During mammalian development, electrical activity promotes the calcium-dependent survival of neurons that have made appropriate synaptic connections. However, the mechanisms by which calcium mediates neuronal survival during development are not well characterized. A transcription-dependent mechanism was identified by which calcium influx into neurons promoted cell survival. The transcription factor MEF2 was selectively expressed in newly generated postmitotic neurons and was required for the survival of these neurons. Calcium influx into cerebellar granule neurons led to activation of p38 mitogen-activated protein kinase-dependent phosphorylation and activation of MEF2. Once activated, MEF2 regulated neuronal survival by stimulating MEF2-dependent gene transcription. These findings demonstrate that MEF2 is a calcium-regulated transcription factor and define a function for MEF2 during nervous system development that is distinct from previously well-characterized functions of MEF2 during muscle differentiation.

SUMMARY Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter of which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor neuron death, and hepatitis C infection. We find little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.

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The pathway determining malignant cellular transformation, which depends upon mutation of the BRCA1 tumor suppressor gene, is poorly defined. A growing body of evidence suggests that promotion of DNA double-strand break repair by homologous recombination (HR) may be the means by which BRCA1 maintains genomic stability, while a role of BRCA1 in error-prone nonhomologous recombination (NHR) processes has just begun to be elucidated. The BRCA1 protein becomes phosphorylated in response to DNA damage, but the effects of phosphorylation on recombinational repair are unknown. In this study, we tested the hypothesis that the BRCA1-mediated regulation of recombination requires the Chk2-and ATM-dependent phosphorylation sites. We studied Rad51-dependent HR and random chromosomal integration of linearized plasmid DNA, a subtype of NHR, which we demonstrate to be dependent on the Mre11-Rad50-Nbs1 complex. Prevention of Chk2-mediated phosphorylation via mutation of the serine 988 residue of BRCA1 disrupted both the BRCA1-dependent promotion of HR and the suppression of NHR. Similar results were obtained when endogenous Chk2 kinase activity was inhibited by expression of a dominant-negative Chk2 mutant. Surprisingly, the opposing regulation of HR and NHR did not require the ATM phosphorylation sites on serines 1423 and 1524. Together, these data suggest a functional link between recombination control and breast cancer predisposition in carriers of Chk2 and BRCA1 germ line mutations. We propose a dual regulatory role for BRCA1 in maintaining genome integrity, whereby BRCA1 phosphorylation status controls the selectivity of repair events dictated by HR and error-prone NHR.The tumor suppressor gene BRCA1 is mutated in up to 50% of cases of familial early-onset breast cancer and in most families with hereditary breast and ovarian cancer (50). BRCA1 is necessary for cellular processes ranging from cell cycle checkpoint control, DNA repair, regulation of transcription, protein ubiquitination, and apoptosis to chromatin remodeling (28,48,54,63). Dysfunction of many or all of these BRCA1 properties may be invoked in cancer development.Of the many types of DNA damage, DNA double-strand breaks (DSBs) represent a particularly dangerous form of damage. If not properly repaired, a DSB causes genetic changes and/or cell death. DSBs can arise spontaneously or may be induced by exogenous DNA damaging agents. The cell utilizes two principal pathways for the repair of DSBs: homologous recombination (HR) and nonhomologous recombination (NHR) (29, 62). Homology-mediated repair requires an undamaged template molecule that contains a homologous DNA sequence ordinarily on a sister chromatid or a homologous chromosome. HR is mediated through multiple proteins, including the Rad51/Rad52 recombinases and BRCA2. Several lines of evidence have indicated a role of BRCA1 in the HR pathway. BRCA1 colocalizes with BRCA2 and Rad51 (10, 41, 52, 70) and forms ionizing radiation (IR)-induced subnuclear foci (IRIF) containing Rad51 protein. Rad51 IRIF are reduc...

Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP ؊/؊ cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.Among the regulators of programmed cell death, or apoptosis (1), Bcl-2 proteins (2) control the release of apoptogenic proteins from mitochondria, notably cytochrome c (3), whereas members of the inhibitor of apoptosis (IAP) 1 gene family act as endogenous inhibitors of caspases (4), the enzymatic effectors of apoptosis (1). The structural requirements of IAP-caspase(s) complexes have been defined in considerable detail (5).Survivin is a structurally unique IAP protein that has been implicated in protection from apoptosis and regulation of mitosis (6). A role of survivin in cell division has been linked to assembly/stability of metaphase and anaphase microtubules (7) and spindle checkpoint function (8). In contrast, despite its ability to counteract apoptosis in vitro, and in transgenic animals (6), the mechanism(s) by which survivin inhibits apoptosis has remained elusive. This is important because IAPs, especially XIAP (9) and survivin (6), have emerged as critical regulators of cell survival in tumors and promising targets for rational anti-cancer therapy (10,11).In this study, we investigated the mechanism(s) of survivin cytoprotection. We found that in response to cell death stimulation, survivin physically associates with XIAP, and this complex promotes enhanced XIAP stability and synergistic inhibition of caspase-9 activation. MATERIALS AND METHODSCell Culture-Breast carcinoma MCF-7, lymphoblastoid Raji, and kidney embryonic HEK293T cells were from the American Type Culture Collection (ATCC, Manassas, VA). Wild type (WT) or XIAP Ϫ/Ϫ mouse embryonic fibroblasts (MEF) (12) were the gift of Dr. C. Duckett (University of Michigan).Protein-Protein Interactions-Affinity fractionation and immunoprecipitation experiments were carried out as described (10, 13). Fulllength survivin, truncated survivin BIR-(1-87), full-length XIAP, cIAP1, cIAP2, or the three isolated XIAP, BIR1-(1-123), BIR2-(123-259) and BIR3-(260 -336) were expressed as GST fusion proteins (14). Replication-deficient adenoviruses encoding GFP (pAd-GFP) or survivin (pAd-survivin) were described (15). Pull-down experiments with recombinant survivin (0.1-0.4 g) and GST, GST-XIAP, GST-cIAP1, GST-cIAP2 (8 g), or the individual GST-BIR1, -BIR2, or -BIR3 of XIAP (10 g) bound to glutathione beads (100 l) were as described (13). Alternatively, XIAP was translated in vitro in the presence of [ 35 S]methionine (Amersham Sciences), mixed with 5 g of GST or GST-survivin, and u...