify (Fig. 1). Relative divergence does not differ between core feedback-loop genes and post-translational modifiers of those genes in a Sabethine species (W. smithii) that has experienced a continual northward dispersal into temperate regions of progressively longer summer day lengths as compared with six other insect taxa. An associated microarray platform derived from this work will enable the investigation of functional genomics of circadian rhythmicity, photoperiodic time measurement, and diapause along a photic and seasonal geographic gradient. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1937-y) contains supplementary material, which is available to authorized users.

Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSC), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Furthermore, CSCs are believed to contribute to chemo- and radioresistance. In this study, we used the MMTV-Neu-Tg mouse mammary tumor model to identify potential new strategies for eliminating CSCs. We found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which facilitate oncogenic transformation and tumorigenic potential. A combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate markedly reduced CSC abundance and increased the overall survival in this mouse model. RNA-seq analysis of CSCs treated with 5-azacytidine plus butyrate provided evidence that inhibition of chromatin modifiers blocks growth-promoting signaling molecules such as RAD51AP1 and SPC25, which play key roles in DNA damage repair and kinetochore assembly. Moreover, RAD51AP1 and SPC25 were significantly overexpressed in human breast tumor tissues and were associated with reduced overall patient survival. In conclusion, our studies suggest that breast CSCs are intrinsically sensitive to genetic and epigenetic modifications and can therefore be significantly affected by epigenetic-based therapies, warranting further investigation of combined DNMT and HDAC inhibition in refractory or drug-resistant breast cancer. Cancer Res; 76(11); 3224–35. ©2016 AACR.

It is well known that cancer cells are diverse and heterogeneous among patients, and that this heterogeneity makes cancer diagnosis and cure difficult. Intra-tumoral heterogeneity has very recently become important because a small proportion of drug-resistant or tumor-initiating cells can ultimately determine a patient's outcome. In this study, we propose new approaches to use variance tests, including the Lepage test, to identify inter-patient cancer heterogeneity, as well as multinomial logistic regression to identify intra-patient cancer heterogeneity, i.e., different epiallele composition. We conduct experiments to show the performance of the proposed approaches.

The Fas–FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colon carcinoma often uses silencing Fas expression as a mechanism of immune evasion. The molecular mechanism under FAS transcriptional silencing in human colon carcinoma is unknown. We performed genome-wide chromatin immunoprecipitation sequencing analysis and identified that the FAS promoter is enriched with H3K9me3 in metastatic human colon carcinoma cells. The H3K9me3 level in the FAS promoter region is significantly higher in metastatic than in primary cancer cells, and it is inversely correlated with Fas expression level. We discovered that verticillin A is a selective inhibitor of histone methyltransferases SUV39H1, SUV39H2, and G9a/GLP that exhibit redundant functions in H3K9 trimethylation and FAS transcriptional silencing. Genome-wide gene expression analysis identified FAS as one of the verticillin A target genes. Verticillin A treatment decreased H3K9me3 levels in the FAS promoter and restored Fas expression. Furthermore, verticillin A exhibited greater efficacy than decitabine and vorinostat in overcoming colon carcinoma resistance to FasL-induced apoptosis. Verticillin A also increased DR5 expression and overcame colon carcinoma resistance to DR5 agonist drozitumab-induced apoptosis. Interestingly, verticillin A overcame metastatic colon carcinoma resistance to 5-fluorouracil in vitro and in vivo. Using an orthotopic colon cancer mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL+ and that FasL-mediated cancer immune surveillance is essential for colon carcinoma growth control in vivo. Our findings determine that H3K9me3 of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant colon carcinoma immune evasion and progression.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

DNA methylation is the most studied epigenetic event in cancer, with focus being placed on studying the entire DNA methylation landscape in specific cancers. Due to the recent advances of next-generation sequencing technology, several effective methods have been developed for high-throughput analysis of DNA methylation, enabling DNA methylation markers to be innovative diagnostic and therapeutic strategies in cancer. In this review, we discuss various current and emerging technologies in DNA methylation analysis that integrate next-generation sequencing with the basic principles of methylation detections including methylation sensitive restriction enzyme digestion, affinity purification with antibody or binding proteins, and bisulfite treatment. Variations to these described methods have also allowed for detection of 5-hydroxymethylcytosine marks on a genome-wide scale. We also describe several of the bioinformatic tools used to properly analyze methylome-sequencing data. Finally, recently developed artificial transcription-factor (ATF) targeting tools may provide flexible manipulation of DNA methylation events in specific gene regions, revealing the functional consequences of particular DNA methylation events.

