Ofra Sabag

Establishment of methylation patterns in ES cells

After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at implantation. We have identified a demethylation pathway in mouse embryonic cells that uses hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this demethylation system is not necessary for generating the overall bimodal methylation pattern but does appear to be involved in resetting methylation patterns during somatic-cell reprogramming.

Gender-specific postnatal demethylation and establishment of epigenetic memory

DNA methylation patterns are set up in a relatively fixed programmed manner during normal embryonic development and are then stably maintained. Using genome-wide analysis, we discovered a postnatal pathway involving gender-specific demethylation that occurs exclusively in the male liver. This demodification is programmed to take place at tissue-specific enhancer sequences, and our data show that the methylation state at these loci is associated with and appears to play a role in the transcriptional regulation of nearby genes. This process is mediated by the secretion of testosterone at the time of sexual maturity, but the resulting methylation profile is stable and therefore can serve as an epigenetic memory even in the absence of this inducer. These findings add a new dimension to our understanding of the role of DNA methylation in vivo and provide the foundations for deciphering how environment can impact on the epigenetic regulation of genes in general.

Therapeutic potency of IL2–caspase 3 targeted treatment in a murine experimental model of inflammatory bowel disease

Background: Inflammatory bowel disease (IBD) comprises primarily the two disorders – ulcerative colitis and Crohn’s disease – that involve deregulated T cell responses. The ever-increasing incidence rate of Crohn’s disease and ulcerative colitis during recent decades, combined with the limited efficacy and potential adverse effects of current treatments, explain the real need for seeking more specific and selective methods for treating these diseases. Aim: To investigate the ability of interleukin 2 (IL2)–caspase 3 chimeric protein, designed to target activated T lymphocytes that express the high-affinity IL2 receptor, to ameliorate the clinical symptoms of acute murine experimental colitis, using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Methods: Mice with DSS-induced colitis were treated with IL2–caspase 3 for 7 days and disease severity was assessed in parallel to control, non-treated mice, receiving only daily injections of phosphate-buffered saline. IL2–caspase 3 was tested both for its ability to prevent the development of colitis, and for its therapeutic potential to cure on-going, active acute disease. In addition, colon tissue samples were used for myeloperoxidase assays and RNA isolation followed by polymerase chain reaction to determine mRNA expression levels of specific genes. Results: Treatment with IL2–caspase 3 dose-dependently ameliorated the disease activity index (DAI) of mice colitis. We achieved up to 78% improvement in DAI with intravenous injections of 15 μg/mouse/day. Furthermore, IL2–caspase 3 decreased neutrophil and macrophage infiltration to the inflamed tissue by up to 57%. IL2–caspase 3 was proven as a therapeutic reagent in another model, where treatment begins only after disease onset. Here we demonstrated a 70% decrease in DAI when compared to non-treated sick mice. A reduction in mRNA expression levels of both IL1β and tumour necrosis factor α was found in lysates of total colon tissue of treated mice, as compared to sick, untreated mice. We also found that expression levels of Bcl2 were significantly decreased after treatment, while Bax was upregulated in comparison to non-treated mice. Moreover, the Bcl2/Bax ratio, which is elevated in both experimental colitis and in human Crohn’s disease, decreased dramatically after treatment. Conclusions: IL2–caspase 3 chimeric protein may provide a novel approach to the therapy of human IBD, and a possible suggested treatment for other pathological conditions that involve uncontrolled expansion of activated T cells.

Soluble CD40 ligand (sCD40L) provides a new delivery system for targeted treatment: sCD40L-caspase 3 chimeric protein for treating B-cell malignancies.

A wide range of hematologic malignancies arises from numerous cell types. In an attempt to offer a new target for treating B‐cell malignancies, in this study, the authors tested the possibility of using the CD40/CD40L system as a common targeting system for the various malignancies in this group.

Postnatal DNA demethylation and its role in tissue maturation

Development in mammals is accompanied by specific de novo and demethylation events that are thought to stabilize differentiated cell phenotypes. We demonstrate that a large percentage of the tissue-specific methylation pattern is generated postnatally. Demethylation in the liver is observed in thousands of enhancer-like sequences associated with genes that undergo activation during the first few weeks of life. Using a conditional gene ablation strategy we show that the removal of these methyl groups is stable and necessary for assuring proper hepatocyte gene expression and function through its effect on chromatin accessibility. These postnatal changes in methylation come about through exposure to hormone signaling. These results define the molecular rules of 5-methyl-cytosine regulation as an epigenetic mechanism underlying cellular responses to a changing environment.Here the authors show that a large fraction of the tissue-specific methylation pattern is generated postnatally. These changes, which occur in response to hormone signaling, appear to play a major role in the regulation of gene expression and tissue maturation in the liver.

