Global Epigenetic Changes Research Articles

Abstract 2424: The in-parallel whole-genome ChIP-Seq analysis of primary tissues, patient derived xenografts, and cancer cell lines from HPV-relative HNSCC samples

Abstract This project develops a novel experimental technique to perform ChIP-Seq (chromatin immunoprecipitation with massively parallel DNA sequencing) analysis of chromatin structure in primary tumor tissues from high risk HPV-related head and neck squamous cell carcinomas (HPV+ HNSCC). Recent data suggest that chromatin structure is the central regulator and predictor of cancer-specific expression and mutagenesis landscape of diseased cells. Genome-wide gene expression dysregulation in many tumors, including HPV+ HNSCC, are incompletely described by current knowledge. Methods for study of chromatin structure in primary tumor tissue are needed to better understand the role global epigenetic changes may play in these tumors. However, ChIP-Seq, which is the state-of-the-art method of elucidating chromatin structure, until now, has not been reliably performed on any HNSCC samples. Because chromatin structure is disrupted at room temperature, ChIP-Seq is especially complicated for primary patient tissues, which are primarily obtained as surgical waste after pathology review. Snap freezing of leftover waste surgical tissues and further tissue thawing for the analysis decreases chromatin structure integrity necessary for highly sensitive ChIP-Seq methodology, especially for tumor samples with chromatin structure deformed during carcinogenesis. To improve the chromatin structure integrity in tumor sample we added a xenografting step and minimized the exposure of cancer tissue to room temperature conditions after mouse surgery. We also minimized patient non-cancer tissue preservation at ambient temperature after patient surgery. We successfully performed ChIP-Seq for H3K4me3, H3K9me3, and H3K9ac on frozen uvulopalatopharyngoplasty (UPPP) primary tissues, frozen patient derived xenograft tissues, and freshly-cultured head and neck squamous cell carcinoma cell lines, revealing comparable success rates between tissue type and sample preservation techniques. ChIP-Seq techniques were performed and cross validated using tried and true qRT-PCR methods to demonstrate data reproducibility. The biological relevance of the ChIP-Seq data was confirmed through massive RNA-Seq analysis of 47 HPV+ HNSCC samples and 25 non-cancer controls. Analysis revealed that most H3K9ac and H3K9me3 enrichment is similar in primary tissues, regardless of disease status. Only small portion of them showed differential histone enrichment, which correlated with differential expression of corresponding genes. On the other hand, H3K4me3 showed strong tissue specificity and were found differentially enriched especially in tumor samples. The proposed experimental pipeline demonstrates high reproducibility between biological replicates, diversity of tissue models, and low dependence of ChIP-Seq analysis on tissue preservation techniques. Citation Format: Dylan Z. Kelley, Emily L. Flam, Hildegard A. Wulf, Theresa Guo, Evgeny Izumchenko, Dzov A. Singman, Ludmila V. Danilova, Elena D. Stavrovskaya, Michael Considine, Justin A. Bishop, William H. Westra, Zubair Khan, Wayne M. Koch, David Sidransky, Sarah Wheelan, Joseph A. Califano, Alexander V. Favorov, Elana J. Fertig, Daria A. Gaykalova. The in-parallel whole-genome ChIP-Seq analysis of primary tissues, patient derived xenografts, and cancer cell lines from HPV-relative HNSCC samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2424. doi:10.1158/1538-7445.AM2017-2424

ITIH5 mediates epigenetic reprogramming of breast cancer cells

BackgroundExtracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive.MethodsITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement.ResultsITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility.ConclusionsOur results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.

Nuclear Localization of Mitochondrial TCA Cycle Enzymes as a Critical Step in Mammalian Zygotic Genome Activation

Transcriptional control requires epigenetic changes directed by mitochondrial tricarboxylic acid (TCA) cycle metabolites. In the mouse embryo, global epigenetic changes occur during zygotic genome activation (ZGA) at the 2-cell stage. Pyruvate is essential for development beyond this stage, which is at odds with the low activity of mitochondria in this period. We now show that a number of enzymatically active mitochondrial enzymes associated with the TCA cycle are essential for epigenetic remodeling and are transiently and partially localized to the nucleus. Pyruvate is essential for this nuclear localization, and a failure of TCA cycle enzymes to enter the nucleus correlates with loss of specific histone modifications and a block in ZGA. At later stages, however, these enzymes are exclusively mitochondrial. In humans, the enzyme pyruvate dehydrogenase is transiently nuclear at the 4/8-cell stage coincident with timing of human embryonic genome activation, suggesting a conserved metabolic control mechanism underlying early pre-implantation development.

Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies.

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). Here we characterize global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Bx. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and we identify different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, we find that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. Our results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs.

Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis.

Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients.

Abstract 1501: Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer

Abstract Gastric cancer is the fourth leading cause of cancer-related death worldwide. Previous studies demonstrated that activation of the JAK/STAT3 signaling pathway is frequently observed in H. pylori infected gastric cancer. However, the role of aberrant JAK/STAT signaling in the global epigenetic changes remains unclear. In this regard, we compared the global methylomic changes in AGS gastric cancer cells showing constitutive activation of STA3 vs its STAT3 knock-down subclone by MBDcap-Seq followed by PrEMeR-CG analysis. Together with RNA-Seq, we identified 97 targets showing concomitant hypomethylation and over-expression while 76 targets showing concomitant hypermethylation and down-regulation after STAT3 knock down. Genes showing hypomethylaton/over-expression were subjected to transcription factor binding site analysis by MEME CentriMo. Interestingly, the transcriptional repressors binding site for ETS1 (p = 2.90E-06) and EHF (p = 3.50E-06) were overrepresented in those identifed STAT3 targets suggesting the cooperative binding with STAT3 in the epigenetic silencing of the targets. Further gene ontology analysis by DAVID showed that genes involved in cell cycle and apoptosis were significantly enriched in the hypomethylated/over-expressed targets while genes involved in protein degradation and ubiquitination were found among the hypermethylation/down-regulated targets. To experimentally confirm our result, we analyzed the functional role of one of the hypomethylated targets, miR-193a in gastric cancer. Concomitant with MBDcap-Seq, bisulphite pyrosequencing confirmed that promoter region of miR-193a was hypomethylated in AGS cells depleted with STAT3 but hypermethylated in MKN28 gastric cancer cells overexpressed with constitutive activated STAT3. Cell lines studies also found that promoter region of miR-193a was hypermethylated in gastric cancer cells which did not express miR-193a. Over-expression of miR-193a in AGS cells inhibited cell proliferation (p<0.001) and migration (p <0.01) by colony formation assay and wound-healing assay respectively. Clinically, significantly higher promoter methylation of miR-193a was observed in gastric cancer patient samples (Hong Kong, n = 70; Taiwan, n = 38) as compared to gastritis (n = 9, p<0.05). Interestingly, gastritis with H. pylori infection (p <0.05) had higher methylation of miR-193a than that without H. pylori infection. Patients with higher methylation of miR-193a tended to have shorter overall survival. Importantly, overexpression of miR-193a suppressed the expression of a predicted miR-193a target, YWHAZ (14-3-3ζ). As YWHAZ has been previously found to be a positive regulator in TGF-β-mediated EMT in human cancer, the role of JAK/STAT signaling in promoting TGF-β-mediated EMT program deserves further investigation. Citation Format: Jora M.j. Lin, Jiang-Liang Chou, David E. Frankhouser, Yu-Ming Chuang, Alex Liang-Yu Chang, Li-Han Zeng, Szu-Shan Chen, Ru-Inn Lin, Cheng-Shyong Wu, Kuo-Liang Wei, Enders K.W. Ng, Pearlly S. Yan, Alfred S.L. Cheng, Chin Li, Michael W. Y. Chan. Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer. [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 1501.

Transition of differential histone H3 methylation in photoreceptors and other retinal cells during retinal differentiation.

To analyze cell lineage-specific transitions in global transcriptional and epigenetic changes during retinogenesis, we purified retinal cells from normal mice during postnatal development into two fractions, namely, photoreceptors and other retinal cells, based on Cd73 expression, and performed RNA sequencing and ChIP sequencing of H3K27me3 and H3K4me3. Genes expressed in the photoreceptor lineage were marked with H3K4me3 in the Cd73-positive cell fraction; however, the level of H3K27me3 was very low in both Cd73-positive and -negative populations. H3K27me3 may be involved in spatio-temporal onset of a subset of bipolar-related genes. Subsets of genes expressed in amacrine and retinal ganglion cells, which are early-born retinal cell types, were suggested to be maintained in a silent state by H3K27me3 during late-stage retinogenesis. In the outer nuclear layer, upregulation of Rho and rod-related genes were observed in Ezh2-ablated retina, suggesting a role for H3K27me3 in the maintenance of proper expression levels. Taken together, our data on the transition of lineage-specific molecular signatures during development suggest that histone methylation is involved in retinal differentiation and maintenance through cell lineage-specific mechanisms.

