Peroxisome Proliferator-Activated Receptor alpha (PPAR alpha) down-regulation in cystic fibrosis lymphocytes

Background: PPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response. Methods: PPARα, β and γ mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARα protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARα was analyzed by gel shift assay. Results: In lymphocytes, the expression of PPARα mRNA, but not of PPARβ, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARα was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARα and PPARβ mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARγ mRNA levels were below the detection limit. Conclusion: Lymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARα may therefore contribute to the inflammatory processes that are observed in CF

Comparative analysis of sequence characteristics of imprinted genes in human, mouse, and cattle

Genomic imprinting is an epigenetic mechanism that results in monoallelic expression of genes depending on parent-of-origin of the allele. Although the conservation of genomic imprinting among mammalian species has been widely reported for many genes, there is accumulating evidence that some genes escape this conservation. Most known imprinted genes have been identified in the mouse and human, with few imprinted genes reported in cattle. Comparative analysis of genomic imprinting across mammalian species would provide a powerful tool for elucidating the mechanisms regulating the unique expression of imprinted genes. In this study we analyzed the imprinting of 22 genes in human, mouse, and cattle and found that in only 11 was imprinting conserved across the three species. In addition, we analyzed the occurrence of the sequence elements CpG islands, C + G content, tandem repeats, and retrotransposable elements in imprinted and in nonimprinted (control) cattle genes. We found that imprinted genes have a higher G + C content and more CpG islands and tandem repeats. Short interspersed nuclear elements (SINEs) were notably fewer in number in imprinted cattle genes compared to control genes, which is in agreement with previous reports for human and mouse imprinted regions. Long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) were found to be significantly underrepresented in imprinted genes compared to control genes, contrary to reports on human and mouse. Of considerable significance was the finding of highly conserved tandem repeats in nine of the genes imprinted in all three species

Short Interspersed Element (SINE) Depletion and Long Interspersed Element (LINE) Abundance Are Not Features Universally Required for Imprinting

Genomic imprinting is a form of gene dosage regulation in which a gene is expressed from only one of the alleles, in a manner dependent on the parent of origin. The mechanisms governing imprinted gene expression have been investigated in detail and have greatly contributed to our understanding of genome regulation in general. Both DNA sequence features, such as CpG islands, and epigenetic features, such as DNA methylation and non-coding RNAs, play important roles in achieving imprinted expression. However, the relative importance of these factors varies depending on the locus in question. Defining the minimal features that are absolutely required for imprinting would help us to understand how imprinting has evolved mechanistically. Imprinted retrogenes are a subset of imprinted loci that are relatively simple in their genomic organisation, being distinct from large imprinting clusters, and have the potential to be used as tools to address this question. Here, we compare the repeat element content of imprinted retrogene loci with non-imprinted controls that have a similar locus organisation. We observe no significant differences that are conserved between mouse and human, suggesting that the paucity of SINEs and relative abundance of LINEs at imprinted loci reported by others is not a sequence feature universally required for imprinting

DNA Methylation Dynamics in Human Induced Pluripotent Stem Cells over Time

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the “convergence” of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs

The Drosophila homolog of the mammalian imprint regulator, CTCF, maintains the maternal genomic imprint in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>CTCF is a versatile zinc finger DNA-binding protein that functions as a highly conserved epigenetic transcriptional regulator. CTCF is known to act as a chromosomal insulator, bind promoter regions, and facilitate long-range chromatin interactions. In mammals, CTCF is active in the regulatory regions of some genes that exhibit genomic imprinting, acting as insulator on only one parental allele to facilitate parent-specific expression. In <it>Drosophila</it>, CTCF acts as a chromatin insulator and is thought to be actively involved in the global organization of the genome.</p> <p>Results</p> <p>To determine whether CTCF regulates imprinting in <it>Drosophila</it>, we generated <it>CTCF </it>mutant alleles and assayed gene expression from the imprinted <it>Dp(1;f)LJ9 </it>mini-X chromosome in the presence of reduced <it>CTCF </it>expression. We observed disruption of the maternal imprint when <it>CTCF </it>levels were reduced, but no effect was observed on the paternal imprint. The effect was restricted to maintenance of the imprint and was specific for the <it>Dp(1;f)LJ9 </it>mini-X chromosome.</p> <p>Conclusions</p> <p>CTCF in <it>Drosophila </it>functions in maintaining parent-specific expression from an imprinted domain as it does in mammals. We propose that <it>Drosophila </it>CTCF maintains an insulator boundary on the maternal X chromosome, shielding genes from the imprint-induced silencing that occurs on the paternally inherited X chromosome.</p> <p>See commentary: <url>http://www.biomedcentral.com/1741-7007/8/104</url></p

Repressor element-1 silencing transcription factor (REST)-dependent epigenetic remodeling is critical to ischemia-induced neuronal death.

