Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

The functional importance of gene enhancers in regulated gene expression is well established(1–3). In addition to widespread transcription of long non-coding RNAs (ncRNA) in mammalian cells(4–6), bidirectional ncRNAs referred to as eRNAs are transcribed on enhancers(7–9). However, it has remained unclear whether these eRNAs are functional, or merely a reflection of enhancer activation. Here, we report that 17β-estradiol (E2)-bound estrogen receptor α (ERα) on enhancers causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, appear to exert important roles for the observed ligand-dependent induction of target coding genes, causing an increased strength of specific enhancer:promoter looping initiated by ERα binding. Cohesin, present on many ERα-regulated enhancers even prior to ligand treatment, apparently contributes to E2-dependent gene activation, at least in part, by stabilizing E2/ERα/eRNA-induced enhancer:promoter looping. Our data indicate that eRNAs are likely to exert important functions in many regulated programs of gene transcription.

Summary Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded-AR first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic machinery induced by genotoxic stress and liganded-AR, including Activation-Induced Cytidine Deaminase (AID) and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymatic machineries synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by Non-Homologous Ending Joining (NHEJ) pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded-nuclear receptor and genotoxic stress underlie non-random tumor translocations, which may function in many types of tumors and pathological processes.

Helper T cells coordinate immune responses through the production of cytokines. Th2 cells express the closely linked Il4, Il13, and Il5 cytokine genes, whereas these same genes are silenced in the Th1 lineage. The Th1/Th2 lineage choice has become a textbook example for the regulation of cell differentiation, and recent discoveries have further refined and expanded our understanding of how Th2 differentiation is initiated and reinforced by signals from antigen-presenting cells and cytokine-driven feedback loops. Epigenetic changes that stabilize the active or silent state of the Il4 locus in differentiating helper T cells have been a major focus of recent research. Overall, the field is progressing toward an integrated model of the signaling and transcription factor networks, cis-regulatory elements, epigenetic modifications, and RNA interference mechanisms that converge to determine the lineage fate and gene expression patterns of differentiating helper T cells.

While recent studies indicated roles of long non-coding RNAs (lncRNAs) in physiologic aspects of cell-type determination and tissue homeostasis1 yet their potential involvement in regulated gene transcription programs remain rather poorly understood. Androgen receptor (AR) regulates a large repertoire of genes central to the identity and behavior of prostate cancer cells2, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy3. Here, we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 and PCGEM1, bind successively to the AR and strongly enhance both ligand-dependent and ligand-independent AR-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the C-terminally acetylated AR on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the DOT1L-mediated methylated AR N-terminus. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited Pygopus2 PHD domain proves to enhance selective looping of AR-bound enhancers to target gene promoters in these cells. In “resistant” prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full length AR, causing ligand-independent activation of the AR transcriptional program and cell proliferation. Conditionally-expressed short hairpin RNA (shRNA) targeting of these lncRNAs in castration-resistant prostate cancer (CRPC) cell lines strongly suppressed tumor xenograft growth in vivo. Together, these results suggest that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumors.

Genome wide association studies (GWAS) have identified SNPs in the 9p21 gene desert associated with coronary artery disease (CAD)1–4 and Type 2 diabetes (T2D)5–7. Despite evidence for a role of the associated interval in neighboring gene regulation8–10, the biological underpinnings of these genetic associations to CAD or T2D have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene-desert for predicted enhancers and 6 times denser than the whole genome (p<6.55 10−33). The CAD risk alleles of SNPs rs10811656/rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines (LCL) homozygous for the CAD risk haplotype exhibit no binding of STAT1, and in LCL homozygous for the CAD non-risk haplotype binding of STAT1 inhibits CDKN2BAS expression, which is reversed by siRNA knock-down of STAT1. Using a new, open-ended approach to detect long-distance interactions (3D-DSL), we find that in human vascular endothelium cells (HUVEC) the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of INFA21. In HUVEC, IFNγ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1 binding, long-range enhancer interactions and altered expression of neighboring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signaling in a vascular cell type and thus demonstrate the utility of GWAS findings to direct studies to novel genomic loci and biological processes important for disease etiology.

While reversible histone modifications are linked to an ever-expanding range of biological functions1–5, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here, we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while utilizing multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1/S transition in conjunction with E2F1, HCF-1 and Set1A, at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the Condensin II loading process. Accordingly, the HEAT repeat clusters in two non-SMC Condensin II subunits, N-CAPD3 and N-CAPG2, are capable of recognizing H4K20me1, and ChIP-seq. analysis demonstrate a significant overlap of Condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of the first H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression.

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.