Motivation: Gene clusters are arrangements of functionally related genes on a chromosome. In bacteria, it is expected that evolutionary pressures would conserve these arrangements due to the functional advantages they provide. Visualization of conserved gene clusters across multiple genomes provides key insights into their evolutionary histories. Therefore, a software tool that enables visualization and functional analyses of gene clusters would be a great asset to the biological research community. Results: We have developed GeneclusterViz, a Java-based tool that allows for the visualization, exploration and downstream analyses of conserved gene clusters across multiple genomes. GeneclusterViz combines an easy-to-use exploration interface for gene clusters with a host of other analysis features such as multiple sequence alignments, phylogenetic analyses and integration with the KEGG pathway database. Availability: http://biohealth.snu.ac.kr/GeneclusterViz/; http://microbial.informatics.indiana.edu/GeneclusterViz/ Contact: sunkim.bioinfo@snu.ac.kr; ybrun@indiana.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Background Although Daphnia is increasingly recognized as a model for ecological genomics and biomedical research, there is, as of yet, no high-resolution genetic map for the genus. Such a map would provide an important tool for mapping phenotypes and assembling the genome. Here we estimate the genome size of Daphnia magna and describe the construction of an SNP array based linkage map. We then test the suitability of the map for life history and behavioural trait mapping. The two parent genotypes used to produce the map derived from D. magna populations with and without fish predation, respectively and are therefore expected to show divergent behaviour and life-histories. Results Using flow cytometry we estimated the genome size of D. magna to be about 238 mb. We developed an SNP array tailored to type SNPs in a D. magna F2 panel and used it to construct a D. magna linkage map, which included 1,324 informative markers. The map produced ten linkage groups ranging from 108.9 to 203.6 cM, with an average distance between markers of 1.13 cM and a total map length of 1,483.6 cM (Kosambi corrected). The physical length per cM is estimated to be 160 kb. Mapping infertility genes, life history traits and behavioural traits on this map revealed several significant QTL peaks and showed a complex pattern of underlying genetics, with different traits showing strongly different genetic architectures. Conclusions The new linkage map of D. magna constructed here allowed us to characterize genetic differences among parent genotypes from populations with ecological differences. The QTL effect plots are partially consistent with our expectation of local adaptation under contrasting predation regimes. Furthermore, the new genetic map will be an important tool for the Daphnia research community and will contribute to the physical map of the D. magna genome project and the further mapping of phenotypic traits. The clones used to produce the linkage map are maintained in a stock collection and can be used for mapping QTLs of traits that show variance among the F2 clones., BMC Genomics, 15, ISSN:1471-2164

Background Cellular differentiation is characterized by the acquisition of specialized structures and functions, cell cycle exit, and global attenuation of the DNA damage response. It is largely unknown how these diverse cellular events are coordinated at the molecular level during differentiation. We addressed this question in a model system of neuroblastoma cell differentiation induced by HOXC9. Results We conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program. Microarray gene expression profiling revealed that HOXC9-induced differentiation was associated with transcriptional regulation of 2,370 genes, characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping by ChIP-seq demonstrated that HOXC9 bound to 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and the DNA damage response. Moreover, we showed that HOXC9 interacted with the transcriptional repressor E2F6 and recruited it to the promoters of cell cycle genes for repressing their expression. Conclusions Our results demonstrate that HOXC9 coordinates diverse cellular processes associated with differentiation by directly activating and repressing the transcription of distinct sets of genes.