Dysregulation of the transcriptional repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor is important in a broad range of diseases, including cancer, diabetes, and heart disease. The role of REST-dependent epigenetic modifications in neurodegeneration is less clear. Here, we show that neuronal insults trigger activation of REST and CoREST in a clinically relevant model of ischemic stroke and that REST binds a subset of "transcriptionally responsive" genes (gria2, grin1, chrnb2, nefh, nf\u3bab2, trpv1, chrm4, and syt6), of which the AMPA receptor subunit GluA2 is a top hit. Genes with enriched REST exhibited decreased mRNA and protein. We further show that REST assembles with CoREST, mSin3A, histone deacetylases 1 and 2, histone methyl-transferase G9a, and methyl CpG binding protein 2 at the promoters of target genes, where it orchestrates epigenetic remodeling and gene silencing. RNAi-mediated depletion of REST or administration of dominant-negative REST delivered directly into the hippocampus in vivo prevents epigenetic modifications, restores gene expression, and rescues hippocampal neurons. These findings document a causal role for REST-dependent epigenetic remodeling in the neurodegeneration associated with ischemic stroke and identify unique therapeutic targets for the amelioration of hippocampal injury and cognitive deficits

Two Different EVI1 Expressing Poor-Risk AML Subgroups with Distinct Epigenetic Signatures Uncovered by Genome Wide DNA Methylation Profiling

Abstract Although the clinical outcome for patients with acute myeloid leukemia (AML) has improved over the years, failure to maintain complete remission remains a major problem with current standard treatments. The development of individually tailored and patient-specific therapy could potentially significantly improve therapeutic efficacy. In particular we are interested in better understanding the biological features associated with aberrant expression of the EVI1 oncogene, which we previously showed is associated with a poor prognosis. Two different EVI1 transcripts have been identified, i.e. a short form (E) and a long form called MDS1-EVI1 (ME) encoding respectively, a 140 kDa and 170 kDa protein. In EVI1 positive AMLs a distinction can be made between patients that express both EVI1 transcripts (E+/ME+) and cases that express the short form solely (E+), since the latter group is exclusively associated with 3q26 chromosomal abnormalities. EVI1 is a nuclear zinc-finger transcriptional repressor oncoprotein that is known to interact with several epigenetic regulators, e.g. HDACs, CtBPs, histone methyl transferases and MBD3. Since EVI1 presumably mediates its effects through aberrant transcriptional repression, we hypothesize that its aberrant expression results in aberrant epigenetic programming of leukemia cells, which might provide an opportunity for epigenetic-targeted therapy in these patients. In order to determine whether EVI1 over-expressing (EVI1+) AMLs display aberrant epigenetic programming we performed HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) DNA methylation assays in 26 EVI1+ AMLs and 8 CD34+ normal bone marrow controls (NBM). Our HELP assay measured the abundance of DNA methylation at ~50,000 CpG sites covering ~13,000 promoter regions. Single locus validation assays using Sequenom Epityping showed that HELP was >95% accurate in quantifying CpG methylation. We found that unsupervised analysis using hierarchical clustering (Pearson correlation distance with Ward’s clustering method) readily separated the EVI1+ AMLs from NBMs. Supervised analysis comparing EVI1+ to NBM identified 303 promoter sequences as being differently methylated (P1.5). Remarkably, 80% of these genes were hypermethylated in EVI1+ patients, while only 20% of genes were hypomethylated. The hypermethylated profile included genes associated with cell death (Caspase-2, MAD1L1) and cell cycle (TNF, JARID1B). The 26 EVI1+ leukemias further segregated into two distinct subgroups in unsupervised analysis: one cluster (n=14) was highly enriched for E+ AML cases carrying 3q26 abnormalities (n=7) while the other one (n=12) mainly harbored the E+/ME+ AMLs (n=10). Supervised analysis of these two EVI1+ clusters revealed that the 3q26-enriched group featured 122-gene signature (P1.5) consisting entirely of hypermethylated genes. When each of the individual EVI1 clusters was independently compared to the NBM samples using supervised analysis we found that the 3q26-enriched group contained a significantly more methylated gene signature containing 429 hypermethylated and 47 hypomethylated HpaII fragments (P1.5). Pathway analysis of the promoter regions differentially methylated in the 3q26-enriched AML group included genes involved in protein degradation and cellular response to therapeutics. In contrast, the E+/ME+ enriched group showed a more balanced distribution of differential methylation when compared to the NBMs (226 hypermethylated and 158 hypomethylated genes). Taken together, our data show that EVI1 overexpression is associated with specific alterations in epigenetic programming vs. normal CD34+ cells. Even more remarkably, we showed that EVI1+ AMLs form two epigenetically distinct AML subtypes. Specifically, the 3q26 subgroup, short EVI1+ isoform AMLs display marked hypermethylation vs. the MDS1-EVI1 expressing patients, involving aberrant methylation of different pathways. This shows that the two forms of EVI1+ AMLs become aberrantly programmed in different ways and are biologically distinct entities, and further suggest distinct mechanisms of action for the different EVI1 isoforms. The marked hypermethylation profile of the short EVI1 isoform AMLs suggests that these patients might benefit from treatment with DNA methyltransferase inhibitors