Somatic Genetic and Epigenetic Architecture of Myelodysplastic Syndromes Arising from GATA2 Deficiency

The emergence of GATA2 deficiency as a germline predisposition to myeloid malignancies raises questions about the nature of acquired secondary genetic and epigenetic events facilitating leukemogenesis. Previously, mutations in ASXL1 were implicated as a possible somatic driver in single cases of GATA2-related MDS. However the landscape of secondary changes had not yet been systematically examined in larger MDS cohorts, and accounting for confounding factors. In this study, we used next-generation genomic platforms to investigate targeted mutational landscape and global epigenetic profiles in patients with GATA2 deficiency.In a large cohort of consecutively diagnosed children with MDS we had initially established that GATA2 deficiency accounts for 7% of primary MDS cases. Exploring the known association between GATA2 mutated (GATA2mut) cases and monosomy 7 (-7), the prevalence of GATA2 deficiency was very high in patients with -7 (37%), reaching its peak in adolescence (>70%). We next tested 60 GATA2-deficient patients with MDS for the presence of secondary mutations using targeted NGS for genes involved in myeloid malignancies. Somatic status was confirmed by matched analysis of fibroblasts, hair follicles or T-cells. Single hematopoietic CFU colonies were sequenced to identify subclonal patterns. For comparison, a GATA2 wildtype (GATA2-WT) cohort of 422 children and adolescents with MDS enrolled in the studies of the European Working Group of Childhood MDS were analyzed by targeted NGS. Somatic mutations were detected in 45% (27/60) of GATA2mut as compared to 19% (82/422) GATA2-WT MDS cases (p<0.0001). Recurrently mutated genes in the GATA2mut group included SETBP1, ASXL1, STAG2, RUNX1, CBL, EZH2, NRAS/KRAS, JAK3, and PTPN11. No mutations were found in TP53, BCOR/BCORL and a number of other oncogenes. Because -7 karyotype was significantly overrepresented in GATA2mut cases with somatic mutations (78%), we next focused on this cytogenetic category. Within the -7 subgroup the rate of somatic mutations was the same in GATA2mut (56%) and GATA2-WT (58%) subgroups. However, hotspot SETBP1 mutations were overrepresented in GATA2-deficient patients with -7 (50%) vs. GATA2-WT MDS cohort (22%, p<0.05). Furthermore, STAG2 mutations were found frequently in the GATA2mut group (10%, 6/60) as opposed to only 0.2% (1/422) of the total GATA2-WT cohort (p<0.0001). Next, we aimed to define the clonal hierarchy of concurrent mutations by longitudinal NGS-analysis during disease course in selected patients. Our results indicate that somatic SETBP1 lesions precede the development of ASXL1 mutations. Remarkably, this model of clonal evolution does not depend on preexisting germline GATA2 lesion, as confirmed by sequencing of single CFU colonies cultivated from the bone marrow of 3 GATA2mut and 3 GATA2-WT MDS patients. Finally, to elucidate the epigenetic effects, we compared methylation patterns using methyl-CpG-immunoprecipitation and Illumina-NGS in 25 GATA2mut to 17 GATA2-WT patients and 10 healthy controls. Based on the degree of global methylation, there were no significant alterations allowing for the discrimination of GATA2-deficient patients from the total MDS cohort, when accounted for bias arising from cytogenetic and morphologic subgroups.In summary, somatic SETBP1 and STAG2 mutations are associated with MDS arising from GATA2 deficiency. The remaining targeted clonal landscape is essentially determined by the presence of monosomy 7. Similarly, the global epigenetic changes correlate with morphological and cytogenetic subgroups, rather than with germline GATA2 status. The prospect of potential drug targetability of mutations frequently found in children, particularly in the SETBP1 oncogene, and in histone modifiers ASXL1 and EZH2, warrants further biological studies. DisclosuresNo relevant conflicts of interest to declare.

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

SummaryEpigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increased cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.

Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells

Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics.

Abstract 214: Balancing Innate Immunity Activation and Death Signals for Vascular Regeneration