SummaryIncreased activation of the serine-glycine biosynthetic pathway is an integral part of cancer metabolism that drives macromolecule synthesis needed for cell proliferation. Whether this pathway is under epigenetic control is unknown. Here we show that the histone H3 lysine 9 (H3K9) methyltransferase G9A is required for maintaining the pathway enzyme genes in an active state marked by H3K9 monomethylation and for the transcriptional activation of this pathway in response to serine deprivation. G9A inactivation depletes serine and its downstream metabolites, triggering cell death with autophagy in cancer cell lines of different tissue origins. Higher G9A expression, which is observed in various cancers and is associated with greater mortality in cancer patients, increases serine production and enhances the proliferation and tumorigenicity of cancer cells. These findings identify a G9A-dependent epigenetic program in the control of cancer metabolism, providing a rationale for G9A inhibition as a therapeutic strategy for cancer.

Despite sharing much of their genomes, males and females are often highly dimorphic, reflecting at least in part the resolution of sexual conflict in response to sexually antagonistic selection. Sexual dimorphism arises owing to sex differences in gene expression, and steroid hormones are often invoked as a proximate cause of sexual dimorphism. Experimental elevation of androgens can modify behavior, physiology, and gene expression, but knowledge of the role of hormones remains incomplete, including how the sexes differ in gene expression in response to hormones. We addressed these questions in a bird species with a long history of behavioral endocrinological and ecological study, the dark-eyed junco (Junco hyemalis), using a custom microarray. Focusing on two brain regions involved in sexually dimorphic behavior and regulation of hormone secretion, we identified 651 genes that differed in expression by sex in medial amygdala and 611 in hypothalamus. Additionally, we treated individuals of each sex with testosterone implants and identified many genes that may be related to previously identified phenotypic effects of testosterone treatment. Some of these genes relate to previously identified effects of testosterone-treatment and suggest that the multiple effects of testosterone may be mediated by modifying the expression of a small number of genes. Notably, testosterone-treatment tended to alter expression of different genes in each sex: only 4 of the 527 genes identified as significant in one sex or the other were significantly differentially expressed in both sexes. Hormonally regulated gene expression is a key mechanism underlying sexual dimorphism, and our study identifies specific genes that may mediate some of these processes.

The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression., Author Summary Insects use methylation to modulate genome function in a different manner from vertebrates. Here, we quantified the global methylation profile in a parasitic wasp species, Nasonia vitripennis, a model with some advantages over ant and honeybee for functional and genetic analyses of methylation, such as short generation time, inbred lines, and inter-fertile species. Using a highly inbred line permitted us to precisely characterize DNA methylation, which is compared to gene expression variation across developmental stages, and contrasted to other insect species. DNA methylation is almost exclusively on the 5′-most 1 kbp coding exons, and ∼1/3 of protein coding genes are methylated. Methylated genes tend to occur in small clusters in the genome. Unlike many organisms, Nasonia leaves nearly all transposable element genes non-methylated. Methylated genes exhibit more uniform expression across developmental stages for both moderately and highly expressed genes, suggesting that DNA methylation is marking the genes for constitutive expression. Among pairs of differentially methylated duplicated genes, the paralogs that lose DNA methylation after duplication in the Nasonia lineage show lower expression and greater specialization of expression. Finally, by comparative analysis, we show that methylated genes are more conserved at three different time scales during evolution.

