Michal Lichtenstein

B cell-specific demethylation: A novel role for the intronic κ chain enhancer sequence

We studied the molecular mechanism of demethylation and its role in kappa chain gene regulation. Following transfection into B cell cultures, this gene undergoes regional demethylation in a process that is developmentally regulated in a lineage- and stage-specific manner. Although a germline V kappa promoter is not required for the demodification activity, a fragment containing the intronic kappa chain transcriptional enhancer and the nearby matrix attachment region is essential. In its natural location downstream to the J kappa 5 sequence, this element induces bidirectional demodification of plasmid constructs in a distance- and orientation-independent manner. When this enhancer is placed in an upstream position, however, the kappa gene remains modified and transcriptionally inactive, demonstrating that demethylation is required for kappa chain activation. These studies suggest that the kappa enhancer plays a dual role in regulating B cell differentiation by inducing demethylation and by promoting tissue-specific transcription.

Tissue-specific DNA demethylation is required for proper B-cell differentiation and function

Significance Even though DNA methylation is known to be correlated with gene repression, it has never been demonstrated that this modification must indeed be removed from a gene in order for it to become activated during cell differentiation in vivo. In this paper, we inactivated the enzymes responsible for the demethylation reaction in the B-cell lineage and in this manner have shown that this epigenetic mark plays a critical role in development, independently of the many specific transcription factors that direct the selection of genes involved in cell differentiation. Our study is the first to our knowledge to causally connect all of the molecular components necessary to prove the link between the Tet enzymes, CpG demethylation, expression, and phenotype. There is ample evidence that somatic cell differentiation during development is accompanied by extensive DNA demethylation of specific sites that vary between cell types. Although the mechanism of this process has not yet been elucidated, it is likely to involve the conversion of 5mC to 5hmC by Tet enzymes. We show that a Tet2/Tet3 conditional knockout at early stages of B-cell development largely prevents lineage-specific programmed demethylation events. This lack of demethylation affects the expression of nearby B-cell lineage genes by impairing enhancer activity, thus causing defects in B-cell differentiation and function. Thus, tissue-specific DNA demethylation appears to be necessary for proper somatic cell development in vivo.

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.

Replacement of the C6ORF66 Assembly Factor (NDUFAF4) Restores Complex I Activity in Patient Cells

Disorders of the oxidative phosphorylation (OXPHOS) system frequently result in a severe multisystem disease with the consequence of early childhood death. Among these disorders, isolated complex I deficiency is the most frequently diagnosed, accounting for one-third of all cases of respiratory chain deficiency. We chose to focus on complex I deficiency, caused by mutation in the assembly factor chromosome 6, open reading frame 66 (C6ORF66; NADH dehydrogenase [ubiquinone] complex I assembly factor 4 [NDUFAF4]) protein. We used the approach of cell- and organelle-directed protein/enzyme replacement therapy, with the transactivator of transcription (TAT) peptide as the moiety delivery system. This step will enable us to deliver the wild-type assembly factor C6ORF66 into patient cells and their mitochondria, leading to the proper assembly and function of complex I and, as a result, to a functional OXPHOS system. We designed and constructed the TAT-ORF fusion protein by gene fusion techniques, expressed the protein in an Escherichia coli expression system and highly purified it. Our results indicate that TAT-ORF enters patients’ cells and their mitochondria rapidly and efficiently. TAT-ORF is biologically active and led to an increase in complex I activity. TAT-ORF also increased the number of patient cells and improved the activity of their mitochondria. Moreover, we observed an increase in ATP production, a decrease in the content of mitochondria and a decrease in the level of reactive oxygen species. Our results suggest that this approach of protein replacement therapy for the treatment of mitochondrial disorders is a promising one.

Control of Nuclear Export of hnRNP A1

mRNA export is mediated by RNA‐binding proteins which shuttle between the nucleus and cytoplasm. Using an in vitro unidirectional export assay, we observe that the shuttling mRNA‐binding protein, hnRNP A1, is exported only extremely slowly unless incubations are supplemented with snRNA‐specific oligonucleotides which inhibit splicing. In vivo microinjection experiments support this conclusion. Like many examples of nucleocytoplasmic transport, export of hnRNP A1 requires energy and is sensitive to the presence of wheat germ agglutinin. It does not, however, require supplementation with cytoplasmic proteins. Although the exportin, Crm1, is needed for export of several varieties of RNA, both the in vitro assay and in vivo assays show that it is not required for export of hnRNP A1. In vitro and in vivo studies also show that inhibition of transcription allows continued shuttling of hnRNP A1 and in fact accelerates its export. Judging from the stimulatory effects of targeted destruction of snRNAs, this is likely to reflect completion of the covalent maturation of the RNAs with which hnRNP A1 associates. These observations therefore provide a simple explanation of why multiple RNA‐binding proteins relocate to the cytoplasm upon inhibition of transcription in vivo.

Clonally stable Vκ allelic choice instructs Igκ repertoire

Although much has been done to understand how rearrangement of the Igκ locus is regulated during B-cell development, little is known about the way the variable (V) segments themselves are selected. Here we show, using B6/Cast hybrid pre-B-cell clones, that a limited number of V segments on each allele is stochastically activated as characterized by the appearance of non-coding RNA and histone modifications. The activation states are clonally distinct, stable across cell division and developmentally important in directing the Ig repertoire upon differentiation. Using a new approach of allelic ATAC-seq, we demonstrate that the Igκ V alleles have differential chromatin accessibility, which may serve as the underlying basis of clonal maintenance at this locus, as well as other instances of monoallelic expression throughout the genome. These findings highlight a new level of immune system regulation that optimizes gene diversity.

IL-2-granzyme A chimeric protein overcomes multidrug resistance (MDR) through a caspase 3-independent apoptotic pathway.

One of the main problems of conventional anticancer therapy is multidrug resistance (MDR), whereby cells acquire resistance to structurally and functionally unrelated drugs following chemotherapeutic treatment. One of the main causes of MDR is overexpression of the P‐glycoprotein transporter. In addition to extruding the chemotherapeutic drugs, it also inhibits apoptosis through the inhibition of caspases. To overcome MDR, we constructed a novel chimeric protein, interleukin (IL)‐2 granzyme A (IGA), using IL‐2 as a targeting moiety and granzyme A as a killing moiety, fused at the cDNA level. IL‐2 binds to the high‐affinity IL‐2 receptor that is expressed in an array of abnormal cells, including malignant cells. Granzyme A is known to cause caspase 3‐independent cell death. We show here that the IGA chimeric protein enters the target sensitive and MDR cancer cells overexpressing IL‐2 receptor and induces caspase 3‐independent cell death. Specifically, after its entry, IGA causes a decrease in the mitochondrial potential, triggers translocation of nm23‐H1, a granzyme A‐dependent DNase, from the cytoplasm to the nucleus, where it causes single‐strand DNA nicks, thus causing cell death. Moreover, IGA is able to overcome MDR and kill cells resistant to chemotherapeutic drugs. We believe that overcoming MDR with targeted molecules such as IGA chimeric protein that causes caspase‐independent apoptotic cell death could be applied to many other resistant types of tumors using the appropriate targeting moiety. Thus, this novel class of targeted molecules could open up new vistas in the fight against human 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.