Stefanie Weber

A Distinct DNA-Methylation Boundary in the 5′- Upstream Sequence of the FMR1 Promoter Binds Nuclear Proteins and Is Lost in Fragile X Syndrome

We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.

Resolution of inflammation by interleukin-9-producing type 2 innate lymphoid cells

Inflammatory diseases such as arthritis are chronic conditions that fail to resolve spontaneously. While the cytokine and cellular pathways triggering arthritis are well defined, those responsible for the resolution of inflammation are incompletely characterized. Here we identified interleukin (IL)-9-producing type 2 innate lymphoid cells (ILC2s) as the mediators of a molecular and cellular pathway that orchestrates the resolution of chronic inflammation. In mice, the absence of IL-9 impaired ILC2 proliferation and activation of regulatory T (Treg) cells, and resulted in chronic arthritis with excessive cartilage destruction and bone loss. In contrast, treatment with IL-9 promoted ILC2-dependent Treg activation and effectively induced resolution of inflammation and protection of bone. Patients with rheumatoid arthritis in remission exhibited high numbers of IL-9+ ILC2s in joints and the circulation. Hence, fostering IL-9-mediated ILC2 activation may offer a novel therapeutic approach inducing resolution of inflammation rather than suppression of inflammatory responses.

Type 2 innate lymphoid cell counts are increased in patients with systemic sclerosis and correlate with the extent of fibrosis

Objective Type 2 innate lymphoid cells (ILC2s), a recently identified population of lymphoid cells lacking lineage-specific receptors, promote type 2 immunity and tissue remodelling. However, the contributive role of ILC2s in the pathogenesis of systemic sclerosis (SSc) is unknown. We aimed to evaluate the levels and correlations with fibrotic manifestations in SSc. Methods 69 patients with SSc and 47 healthy controls were included. Blood samples and skin sections were analysed by flow cytometry and immunohistochemically by staining two complementary panels of markers. Results Dermal and circulating ILC2s were significantly elevated in patients with SSc compared with controls. Dermal, but not circulating ILC2s were activated. Stratification of the SSc population in patients with limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) demonstrated increased levels of ILC2s in both subgroups with significantly higher frequencies in dcSSc compared with lcSSc. Moreover, dermal and circulating ILC2 counts correlated closely with the modified Rodnan skin score and with the presence of pulmonary fibrosis. Conclusions ILC2 counts are elevated in patients with SSc and correlate with the extent of skin fibrosis and the presence of interstitial lung disease providing compelling evidence for profibrotic effect of ILC2s in SSc.

Destabilization of the human epigenome: consequences of foreign DNA insertions

AIM We previously reported changes of DNA methylation and transcription patterns in mammalian cells that carry integrated foreign DNA. Experiments were now designed to assess the epigenetic consequences of inserting a 5.6 kbp plasmid into the human genome. METHODS Differential transcription and CpG methylation patterns were compared between transgenomic and nontransgenomic cell clones by using gene chip microarray systems. RESULTS In 4.7% of the 28.869 gene segments analyzed, transcriptional activities were up- or downregulated in the transgenomic cell clones. Genome-wide profiling revealed differential methylation in 3791 of > 480,000 CpGs examined in transgenomic versus nontransgenomic clones. CONCLUSION The data document genome-wide effects of foreign DNA insertions on the epigenetic stability of human cells. Many fields in experimental biology and medicine employ transgenomic or otherwise genome-manipulated cells or organisms without considering the epigenetic consequences for the recipient genomes.

Epigenetic analysis of HIV-1 proviral genomes from infected individuals: Predominance of unmethylated CpG's

Efforts to cure HIV-1 infections aim at eliminating proviral DNA. Integrated DNA from various viruses often becomes methylated de novo and transcriptionally inactivated. We therefore investigated CpG methylation profiles of 55 of 94 CpGs (58.5%) in HIV-1 proviral genomes including ten CpGs in each LTR and additional CpGs in portions of gag, env, nef, rev, and tat genes. We analyzed 33 DNA samples from PBMCs of 23 subjects representing a broad spectrum of HIV-1 disease. In 22 of 23 HIV-1-infected individuals, there were only unmethylated CpGs regardless of infection status. In one long term nonprogressor, however, methylation of proviral DNA varied between 0 and 75% over an 11-year period although the CD4+ counts remained stable. Hence levels of proviral DNA methylation can fluctuate. The preponderance of unmethylated CpGs suggests that proviral methylation is not a major factor in regulating HIV-1 proviral activity in PBMCs. Unmethylated CpGs may play a role in HIV-1 immunopathogenesis.

Cutting Edge: Homeostasis of Innate Lymphoid Cells Is Imbalanced in Psoriatic Arthritis

Innate lymphoid cells (ILC) have a high potency for cytokine production independent of specific Ag stimulation. Imbalance of ILC subsets may influence cytokine production in humans and hence be associated with the development of inflammatory disease. Evidence for an imbalance of ILC homeostasis in human disease, however, is very limited to date. In this study we show that psoriatic arthritis (PsA), a severe disease of the joints depending on the activation of the IL-23/IL-17 pathway, is characterized by a skewed ILC homeostasis. Circulating ILC3s as potent source of IL-17/IL-22 were elevated in active PsA, whereas ILC2s, which produce proresolving cytokines, were decreased. The ILC2/ILC3 ratio was significantly correlated with clinical disease activity scores and the presence of imaging signs of joint inflammation and bone damage. Multivariable analysis showed that a high ILC2/ILC3 ratio is associated with remission in PsA, suggesting that specific alterations of ILC homeostasis control disease activity in PsA.