Chronic lymphocytic leukemia (CLL) is a malignancy characterized by the progressive accumulation of CD19+ B-cells capable of overcoming their regulated life cycle. Because this disease is highly heterogeneous and utilizes several mechanisms to resist apoptotic death, CLL currently remains incurable. Based on the ongoing efforts to interpret the transcriptional dynamics of CLL pathogenicity as well as prior reports demonstrating the efficacy of cyclin-dependent kinase (CDK) transcriptional inhibitors, we sought to characterize the molecular response of CLL cells treated with the covalent CDK7 inhibitor THZ1 and determine the therapeutic potential of CDK7 inhibition in CLL. We first observed that THZ1 was able to inhibit growth in malignant B-cell lines MEC1 and MEC2 in both a dose dependent (IC50: .045uM & .030uM, respectively) and time dependent manner. The inhibition in cell growth corresponded with both G1-mediated cell cycle arrest as well as apoptosis in the respective cell lines. We also observed dose-dependent reduction in cell viability through apoptosis in patient-derived CLL B-cells after 24 hours. Because of the observed outcomes of THZ1-treated CLL cells, we then wanted to determine the specific transcriptional targets significantly suppressed by THZ1 treatment. To identify the THZ1-sensitive transcripts, we performed a time course RNAseq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of covered reads over a 12-hour period, we determined that the top 50 transcripts greatly diminished in both MEC1 and MEC2 contain significant enrichment in genes attributed to glycolytic metabolism (ex: ALDOC, SLC2A1, TPI1, GAPDH, ENO2). Along with the overlapping comparison between the two cell lines, we next compared the downregulated transcripts from the treated cell lines against the RNAseq expression profile of 47 CLL patients and 5 healthy donors. We observed that THZ1 was able to suppress transcripts upregulated in CLL patient samples including ENO2, FGR, WNT10A, and CBX7 in MEC1 and ENO2, MALAT1, CCR7, and PLCG1 in MEC2. Finally, we performed H3K27Ac ChIPseq in MEC1 to identify super enhancers that may overlap with THZ1-sensitive transcripts. From this comparison, we determined that super enhancers exist within reported oncogenic drivers CBX7, WNT10A, and FGR. Overall, we observe that THZ1 can effectively overcome the anti-apoptotic phenotype of CLL B-cells as well as directly suppress transcription of genes that can drive CLL malignancy. With the addition of CDK7 ChIPseq, our ultimate goal is to provide clarity regarding CDK7-mediated transcriptional regulation in CLL and demonstrate the molecular consequences of therapeutic CDK7 inhibition in CLL. Citation Format: Austin Young Shull, Jeong-Hyeon Choi, Jordan Bauer, Lirong Pei, Farrukh T. Awan, Huidong Shi. The covalent CDK7 inhibitor THZ1 can counteract apoptotic resistance and suppress the transcription of genes attributed to chronic lymphocytic leukemia malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4697.

Background With the advent of next-generation sequencing (NGS) technologies, full cDNA shotgun sequencing has become a major approach in the study of transcriptomes, and several different protocols in 454 sequencing have been invented. As each protocol uses its own short DNA tags or adapters attached to the ends of cDNA fragments for labeling or sequencing, different contaminants may lead to mis-assembly and inaccurate sequence products. Results We have designed and implemented a new program for raw sequence cleaning in a graphical user interface and a batch script. The cleaning process consists of several modules including barcode trimming, sequencing adapter trimming, amplification primer trimming, poly-A tail trimming, vector screening and low quality region trimming. These modules can be combined based on various sequencing applications. Conclusions ESTclean is a software package not only for cleaning cDNA sequences, but also for helping to develop sequencing protocols by providing summary tables and figures for sequencing quality control in a graphical user interface. It outperforms in cleaning read sequences from complicated sequencing protocols which use barcodes and multiple amplification primers.

DNA methylation, mediated by the combined action of three DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), is essential for mammalian development and is a major contributor to cellular transformation. To elucidate how DNA methylation is targeted, we mapped the genome-wide localization of all DNMTs and methylation, and examined the relationships among these markers, histone modifications, and nucleosome structure in a pluripotent human tumor cell line in its undifferentiated and differentiated states. Our findings reveal a strong link between DNMTs and transcribed loci, and that DNA methylation is not a simple sum of DNMT localization patterns. A comparison of the epigenomes of normal and cancerous stem cells, and pluripotent and differentiated states shows that the presence of at least two DNMTs is strongly associated with loci targeted for DNA hypermethylation. Taken together, these results shed important light on the determinants of DNA methylation and how it may become disrupted in cancer cells., National Institutes of Health (U.S.) (Grant RC1HG005334), National Science Foundation (U.S.) (Postdoctoral Fellowship 0905968)

We conducted a genome-wide DNA methylation analysis in CD19 (+) B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8-2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3'-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.

