Transcriptional programs guiding lymphocyte differentiation depend on precise expression and timing of transcription factors (TFs). BACH2 is a TF essential for T- and B-lymphocytes and is associated with an archetypal super-enhancer (SE). Single nucleotide variants in the BACH2 locus associate with multiple autoimmune diseases but BACH2 mutations causing Mendelian monogenic primary immunodeficiency have not previously been identified. We describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) resulting from BACH2 haploinsufficiency. Patients had lymphocyte maturation defects, causing immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. Analogous lymphocyte defects existed in Bach2 heterozygous mice. More generally, we found that genes causing monogenic haploinsufficient diseases are substantially enriched for TFs and SE-architecture. These observations show a new feature of SE-architecture in Mendelian diseases of immunity, that heterozygous mutations in SE-regulated genes identified on whole exome/genome sequencing may have greater significance than recognized.

IntroductionSignal transducers and activators of transcription 1 (STAT1) is critical for interferon-gamma (IFN-␥)-mediated immune responses, and its activation requires phosphorylation on tyrosine 701 by the receptorassociated Janus kinases (JAKs). 1 The function of STAT1 is also modulated by other posttranslational modifications, such as phosphorylation of Ser727 and, more recently, sumoylation at Lys703. [2][3][4] Small ubiquitin-like modifier (SUMO)-1, -2, -3, and -4 are protein moieties covalently conjugated to specific lysine residues on substrate proteins through an enzymatic pathway. 5 Recently, the regulatory enzymes in these reactions have been characterized, and protein inhibitors of activated STAT (PIAS) proteins, initially identified as regulators of STAT and androgen receptor activation, were shown to function as E3-type SUMO ligases. 6,7 Sumoylation mediates divergent effects on transcription factors and can cause transcriptional repression or enhanced activation. 8 Previously, we and others showed that PIAS proteins promote the sumoylation of STAT1 at Lys703, but the effect of the Lys703 mutation on reporter gene responses varied, leaving the functional role of this modification elusive. 3,4 This study aimed to analyze the functional role of sumoylation in STAT1, and our results indicate that sumoylation mediates a negative, promoter-dependent regulatory function in STAT1-mediated transcription. Study design Reagents PlasmidsThe SUMO-1, STAT1-wild-type-HA (STAT1-WT-HA), and STAT1-KR-HA plasmids have been previously described. 3 STAT1-I702R-HA (Ile3Arg) and STAT1-E705A-HA (Glu3Ala) were constructed from STAT1-WT-HA using polymerase chain reaction (PCR) mutagenesis with the following primers: 5Ј-GGAACTGGATAT-AGGAAGACTGAGTTGATTTCTGTGTCTGAA-3Ј and 5Ј-TTCAGA-CACAGAAATCAACTCAGTCTTCCTATATCCAGTTCC-3Ј; 5Ј-GGA-ACTGGATATATCAAGACTGCGTTGATTTCTGTGTCTGAA-3Ј and 5Ј-TTCAGACACAGAAATCAACGCAGTCTTGATATATCCAGTTCC-3Ј. Transfections and immunodetectionCOS-7 cells were transfected using Fugene6 reagent and lysed in Triton-X lysis buffer supplemented with 5 mM NEM (N-ethylmaleimide; SigmaAldrich). HeLa cells were transfected using the calcium phosphate method. Immunodetection was performed as described. 3 Quantitative RT-PCR and EMSATotal RNA was extracted using TRIZOL (Gibco-BRL, Carlsbad, CA). Reverse transcription (RT) was performed using a First strands cDNA synthesis kit (MBI Fermentas, Burlington, ON, Canada). The primers for For personal use only. on May 9, 2018. by guest www.bloodjournal.org From glyceraldehyde phosphate dehydrogenase (GAPDH), interferon regulatory factor 1 (IRF1), guanylate-binding protein 1 (GBP1), and transporters associated with antigen presentation 1 (TAP1) real-time PCR were previously described. 2 Detection of IFN-␥-activated sequence (GAS)-binding proteins by electrophoretic mobility shift assay (EMSA) has been previously described. 9 Immunofluorescence detectionStably transfected U3A clones were serum starved and stimulated with 100 ng/mL huIFN-␥. Cells were fixed in p-formaldehyde (4%) a...

JAK/STAT signaling pathway is evolutionarily conserved and tightly regulated. We carried out a reporter-based genome-wide RNAi in vitro screen to identify genes that regulate Drosophila JAK/STAT pathway and found 5 novel regulators. Of these, CG14225 is a negative regulator structurally related to the Drosophila JAK/STAT pathway receptor Domeless, especially in the extracellular domain, and to the mammalian IL-6 receptor and the signal transducer gp130. CG14225 coimmunoprecipitates with Domeless and its associated kinase hopscotch in S2 cells. CG14225 RNAi caused hyperphosphorylation of the transcription factor Stat92E in S2 cells on stimulation with the Drosophila JAK/STAT pathway ligand unpaired. CG14225 RNAi in vivo hyperactivated JAK/STAT target genes on septic injury and enhanced unpaired-induced eye overgrowth, and was thus named the eye transformer (ET). In the gastrointestinal infection model, where JAK/STAT signaling is important for stem cell renewal, CG14225/ET RNAi was protective in vivo. In conclusion, we have identified ET as a novel negative regulator of the Drosophila JAK/STAT pathway both in vitro and in vivo, and it functions in regulating Stat92E phosphorylation.

Understanding the control of antigen restimulation-induced T cell death (RICD), especially in cancer immunotherapy where highly proliferating T cells will encounter potentially large amounts of tumor antigens, is important now more than ever. It has been known that growth cytokines make T cells susceptible to RICD, but the precise molecular mediators that govern this in T cell subsets is unknown until now. Signal transducer and activator transcription (STAT) proteins are a family of transcription factors that regulate gene expression programs underlying key immunological processes. In particular, STAT5 is known to favor the generation and survival of memory T cells. Here we report an unexpected role for STAT5 signaling in the death of effector memory T (TEM) cells in mice and humans. TEM cell death was prevented with neutralizing anti-IL-2 antibody or STAT5/JAK3 inhibitors, indicating that STAT5 signaling drives RICD in TEM cells. Moreover, we identified a unique patient with a heterozygous missense mutation in the coiled-coil (CC) domain of STAT5B that presented with autoimmune lymphoproliferative syndrome (ALPS)-like features. Similar to Stat5b−/− mice, this patient exhibited increased CD4+ TEM cells in the peripheral blood. The mutant STAT5B protein dominantly interfered with STAT5-driven transcriptional activity, leading to global downregulation of STAT5-regulated genes in patient T cells upon IL-2 stimulation. Notably, CD4+ TEM cells from the patient were strikingly resistant to cell death by in vitro TCR restimulation, a finding that was recapitulated in Stat5b−/− mice. Hence, STAT5B is a crucial regulator of RICD in memory T cells in mice and humans.