Combining flagellin and human β-defensin-3 to combat bacterial infections.

The discovery and therapeutic use of antibiotics made a major contribution to the reduction of human morbidity and mortality. However, the growing resistance to antibiotics has become a matter of huge concern. In this study we aimed to develop an innovative approach to treat bacterial infections utilizing two components: the human antibacterial peptide β-defensin-3 (BD3) and the bacterial protein flagellin (F). This combination was designed to provide an efficient weapon against bacterial infections with the peptide killing the bacteria directly, while the flagellin protein triggers the immune system and acts against bacteria escaping from the peptide’s action. We designed, expressed and purified the fusion protein flagellin BD3 (FBD3) and its two components, the F protein and the native BD3 peptide. FBD3 fusion protein and native BD3 peptide had antibacterial activity in vitro against various bacterial strains. FBD3 and F proteins could also recognize their receptor expressed on target cells and stimulated secretion of IL-8. In addition, F and FBD3 proteins had a partial protective effect in mice infected by pathogenic Escherichia coli bacteria that cause a lethal disease. Moreover, we were able to show partial protection of mice infected with E. coli using a flagellin sequence from Salmonella. We also explored flagellin’s basic mechanisms of action, focusing on its effects on CD4+ T cells from healthy donors. We found that F stimulation caused an increase in the mRNA levels of the Th1 response cytokines IL12A and IFNγ. In addition, F stimulation affected its own receptor.

Role of transcription complexes in the formation of the basal methylation pattern in early development

Significance This paper reveals the molecular logic for generating the basal methylation pattern in each individual following erasure of the gametic profile in the preimplantation embryo. The results show that transcription factors and the RNA polymerase complex play a major role in protecting recognized regions from de novo methylation by recruiting the H3K4 methylation machinery. Because methylation is stably maintained through development, this mechanism serves to perpetuate the activity state present in the early embryo. This model may also help explain how transient factors from the gametes may influence methylation patterns in the offspring and, thereby, contribute to intergenerational epigenetic inheritance. Following erasure in the blastocyst, the entire genome undergoes de novo methylation at the time of implantation, with CpG islands being protected from this process. This bimodal pattern is then preserved throughout development and the lifetime of the organism. Using mouse embryonic stem cells as a model system, we demonstrate that the binding of an RNA polymerase complex on DNA before de novo methylation is predictive of it being protected from this modification, and tethering experiments demonstrate that the presence of this complex is, in fact, sufficient to prevent methylation at these sites. This protection is most likely mediated by the recruitment of enzyme complexes that methylate histone H3K4 over a local region and, in this way, prevent access to the de novo methylation complex. The topological pattern of H3K4me3 that is formed while the DNA is as yet unmethylated provides a strikingly accurate template for modeling the genome-wide basal methylation pattern of the organism. These results have far-reaching consequences for understanding the relationship between RNA transcription and DNA methylation.

Eliminating the six N‐terminal amino acids of the caspase 3 large subunit improved production of a biologically active IL2‐Caspase3 chimeric protein

Designing a chimeric protein and developing a procedure for its stable production as a biologically active protein, are key steps in its potential application to clinical trails. IL2‐Caspase3 chimeric protein designed to target activated T lymphocytes was found to be a promising molecule for targeted treatment, however was found to be difficult to produce as a biological active molecule. Thus, we designed a new version of the molecule, IL2‐Caspase3s, in which six amino acids (aa 29–34) from the N‐terminus of the large subunit of caspase 3 were excluded. Repeated expressions, productions, and partial purifications of the IL2‐Caspase3s yielded reproducible batches with consistent results. We found that IL2‐Caspase3s causes cell death in a specific, dose‐, and time‐dependent manner. Cell death due to IL2‐Caspase3s is caused by apoptosis. This improved and biologically stable IL2‐Caspase3s chimeric protein may be developed in the future for clinical trails as a promising therapy for several pathologies involving activated T‐cells. Moreover, this truncated caspase 3 sequence, lacking the N‐terminal six amino acids of its large subunit, may be used in other caspase 3‐based chimeric proteins targeted against various human diseases, using the appropriate targeting moiety.