Transcriptome sequencing from cDNA libraries has been extensively and efficiently used to analyze sequence variation in protein-coding genes (Expressed Sequence Tags) in eukaryote species. Rapid advances in next-generation sequencing (NGS) technology, in terms of cost, speed, and throughput, allow us to address previously unanswerable questions in the fields of ecology, evolution, and systematics using these genomic tools. Transcriptome sequencing from individuals across different populations and species enables researchers to study the evolution of gene sequence variation at a population genomics level. In this chapter, we describe a customized protocol that has been successfully optimized for the development of normalized cDNA libraries in eukaryote systems suitable for Roche 454 GS FLX sequencing, requiring only small quantities of starting material.

Bisulfite sequencing (BS-seq) is one of the most important sequencing protocols for genome-wide DNA methylation studies. However, it causes significant challenges in computation because it converts unmethylated cytosines to thymines. This paper presents a software package to address those challenges and to provide various kinds of analysis modules, from mapping to comparing multiple samples.

Background Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. Methodology/Principal Findings We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. Conclusions/Significance This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

We report the properties of a draft genome sequence of the bacterium Arsenophonus nasoniae, son-killer bacterium of Nasonia vitripennis. The genome sequence data from this study are the first for a male-killing bacterium, and represent a microorganism that is unusual compared with other sequenced symbionts, in having routine vertical and horizontal transmission, two alternating hosts, and being culturable on cell-free media. The resulting sequence totals c. 3.5 Mbp and is annotated to contain 3332 predicted open reading frames (ORFs). Therefore, Arsenophonus represents a relatively large genome for an insect symbiont. The annotated ORF set suggests that the microbe is capable of a broad array of metabolic functions, well beyond those found for reproductive parasite genomes sequenced to date and more akin to horizontally transmitted and secondary symbionts. We also find evidence of genetic transfer from Wolbachia symbionts, and phage exchange with other gammaproteobacterial symbionts. These findings reflect the complex biology of a bacterium that is able to live, invade and survive multiple host environments while resisting immune responses.

Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2015-4060

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumours, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumours and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment.

One of the main issues in comparative genomics is the study of chromosomal gene order in one or more related species. Recently identifying sets of orthologous genes in several genomes has become getting important, since a cluster of similar genes helps us to predict the function of unknown genes. For this purpose, the whole genome alignment is usually used to determine horizontal gene transfer, gene duplication, and gene loss between two related genomes. Also it is well known that a novel visualization tool of the whole genome alignment would be very useful for understanding genome organization and the evolutionary process. In this paper, we propose a method for identifying and visualizing the alignment of the whole genome alignment, especially for detecting gene clusters between two aligned genomes. Since the current rigorous algorithm for finding gene clusters has strong and artificial constraints, they are not useful for coping with “noisy” alignments. We developed the system AlignScope to provide a simplified structure for genome alignment at any level, and also to help us to find gene clusters. In this experiment, we have tested AlignScope on several microbial genomes. Alignment is a procedure that compares two or more sequences by searching for a series of individual characters or character patterns that are in the same order in the sequences. This procedure assists in designating the functions of unknown proteins, determining the relatedness of organisms, and identifying structurally and functionally important elements and other useful functions. 9;12 Many widely divergent organisms are descended from a common ancestor through a process called evolution. The inheritance patterns and diversities of these organisms have significant information regarding the nature of small and large-scale evolutionary events. The complexity and the size of the genome make it difficult to analyze. Because the large amount of biological noises is present when visualizing genomes, it is not enough to simply draw the aligned pairs of various genomes. Therefore an alignment visualization tool needs to provide a method for viewing the global structure of whole genome alignment in a simplified form at any level of detail. Figure-1 clearly illustrates this problem. In Figure-1, the resolution of the snapshot is 800 by 600 pixels, so one pixel corresponds about 6000 bases of a given genome sequence. Currently there are several systems for visualizing the alignment of genomes. The NCBI Map Viewer 14 provides graphical displays of biological features on NCBI’s as