Background: Our recent discoveries have shown that innate signaling supports effective nuclear reprogramming (Lee &amp; Sayed et al. Cell) and transdifferentiation to ECs (Sayed et al. Circ). By activating innate immunity (via TLR3), viral vectors used for reprogramming cause global epigenetic changes that favor an environment conducive for reprogramming. Indeed, activation of innate transcription factors (TFs) enhances EC regeneration, however, activation of the same pathway could induce: 1) apoptosis via activation of Casp8; and 2) programmed necrosis by receptor-interacting protein kinase 3 (RIP3), when Casp8 is absent. Hypothesis: We speculate that death pathways that are activated by these TFs compromise reprogramming. We previously showed that disruption of Casp8 led to mid-gestational death of mice due to unleashing of RIP3-dependent death pathways, preventing the formation of vascular endothelium and hematopoietic cells. By contrast, when Casp8 -/- mice were crossed with Rip3 -/- , the DKO mice developed normally with no stem cell defect. We hypothesize that these pathways exist as biological constraints on epigenetic plasticity and that pharmacological or genetic ablation could enhance EC reprogramming. Results: To test our hypothesis, we transdifferentiated WT and Casp8 -/- Rip3 -/- DKO MEFs using our protocol, employing an innate immunity modifier and EC growth factors. Intriguingly, our preliminary data showed that DKO MEFs transdifferentiated to induced-EC (iECs) with &gt;10-fold higher yield when compared to WT (yields ~30%). Genetic and functional assays showed that iECs generated from DKO MEFs were comparable to WT, indicating that removal of apoptotic pathways did not lead to aberrant reprogramming. Moreover, pharmacological inhibitors of death receptors when combined with our small molecule cocktail showed a similar increase in iEC generation in human fibroblasts. Conclusion: This study is a first step toward development of a regenerative strategy for PAD on the use of ECs derived from small molecules and growth factors without use of viral vectors encoding TFs. We intend to derive an effective and feasible technology for therapeutic transdifferentiation for ischemic syndromes to promote healing with tissue rather than a scar.

Transcriptome Dynamics of the Stomatal Lineage: Birth, Amplification, and Termination of a Self-Renewing Population

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes.

Mouse BRWD1 is critical for spermatid postmeiotic transcription and female meiotic chromosome stability.

Postmeiotic gene expression is essential for development and maturation of sperm and eggs. We report that the dual bromodomain-containing protein BRWD1, which is essential for both male and female fertility, promotes haploid spermatid-specific transcription but has distinct roles in oocyte meiotic progression. Brwd1 deficiency caused down-regulation of ∼300 mostly spermatid-specific transcripts in testis, including nearly complete elimination of those encoding the protamines and transition proteins, but was not associated with global epigenetic changes in chromatin, which suggests that BRWD1 acts selectively. In females, Brwd1 ablation caused severe chromosome condensation and structural defects associated with abnormal telomere structure but only minor changes in gene expression at the germinal vesicle stage, including more than twofold overexpression of the histone methyltransferase MLL5 and LINE-1 elements transposons. Thus, loss of BRWD1 function interferes with the completion of oogenesis and spermatogenesis through sexually dimorphic mechanisms: it is essential in females for epigenetic control of meiotic chromosome stability and in males for haploid gene transcription during postmeiotic sperm differentiation.

Primordial germ cell specification: a context-dependent cellular differentiation event [corrected

During embryonic development, the foundation of the germline is laid by the specification of primordial germ cells (PGCs) from the postimplantation epiblast via bone morphogenetic protein (BMP) and WNT signalling. While the majority of epiblast cells undergo differentiation towards somatic cell lineages, PGCs initiate a unique cellular programme driven by the cooperation of the transcription factors BLIMP1, PRDM14 and AP2γ. These factors synergistically suppress the ongoing somatic differentiation and drive the re-expression of pluripotency and germ cell-specific genes accompanied by global epigenetic changes. However, an unresolved question is how postimplantation epiblast cells acquire the developmental competence for the PGC fate downstream of BMP/WNT signalling. One emerging concept is that transcriptional enhancers might play a central role in the establishment of developmental competence and the execution of cell fate determination. Here, we discuss recent advances on the specification and reprogramming of PGCs thereby highlighting the concept of enhancer function.

Functional normalization of 450k methylation array data improves replication in large cancer studies.

We propose an extension to quantile normalization that removes unwanted technical variation using control probes. We adapt our algorithm, functional normalization, to the Illumina 450k methylation array and address the open problem of normalizing methylation data with global epigenetic changes, such as human cancers. Using data sets from The Cancer Genome Atlas and a large case–control study, we show that our algorithm outperforms all existing normalization methods with respect to replication of results between experiments, and yields robust results even in the presence of batch effects. Functional normalization can be applied to any microarray platform, provided suitable control probes are available.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0503-2) contains supplementary material, which is available to authorized users.

A proteomic glimpse into the initial global epigenetic changes during HIV infection.

