Stimulation of immune cells triggers Ca2؉ entry through store-operated Ca 2؉ release-activated Ca 2؉ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca 2؉ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca 2؉ release-activated Ca 2؉ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca 2؉ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca 2؉ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells. Ca2ϩ is a key second messenger in intracellular signaling pathways. In lymphocytes, specialized store-operated Ca 2ϩ channels known as CRAC 5 channels are required for sustained Ca 2ϩ influx across the plasma membrane (1). The resulting prolonged elevation of intracellular free Ca 2ϩ entry is essential for sustained nuclear translocation of the transcription factor NFAT, a small family of proteins whose activation is critical for a productive immune response (2). NFAT proteins reside in the cytoplasm of resting lymphocytes in a highly phosphorylated form and translocate to the nucleus upon dephosphorylation by the Ca 2ϩ /calmodulin-dependent serine/threonine phosphatase calcineurin (2, 3). In the nucleus, NFAT proteins bind to promoters and regulatory regions of a large number of cytokine genes and other activation-associated genes, thereby mediating the activation, proliferation, and differentiation of T cells, B cells, and other immune system cells.Although the notion of Ca 2ϩ influx through "store-operated" Ca 2ϩ channels was first proposed in 1986 (4, 5), the molecular identity of the proteins involved in this process remained unknown until the advent of large-scale RNAi-based screens. The first components of the pathway to be identified were Drosophila Stim (dStim) and its human homologues STIM1 and STIM2 through large-scale (albeit not genome-wide) RNAibased screens ...

ORAI1 is a pore subunit of the store-operated Ca 2؉ release-activated Ca 2؉ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1 ؊/؊ mice. Orai1 ؊/؊ mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1 ؊/؊ mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1 ؊/؊ mice, but B cells showed a substantial decrease in Ca 2؉ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1 ؊/؊ T cells showed substantial reductions in store-operated Ca 2؉ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.Ca 2ϩ is a universal second messenger that regulates a multitude of cellular functions, including secretion, muscle contraction, ion channel function, and gene expression (5). In many nonexcitable cells, Ca 2ϩ influx occurs through "storeoperated" Ca 2ϩ channels which open in response to depletion of endoplasmic reticulum (ER) Ca 2ϩ stores (40). Physiologically, this occurs when ligand binds to receptors, such as G protein-coupled receptors, immunoreceptors, and receptor tyrosine kinases, that are coupled to the activation of phospholipase C. The resulting production of inositol trisphosphate leads to efflux of Ca 2ϩ from the ER through inositol trisphosphate receptors and decreased Ca 2ϩ concentration in the ER lumen. This decrease directly regulates the opening of storeoperated Ca 2ϩ channels in the plasma membrane (26). In lymphocytes and other immune system cells, the major route of Ca 2ϩ influx is through store-operated Ca 2ϩ releaseactivated Ca 2ϩ (CRAC) channels. CRAC currents (I CRAC ) were first identified in T cells and mast cells (20,21,27,53), and Ca 2ϩ influx through CRAC channels is known to be essential for T-cell activation (8, 25). Mutant Jurkat tumor T-cell lines lacking functional CRAC channels cannot be activated properly (7); moreover, T cells obtained from three independent families of patients with hereditary severe combined immunodeficiency (SCID) were shown to be severely deficient in store-operated Ca 2ϩ entry and the CRAC channel current, I CRAC (10,13,23,36). T-cell responses, particularly proliferation and cytokine production in vitro in response to T-cell receptor stimulation, were strongly impaired in patients from two of these families, explaining their SC...

The transition from naïve to activated T cells is marked by alternative splicing of pre-mRNA encoding the transmembrane phosphatase CD45. Using a short hairpin RNA interference screen, we identified heterogeneous ribonucleoprotein L-like (hnRNPLL) as a critical inducible regulator of CD45 alternative splicing. HnRNPLL was up-regulated in stimulated T cells, bound CD45 transcripts, and was both necessary and sufficient for CD45 alternative splicing. Depletion or overexpression of hnRNPLL in B and T cell lines and primary T cells resulted in reciprocal alteration of CD45RA and RO expression. Exon array analysis suggested that hnRNPLL acts as a global regulator of alternative splicing in activated T cells. Induction of hnRNPLL during hematopoietic cell activation and differentiation may allow cells to rapidly shift their transcriptomes to favor proliferation and inhibit cell death.It is estimated that greater than 75% of genes yield alternative transcripts, contributing to considerable functional diversity within the genome (1,2). SR (serine-arginine rich) proteins are key positive regulators of alternative splicing that bind enhancer sequences on nascent transcripts and recruit spliceosomal proteins to weak splice sites, thereby facilitating proximal spliceo-some assembly (3). Heterogeneous nuclear ribonucleoproteins (hnRNPs) also regulate splicing by binding negative cis-regulatory elements and causing exon exclusion from mature mRNA (3). SR proteins and hnRNPs function as antagonists in alternative splicing, with binding of hnRNPs to silencer sequences inhibiting SR protein binding, thus forcing a shift of splicing to distal splice sites (4).CD45 is an abundant transmembrane protein tyrosine phosphatase expressed at the surface of T cells, B cells, and other hematopoietic cells (5). CD45 transcripts undergo extensive alternative splicing in which exons 4, 5, and 6 are variably excluded (Fig. 1A) (6). Primary naive T cells and B cells express the larger isoforms and are referred to as RA + . In contrast, activated and memory T cells express the shortest isoform, CD45RO (5). CD45 initiates signaling through antigen receptors by dephosphorylating the inhibitory tyrosine on Src-family kinases (5), but attributing specific functions to individual CD45

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure.

c Nuclear lamins play important roles in the organization and structure of the nucleus; however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. The S phase delay appears to be due to the stalling and collapse of replication forks. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions. The lamins are type V intermediate-filament proteins found primarily within the nuclei of metazoan cells. The lamins play important roles in providing mechanical support and shape to the nucleus and also participate in various chromatin-associated processes, including DNA replication, polymerase (Pol) II transcription, DNA repair, mitotic-spindle formation, response to oxidative stress, and chromosome positioning (1). However, the exact mechanisms by which lamins are involved in these pathways remain largely unclear. Vertebrate cells express two types of lamins, the A types, lamins A and C (LA and LC), and the B types, lamin B1 (LB1) and lamin B2 (LB2). LA and LC are expressed in developmentally regulated patterns from a single gene, LMNA, by alternative splicing. In contrast, LB1 and LB2 are expressed from two different genes, with at least one B-type lamin being expressed in all cell types throughout development and differentiation (2).A-type lamins have received the most attention in recent years because of the hundreds of mutations identified in LMNA that cause a spectrum of rare diseases known as laminopathies (3). The altered lamins produced because of these mutations have been shown to affect interactions with lamin-binding proteins, cause telomere dysfunction, disrupt the epigenetic regulation and organization of chromatin, and alter gene expression (4, 5). Accumulation of the unprocessed form of LA, called pre-LA, is also linked to the activation of DNA repair-regulating factors and checkpoint kinases, which possibly contribute to impaired cell cycle progression and replication arrest (6, 7). Pre-LA has also been reported to cause the accumulation of unrepaired DNA because of delayed recruitment of DNA repair proteins to DNA damage sites (8). In contrast to the num...