Summary: COMPAM is a tool for visualizing relationships among multiple whole genomes by combining all pairwise genome alignments. It displays shared conserved regions (blocks) and where these blocks occur (edges) as block relation graphs which can be explored interactively. An unannotated genome, e.g. can then be explored using information from well-annotated genomes, COG-based genome annotation and genes. COMPAM can run either as a stand-alone application or through an applet that is provided as service to PLATCOM, a toolset for whole genome comparative analysis, where a wide variety of genomes can be easily selected. Features provided by COMPAM include the ability to export genome relationship information into file formats that can be used by other existing tools. Availability: Contact: dohhlee@indiana.edu; sunkim2@indiana.edu

It is well-known that functionally related genes occur in a physically clustered form, especially operons in bacteria. By leveraging on this fact, there has recently been an interesting problem formulation known as gene team model, which searches for a set of genes that co-occur in a pair of closely related genomes. However, many gene teams, even experimentally verified operons, frequently scatter within other genomes. Thus, the gene team model should be refined to reflect this observation. In this paper, we generalized the gene team model, that looks for gene clusters in a physically clustered form, to multiple genome cases with relaxed constraints. We propose a novel hybrid pattern model that combines the set and the sequential pattern models. Our model searches for gene clusters with and/or without physical proximity constraint. This model is implemented and tested with 97 genomes (120 replicons). The result was analyzed to show the usefulness of our model. We also compared the result from our hybrid model to those from the traditional gene team model. We also show that predicted gene teams can be used for various genome analysis: operon prediction, phylogenetic analysis of organisms, contextual sequence analysis and genome annotation. Our program is fast enough to provide a service on the web at http://platcom.informatics.indiana.edu/platcom/. Users can select any combination of 97 genomes to predict gene teams.

We have developed a multiple genome alignment algorithm by using a sequence clustering algorithm to combine local pairwisegenome sequence matches produced by pairwise genome alignments, e.g, BLASTZ. Sequence clustering algorithms often generate clusters of sequences such that there exists a common shared region among all sequences in each cluster. To use a sequence clustering algorithm for genome alignment, it is necessary to handle numerous local alignments between a pair of genomes. We propose a multiple genome alignment method that converts the multiple genome alignment problem to the sequence clustering problem. This method does not need to make a guide tree to determine the order of multiple alignment, and it accurately detects multiple homologous regions. As a result, our multiple genome alignment algorithm performs competitively over existing algorithms. This is shown using an experiment which compares the performance of TBA, MultiPipMaker (MPM) and our algorithm in aligning 12 groups of 56 microbial genomes and by evaluating the number of common COGs detected.

DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, 3B, 3L) and maintaining (DNMT1) DNA methylation genome-wide. Aberrant DNA methylation is frequently observed in cancer; however, little is known about how regulation of this modification goes awry. In this study, we aim to understand how DNA methylation is regulated by the DNMTs throughout the genome by identifying specific and broad changes in methylation patterning upon depletion of the DNMTs. We utilize siRNA technology to acutely deplete NCCIT embryonal carcinoma cells of DNMT mRNA (individually and in combination), and then assay the impact on genome-wide DNA methylation patterns using the HumanMethylation450 Bead Chip (450K array). Depletion of DNMT1 (individual/combination) resulted in widespread hypomethylation, most notably in gene bodies, 3′UTRs, and intergenic sequences. DNMT3 knockdown resulted in more specific changes in DNA methylation, but surprisingly, more hypermethylation (predominately in gene bodies) than hypomethylation events occurred. These specific hypermethylation events, particularly in samples with DNMT3B KD, significantly overlapped with sites hypomethylated in DNMT1 KD conditions, indicating a potential cross-regulatory role for DNMT1 and DNMT3B in regulating DNA methylation across gene bodies. To gain a more comprehensive genome-wide view of DNA methylation in the absence of DNMT3B, we performed Methyl-CpG-Binding-Domain(MBD)-seq on DNMT3B KD cells. MBD-seq revealed dynamic changes in methylation with minor hypermethylation in promoters and subtle hypomethylation across gene bodies; however, analysis of only the most significant methylation changes (> 4-fold) revealed that more hypermethylation events occur in intronic sequences, consistent with results obtained using the 450K array. To further investigate the overlap between DNMT1 hypomethylated and DNMT3B hypermethylated sites, we examined DNA methylation in HCT116 colorectal carcinoma cells lacking (KO) or over-expressing (KI) DNMT1/DNMT3B. Interestingly, a marked number of CpG sites that gained methylation in the DNMT3B KO overlapped significantly with sites that became hypermethylated in DNMT1 and DNMT3B KI, and hypomethylated in DNMT1 KO. Additionally, these HCT116 hypermethylated CpG sites gained methylation in NCCIT DNMT3B KD (individual/combination) and lost methylation in DNMT1 KD. Taken together, these results suggest that DNMT1 and DNMT3B co-regulate DNA methylation at conserved loci across cell types in an opposing fashion, providing novel insight into a potential regulatory mechanism for DNA methylation patterning. Further elucidation of this DNMT1 and DNMT3B co-regulation holds the potential to yield novel therapeutic strategies for correcting aberrant methylation events in cancer. Citation Format: Rochelle Tiedemann, Jeong-Hyeon Choi, Keith Robertson. Acute depletion reveals novel co-regulation of DNA methylation at conserved loci by DNMT1 and DNMT3B. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2305. doi:10.1158/1538-7445.AM2014-2305

