Efficient strategies for TALEN-mediated genome editing in mammalian cell lines.

TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.

High frequency targeted mutagenesis using engineered endonucleases and DNA-end processing enzymes.

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.

Differential epigenetic regulation of Aiolos expression in human tumoral cell lines and primary cells.

In order to investigate the epigenetic component of Aiolos regulation, we analyzed the methylation status of its 5' CpG island in relation to histone modifications. Inhibition of CpG methylation restores Aiolos expression, as well as euchromatin-associated markers, in U937 and 1106 mel cell lines. DNA methylation and low levels of euchromatin-associated signatures are observed in U937 and 1106 mel cell lines, while the opposite characterizes Daudi, Jurkat, T and B cells. CpG methylation is not necessary to repress transcription in monocytes and melanocytes where silencing mechanism involves heterochromatin-associated signature. We show that DNA methylation directs Aiolos silencing and chromatin status in tumor cell lines, while in primary cells is mainly regulated by histone modifications.

The Aiolos transcription factor is up-regulated in chronic lymphocytic leukemia.

The Aiolos transcription factor, member of the Ikaros family of zinc finger proteins, plays an important role in the control of mature B lymphocyte differentiation and proliferation, and its function appears to be modulated through alternative splicing. To assess Aiolos isoform role in humans' pathologies, we studied Aiolos variant distribution and expression in mature B lymphoproliferative disorders (chronic lymphocytic leukemia [CLL] and other B-cell lymphomas). We demonstrated that more than 80% of expressed Aiolos in normal as well as in malignant B cells is of the hAio1 type, and we showed for the first time a homogeneous overexpression of the total amounts of Aiolos transcripts in the B cells of CLL patients, independently of ZAP-70 and IgV(H) mutational status prognosis factors. This up-regulation of Aiolos, confirmed at protein level, seems independent of Aiolos promoter H3K9 acetylation and H3K4 trimethylation.

Cascade of transcriptional induction and repression during IL-2 deprivation-induced apoptosis.

Apoptosis of mature T lymphocytes is an essential process for maintaining immune system homeostasis. However, the details of the molecular signaling pathways leading to T cell apoptosis are poorly understood. We used cDNA microarrays containing 15,630 murine genes to study the gene expression profile in T lymphocytes at different time points of IL-2 withdrawal. Comparison of the gene expression profiles revealed that 2% of the genes were affected by cytokine starvation. Interestingly, the apoptotic program rather seems to activate gene expression in the early phase of cell death. On the contrary, transcription was strongly repressed in later stages of apoptosis. Self-organizing map clustering of the 270 differentially expressed transcripts revealed specific temporal expression patterns supporting the idea that IL-2 deprivation triggers a tightly regulated transcriptional program to induce cell death. To validate microarray results, changes in gene expression following IL-2 deprivation were confirmed for selected genes by Northern blot. In addition, the signaling pathways created can explain the molecular events leading to T cell apoptosis, even if the T cell line used in this study might not reflect individual T cell subpopulations expressing different level of IL-2 receptor or IL-2 dependence. Taken together, these results provide novel insights into the temporal regulation of gene expression during T lymphocyte death.

Critical function of Ikaros in controlling Aiolos gene expression.

To characterize the regulation of lymphoid Aiolos transcription factor, we have cloned its promoter. Full promoter and nested deletions were expressed in lymphoid and non-lymphoid cell lines. The minimal promoter activity could be considered as a 172bp upstream from the ATG for Jurkat and HEK293 cells and as a 370bp fragment for U937 cells. Moreover, we have mapped the transcription initiation site. Retardation gels showed binding activity for Ikaros, NFkappaB and AP4 transcription factors and mutations in their binding sites abolish Aiolos promoter activity. Chromatin immunoprecipitation assay revealed that Ikaros, NFkappaB and AP4 are bound to Aiolos promoter. The important function of Ikaros and NFkappaB is underlined by their over expression, which results in the trans-activation of the promoter and drives Aiolos expression in cell lines and in freshly isolated B and T cells, while over expression of a dominant negative Ikaros isoform is able to block Aiolos expression.

Identification of PP1alpha as a caspase-9 regulator in IL-2 deprivation-induced apoptosis.

One of the mechanisms that regulate cell death is the reversible phosphorylation of proteins. ERK/MAPK phosphorylates caspase-9 at Thr(125), and this phosphorylation is crucial for caspase-9 inhibition. Until now, the phosphatase responsible for Thr(125) dephosphorylation has not been described. Here, we demonstrate that in IL-2-proliferating cells, phosphorylated serine/threonine phosphatase type 1alpha (PP1alpha) associates with phosphorylated caspase-9. IL-2 deprivation induces PP1alpha dephosphorylation, which leads to its activation and, as a consequence, dephosphorylation and activation of caspase-9 and subsequent dissociation of both molecules. In cell-free systems supplemented with ATP caspase-9 activation is induced by addition of cytochrome c and we show that in this process PP1alpha is indispensable for triggering caspase-9 as well as caspase-3 cleavage and activation. Moreover, PP1alpha associates with caspase-9 in vitro and in vivo, suggesting that it is the phosphatase responsible for caspase-9 dephosphorylation and activation. Finally, we describe two novel phosphatase-binding sites different from the previously described PP1alpha consensus motifs, and we demonstrate that these novel sites mediate the interaction of PP1alpha with caspase-9.

Modulating apoptosis as a target for effective therapy.

Alterations in cell proliferation and cell death are essential determinants in the pathogenesis and progression of several diseases such as cancer, neurodegenerative disorders or autoimmune diseases among others. Complex networks of regulatory factors determine whether cells proliferate or die. Recent progress in understanding the molecular changes offer the possibility of specifically targeting molecules and pathways to achieve more effective and rational therapies. Drugs that target molecules involved in apoptosis are used as treatment against several diseases. Candidates such as TNF death receptor family, caspase inhibitors, antagonists of the p53-MDM2 interaction, NF-kappaB and PI3K pathways and Bcl-2 family members have been targeted as cancer cell killing agents. Moreover, apoptosis of tumor cells can also be achieved by targeting the inhibitor of apoptosis proteins, IAPs, in addition to the classical antiproliferative approach. Disruption of STAT activation and interferon beta therapy have been used as a treatment to prevent the progression of some autoimmune diseases. In models of Parkinson's, Alzheimer's and amyotrophic lateral sclerosis, blocking of Par-4 expression or function, as well as caspase activation, prevents neuronal cell death. Finally, it has been shown that gene therapy may be an encouraging approach for treatment of neurodegenerative disorders.

Bad-dependent rafts alteration is a consequence of an early intracellular signal triggered by interleukin-4 deprivation.

Many molecules are inducibly localized in lipid rafts, and their alteration inhibits early activation events, supporting a critical role for these domains in signaling. Using confocal microscopy and cellular fractionation, we have shown that the pool of Bad, attached to lipid rafts in proliferating cells, is released when cells undergo apoptosis. Kinetic studies indicate that rafts alteration is a consequence of an intracellular signal triggered by interleukin-4 deprivation. Growth factor deprivation in turn induces PP1alpha phosphatase activation, responsible for cytoplasmic Bad dephosphorylation as well as caspase-9 and caspase-3 activation. Caspases translocate to rafts and induce their modification followed by translocation of Bad from rafts to mitochondria, which correlates with apoptosis. Taken together, our results suggest that alteration of lipid rafts is an early event in the apoptotic cascade indirectly induced by interleukin-4 deprivation via PP1alpha activation, dephosphorylation of cytoplasmic Bad, and caspase activation.