Hepatocyte regeneration is driven by embryo-like DNA methylation reprogramming.

As a result of partial hepatectomy, the remaining liver tissue undergoes a process of renewed proliferation that leads to rapid regeneration of the liver. By following the early stages of this process, we observed dramatic programmed changes in the DNA methylation profile, characterized by both de novo and demethylation events, with a subsequent return to the original adult pattern as the liver matures. Strikingly, these transient alterations partially mimic the DNA methylation state of embryonic hepatoblasts (E16.5), indicating that hepatocytes actually undergo epigenetic dedifferentiation. Furthermore, Tet2/Tet3-deletion experiments demonstrated that these changes in methylation are necessary for carrying out basic embryonic functions, such as proliferation, a key step in liver regeneration. This implies that unlike tissue-specific regulatory regions that remain demethylated in the adult, early embryonic genes are programmed to first undergo demethylation, followed by remethylation as development proceeds. The identification of this built-in system may open targeting opportunities for regenerative medicine.

Complex haploinsufficiency in pluripotent cells yields somatic cells with DNA methylation abnormalities and pluripotency induction defects.

A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog+/-;Sall4+/-, Nanog+/-;Utf1+/-, Nanog+/-;Esrrb+/- and Sox2+/-;Sall4+/-). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.

Pluripotency-independent induction of human trophoblast stem cells from fibroblasts.

Human trophoblast stem cells (hTSCs) can be derived from embryonic stem cells (hESCs) or be induced from somatic cells by OCT4, SOX2, KLF4 and MYC (OSKM). Here we explore whether the hTSC state can be induced independently of pluripotency, and what are the mechanisms underlying its acquisition. We identify GATA3, OCT4, KLF4 and MYC (GOKM) as a combination of factors that can generate functional hiTSCs from fibroblasts. Transcriptomic analysis of stable GOKM- and OSKM-hiTSCs reveals 94 hTSC-specific genes that are aberrant specifically in OSKM-derived hiTSCs. Through time-course-RNA-seq analysis, H3K4me2 deposition and chromatin accessibility, we demonstrate that GOKM exert greater chromatin opening activity than OSKM. While GOKM primarily target hTSC-specific loci, OSKM mainly induce the hTSC state via targeting hESC and hTSC shared loci. Finally, we show that GOKM efficiently generate hiTSCs from fibroblasts that harbor knockout for pluripotency genes, further emphasizing that pluripotency is dispensable for hTSC state acquisition.

Muscle injury causes long-term changes in stem-cell DNA methylation.

Injury to muscle brings about the activation of stem cells, which then generate new myocytes to replace damaged tissue. We demonstrate that this activation is accompanied by a dramatic change in the stem-cell methylation pattern that prepares them epigenetically for terminal myocyte differentiation. These de- and de novo methylation events occur at regulatory elements associated with genes involved in myogenesis and are necessary for activation and regeneration. Local injury of one muscle elicits an almost identical epigenetic change in satellite cells from other muscles in the body, in a process mediated by circulating factors. Furthermore, this same methylation state is also generated in muscle stem cells (MuSCs) of female animals following pregnancy, even in the absence of any injury. Unlike the activation-induced expression changes, which are transient, the induced methylation profile is stably maintained in resident MuSCs and thus represents a molecular memory of previous physiological events that is probably programmed to provide a mechanism for long-term adaptation.

Laminin111-based defined culture promoting self-renewing human pluripotent stem cells with properties of the early post-implantation epiblast.

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.

The role of DNA methylation in genome-wide gene regulation during development.

Although it is well known that DNA methylation serves to repress gene expression, precisely how it functions during the process of development remains unclear. Here, we propose that the overall pattern of DNA methylation established in the early embryo serves as a sophisticated mechanism for maintaining a genome-wide network of gene regulatory elements in an inaccessible chromatin structure throughout the body. As development progresses, programmed demethylation in each cell type then provides the specificity for maintaining select elements in an open structure. This allows these regulatory elements to interact with a large range of transcription factors and thereby regulate the gene expression profiles that define cell identity.

Determining gestational age using genome methylation profile: A novel approach for fetal medicine.

Gestational age determination by traditional tools (last menstrual period, ultrasonography measurements and Ballard Maturational Assessment in newborns) has major limitations and therefore there is a need to find different approaches. In this study, we looked for a molecular marker that can be used to determine the accurate gestational age of the newborn. To this end, we performed reduced representation bisulfite sequencing (RRBS) on 41 cord blood and matching placenta samples from women between 25 and 40 weeks of gestation and generated an epigenetic clock based on the methylation level at different loci in the genome. We identified a set of 332 differentially methylated regions (DMRs) that undergo demethylation in late gestational age in cord blood cells and can predict the gestational age (r = -.7, P = 2E-05). Once the set of 411 DMRs that undergo de novo methylation in late gestational age was used in combination with the first set, it generated a more accurate clock (R = .77, P = 1.87E-05). We have compared gestational age determined by Ballard score assessment with our epigenetic clock and found high concordance. Taken together, this study demonstrates that DNA methylation can accurately predict gestational age and thus may serve as a good clinical predictor.