The Ten-eleven translocation (TET) family of dioxygenases hydroxylate 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. 5mC provides important epigenetic instructions during development, and its aberrant control is a major contributor to cellular transformation; however TET functions in regulating the epigenome, particularly in cancer, remain largely unknown. We targeted TET1, TET2, and TET3 for siRNA-mediated depletion in pluripotent human embryonic carcinoma cells and examined the impact on 5mC and 5hmC genome-wide localization. TET1, TET2, and TET3 co-regulate 5hmC at many sites, and depletion of only one of the TETs is sufficient to reduce 5hmC at these co-regulated sites, suggesting a functional co-dependence for TETs. Depletion of TET1 and TET2 had the greatest impact on 5hmC levels at high and low CpG density promoters, respectively, indicating that TETs exhibit DNA sequence-based functional specificity. All TETs prevent hypermethylation throughout the genome, especially in CpG island shores, where TET depletion resulted in prolific hypermethylation. Promoter hypermethylation resulting from TET depletion was associated with histone H2AK119 monoubiquitination, DNMT1, and DNMT3B occupancy. Surprisingly, TETs also promote cytosine methylation, as many loci became hypomethylated following TET depletion. Induction of differentiation generally caused 5hmC reduction, except at transcriptionally activated genes, which become enriched for 5hmC. Importantly, genes prone to promoter hypermethylation in cancer become depleted of intragenic 5hmC and 5mC with TET deficiency. This study highlights the multi-dimensional functions of TETs in mediating DNA methylation, hydroxymethylation, and gene expression patterns, and the results reveal that chromatin landscape and DNA sequence composition are regulators of TET function. Citation Format: Emily L. Putiri, Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2014-2319

Glioblastoma (GBM) is the most common and most aggressive brain tumor in adults. Its frequent recurrence after resection and dismal prognosis are thought to be due to a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy. In this study, we performed an in depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary GBM tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified two groups hyper- and hypomethylated of genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also be found in primary GBM-derived xenograft tumors suggesting that they were not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM, and PENK. The comparison between the NSC line and the normal brain tissue sample leads to the identification of a group of NSC-specific differentially methylated regions (nsDMRs). Cluster analysis using nsDMRs revealed the presence of stem cell-specific DNA methylation signatures in both primary GBM and GSC lines. Higher expression of genes associated with stem cell-specific DNA methylation signatures such as DNMT3A, STAT5A, CSTB, PMEPA1 and G6PD in GBM patients was found to be associated with poor patient survival. The results from this study demonstrate the utility of using cancer stem cell models for advancing understanding of the pathobiology of gliomas. Citation Format: Eun Joon Lee, Prakash Rath, Jimei Liu, Dungsheng Ryu, Alan Free, Lirong Pei, Douglas C Anthony, Suash Sharma, Mark D Kirk, John J. Laterra, Duck Hwan Ryu, Jeong-Hyeon Choi, Huidong Shi, Douglas C. Miller, N. Scott Litofsky, Qi Feng. Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2014-1379