HIV-1 replication requires the insertion of viral DNA into the host genome, which is catalyzed by HIV-1 integrase. This integration event can lead to vast changes in the chromatin landscape and gene transcription. In this study, we sought to correlate the extensive changes of histone PTM abundances with the equally dynamic shifts in host transcriptional activity. To fully capture the changes that were occurring during the course of HIV-infection, we performed time-courses in which we extracted both histones and mRNA from HIV-infected, UV-inactivated HIV-infected and mock-infected SUP-T1 cells. We then analyzed the alterations to histone PTM profiles using nano-LC-MS/MS, as well as the expression of chromatin-associated enzymes, such as histone deacetylases, acetyltransferases, demethylases, methyltransferases, and histone chaperone proteins. As expected, we observed major changes in histone PTM abundances, which we linked to massive fluctuations in mRNA expression of associated chromatin enzymes. However, we find few differences between HIV and HIVUV (UV-inactivated) infection, which suggests that initial histone PTM changes during HIV infection are from the host in response to the infection, and not due to the HIV virus manipulating the transcriptional machinery. We believe that these preliminary experiments can provide a basis for future forays into targeted manipulations of histone PTM-regulated aspects of HIV progression through its replication cycle.

Influence of relative NK-DC abundance on placentation and its relation to epigenetic programming in the offspring.

Normal placentation relies on an efficient maternal adaptation to pregnancy. Within the decidua, natural killer (NK) cells and dendritic cells (DC) have a critical role in modulating angiogenesis and decidualization associated with pregnancy. However, the contribution of these immune cells to the placentation process and subsequently fetal development remains largely elusive. Using two different mouse models, we here show that optimal placentation and fetal development is sensitive to disturbances in NK cell relative abundance at the fetal–maternal interface. Depletion of NK cells during early gestation compromises the placentation process by causing alteration in placental function and structure. Embryos derived from NK-depleted dams suffer from intrauterine growth restriction (IUGR), a phenomenon that continued to be evident in the offspring on post-natal day 4. Further, we demonstrate that IUGR was accompanied by an overall reduction of global DNA methylation levels and epigenetic changes in the methylation of specific hepatic gene promoters. Thus, temporary changes within the NK cell pool during early gestation influence placental development and function, subsequently affecting hepatic gene methylation and fetal metabolism.

The potential of epigenetic therapies in neurodegenerative diseases.

Available treatments for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, do not arrest disease progression but mainly help keeping patients from getting worse for a limited period of time. Increasing evidence suggests that epigenetic mechanisms such as DNA methylation and histone tail modifications are dynamically regulated in neurons and play a fundamental role in learning and memory processes. In addition, both global and gene-specific epigenetic changes and deregulated expression of the writer and eraser proteins of epigenetic marks are believed to contribute to the onset and progression of neurodegeneration. Studies in animal models of neurodegenerative diseases have highlighted the potential role of epigenetic drugs, including inhibitors of histone deacetylases and methyl donor compounds, in ameliorating the cognitive symptoms and preventing or delaying the motor symptoms of the disease, thereby opening the way for a potential application in human pathology.

Reprogramming Factor Expression Leads to Tumor Development

Abstract Transient reprogramming factor expression leads to global epigenetic changes and tumor formation.

Distinct DNA methylation patterns of cognitive impairment and trisomy 21 in down syndrome

BackgroundThe presence of an extra whole or part of chromosome 21 in people with Down syndrome (DS) is associated with multiple neurological changes, including pathological aging that often meets the criteria for Alzheimer’s Disease (AD). In addition, trisomies have been shown to disrupt normal epigenetic marks across the genome, perhaps in response to changes in gene dosage. We hypothesized that trisomy 21 would result in global epigenetic changes across all participants, and that DS patients with cognitive impairment would show an additional epigenetic signature.MethodsWe therefore examined whole-genome DNA methylation in buccal epithelial cells of 10 adults with DS and 10 controls to determine whether patterns of DNA methylation were correlated with DS and/or cognitive impairment. In addition we examined DNA methylation at the APP gene itself, to see whether there were changes in DNA methylation in this population. Using the Illumina Infinium 450 K Human Methylation Array, we examined more than 485,000 CpG sites distributed across the genome in buccal epithelial cells.ResultsWe found 3300 CpGs to be differentially methylated between the groups, including 495 CpGs that overlap with clusters of differentially methylated probes. In addition, we found 5 probes that were correlated with cognitive function including two probes in the TSC2 gene that has previously been associated with Alzheimer’s disease pathology. We found no enrichment on chromosome 21 in either case, and targeted analysis of the APP gene revealed weak evidence for epigenetic impacts related to the AD phenotype.ConclusionsOverall, our results indicated that both Trisomy 21 and cognitive impairment were associated with distinct patterns of DNA methylation.