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

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.

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. 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. changing environment.

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.

Combining flagellin and human ß-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 ß-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.

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.

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

BACKGROUND: 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. METHODS: Two chimeric proteins, soluble CD40 ligand (sCD40L)-caspase 3 (sCD40L-l-Caspase3) and sCD40L-pseudomonas exotoxin 38 (PE38) (sCD40L-l-PE38), were constructed, expressed, and partially purified. The ability of the chimeric proteins to kill tumor cells that expressed CD40 was tested by using proliferation assays. In addition, the induction of apoptosis in treated cells was followed by measuring expression levels of apoptotic proteins using real-time polymerases chain reaction analysis, caspase 3 enzymatic activity, and tracking changes in the cell cycle with fluorescence-activated cell-sorting analysis. RESULTS: The chimeric proteins exhibited concentration-dependent and time-dependent killing ability. The new chimeric proteins had no effect in several carcinoma cell lines that did not express the CD40 receptor. Treating tumor cells with sCD40L-based chimeric proteins led to internalization of the fusion proteins into the cell cytoplasm of B cells. Shortly after treatment, a sharp rise in B-cell chronic lymphocytic leukemia/lymphoma 2 (Bcl2) expression levels occurred. Approximately 36 hours after the initiation of treatment, Bcl2 levels dropped, whereas Bcl2-associated X protein (Bax) expression levels rose, pushing the cells toward apoptosis. Concomitantly, caspase 3 RNA levels rose. CONCLUSIONS: sCD40L-based chimeric proteins were able to bind and internalize into B cells that expressed the CD40 receptor and specifically and efficiently induced apoptotic death. Moreover, the current results validated for the first time the ability of sCD40L to serve as a direct delivery system for targeted molecules. sCD40L-based chimeric cytotoxic proteins offer a new weapon in the everlasting war against cancer.

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.

α-Synuclein expression selectively affects tumorigenesis in mice modeling Parkinson's disease.

Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn-dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over-expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer.

Successful TAT-mediated enzyme replacement therapy in a mouse model of mitochondrial E3 deficiency.

Medicine today offers no cure for patients suffering from mitochondrial disorders, such as lipoamide dehydrogenase (LAD; also known as E3) deficiency, and treatment is limited to symptomatic care. LAD is one of the components of the α-ketoacid dehydrogenase complexes, which are mitochondrial multienzyme complexes crucial for the metabolism of carbohydrates and amino acids. Recently, we tested the therapeutic approach for treating mitochondrial disorders whereby the activity of multicomponent complexes in the mitochondria is restored by TAT-mediated enzyme replacement therapy (ERT). The LAD deficiency disease was used before as a proof-of-principle in vitro, in patients' cells, utilizing the TAT-LAD fusion protein. In this report, we present successful TAT-mediated ERT in an in vivo mouse model using E3-deficient mice. We demonstrate the delivery of TAT-LAD into E3-deficient mice tissues and that a single administration of TAT-LAD results in a significant increase in the enzymatic activity of the mitochondrial multienzyme complex pyruvate dehydrogenase complex within the liver, heart and, most importantly, the brain of TAT-LAD-treated E3-deficient mice. We believe that this TAT-mediated ERT approach could change the management of mitochondrial disorders and of other metabolic diseases in modern medicine.

Utilizing a GnRH-based chimeric protein for the detection of adenocarcinoma.

Since early diagnosis of many types of cancer greatly improves the chances for successful treatment, high-quality methods for cancer detection are necessary. Our laboratory develops chimeric proteins for targeted therapy, such as gonadotropin releasing hormone (GnRH)-based chimeric proteins for the targeted therapy of adenocarcinomas in humans. For chimeric proteins to cause specific cell death, they must first recognize specific receptors/binding sites expressed on the surface of target cells. Thus, we examined whether we could exploit these binding sites not only as targets for the killing of specific cells but also as a diagnostic marker for identifying adenocarcinomas, using the same chimeric proteins. In this report, we show that one such GnRH-based chimeric protein, GnRH-Caspase3, can indeed serve as a diagnostic tool. GnRH-Caspase3 was able to specifically bind adenocarcinoma cells, as measured by FACS analysis and demonstrated with the aid of confocal microscopy and specific antibodies. Moreover, we found a correlation between cell sensitivity to treatment and the binding level of the chimeric protein to the cells. Hence, we suggest that in addition to their therapeutic potential, GnRH-based chimeric proteins can be used as a diagnostic tool for the detection of adenocarcinomas.