Epigenetic Alterations upon the Insertion of Foreign DNA into Mammalian Genomes: Oncogenesis and Evolution

The fate of foreign DNA in mammalian systems has been a long-term interest in our laboratory [Doerfler (Foreign DNA in mammalian systems. Wiley-VCH, 2000)]. The current overview summarizes an update of the data presented at the Fifth Weissenburg Symposium 2014 (Weissenburg Symposia, 2001–2014). In earlier studies on integrated adenovirus type 12 (Ad12) DNA in Ad12-transformed hamster cells, we discovered that the CpG methylation profiles in some of their ubiquitous endogenous retrotransposon sequences and in several cellular genes were markedly increased. This hypermethylation persisted in revertants of the transformed cells which had lost all Ad12 genomes. Alterations of cellular methylation and transcription profiles were also observed in hamster cells transgenomic for bacteriophage lambda DNA. We have now investigated human HCT116 cells which were transgenomic for a 5.6 kbp bacterial plasmid. In five non-transgenomic HCT116 control clones, transcription and methylation patterns proved similar, if not identical. This finding opened the possibility to compare these patterns between non-transgenomic and transgenomic cell clones. In 4.7 % of the 28,869 gene segments analyzed, the transcriptional activities were upregulated (907 genes) or downregulated (436 genes) in plasmid-transgenomic cell clones in comparison to control clones. Genome-wide methylation profiling was performed for >480,000 CpG sites. In comparisons to methylation levels in five transgenomic versus four non-transgenomic cell clones, 3791 CpGs were differentially methylated, 1504 CpGs were hypermethylated, and 2287 were hypomethylated. The mechanisms underlying the observed epigenetic alterations are unknown. Extent and location of alterations in genome activities and CpG methylation might depend on the site(s) of foreign DNA insertion. Genome manipulations have been an everyday practice in many laboratories. With further refinement of epigenetic technologies, hitherto unsuspected complications in the evaluation of experiments with genome manipulated cells and organisms will become apparent.

DNA methylation and transcription in HERV (K, W, E) and LINE sequences remain unchanged upon foreign DNA insertions

AIM DNA methylation and transcriptional profiles were determined in the regulatory sequences of the human endogenous retroviral (HERV-K, -W, -E) and LINE-1.2 elements and were compared between non-transgenomic and plasmid-transgenomic cells. METHODS DNA methylation profiles in the HERV (K, W, E) and LINE sequences were determined by bisulfite genomic sequencing. The transcription of these genome segments was assessed by quantitative real-time PCR. RESULTS In HERV-K, HERV-W and LINE-1.2 the levels of DNA methylation ranged between 75 and 98%, while in HERV-E they were around 60%. Nevertheless, the HERV and LINE-1.2 sequences were actively transcribed. No differences were found in comparisons of HERV and LINE-1.2 CpG methylation and transcription patterns between non-transgenomic and plasmid-transgenomic HCT116 cells. CONCLUSION The insertion of a 5.6 kbp plasmid into the HCT116 genome had no effect on the HERV and LINE-1.2 methylation and transcription profiles, although other parts of the HCT116 genome had shown marked changes. These repetitive sequences are transcribed, probably because the large number of HERV and LINE-1.2 elements harbor copies with non- or hypo-methylated long terminal repeat sequences.

Epigenetic modifications of HIV proviral LTRs: potential targets for cure

HIV-1 cure remains elusive despite HAART due to the reservoirs of proviral DNA integrated into the human genome. Efforts to cure HIV-1 therefore need to aim at eliminating proviral DNA from cellular reservoirs. The first epigenetic signal identified in virus infected and uninfected cells has been promoter methylation. Compelling evidence confirms that specific promoter methylation can lead to gene silencing. Previous studies have examined HIV-1-epigenetics mostly in vitro.

DNA Methylation Profiles in the 5′-Upstream Region of the Human FMR1 Promoter and in an Adenovirus Transgenome

Publisher Summary This chapter explains that the foreign (Ad12 viral) DNA is integrated into the hamster genome of the Ad12-transformed cell line T637 and of its revertant TR12 in an orientation co-linear with the virion genome. There is only one or a few nucleotides altered at the termini of the integrated Ad12 genome. The insert in cell line TR12 consists of one complete Ad12 genome and an additional 3.9kb fragment of Ad12 DNA, which has been derived from the right viral end and has been flip-flopped. Foreign DNA into established eukaryotic genomes has become an important technique in experimental biology and medicine with the aim to generate transgenic cells or organisms. In spite of the generality of the application of these procedures, little attention has been focused on their consequences for the recipient cell or organism. It is often implicitly assumed that foreign DNA insertion into a genome might not have consequences other than the ones aspired to by the experimenter.