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Efforts to identify lupus-associated causal variants in the FAM167A/BLK locus on 8p21 are hampered by highly associated noncausal variants. In this report, we used a trans-population mapping and sequencing strategy to identify a common variant (rs922483) in the proximal BLK promoter and a tri-allelic variant (rs1382568) in the upstream alternative BLK promoter as putative causal variants for association with systemic lupus erythematosus. The risk allele (T) at rs922483 reduced proximal promoter activity and modulated alternative promoter usage. Allelic differences at rs1382568 resulted in altered promoter activity in B progenitor cell lines. Thus, our results demonstrated that both lupus-associated functional variants contribute to the autoimmune disease association by modulating transcription of BLK in B cells and thus potentially altering immune responses.

ObjectiveSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and altered type I interferon expression. Genetic surveys and genome-wide association studies have identified >30 SLE susceptibility genes. One of these genes, TNIP1, encodes the ABIN1 protein. ABIN1 functions in the immune system by restricting NF-κB signaling. The present study was undertaken to investigate the genetic factors that influence association with SLE in genes that regulate the NF-κB pathway.MethodsWe analyzed a dense set of genetic markers spanning TNIP1 and TAX1BP1, as well as the TNIP1 homolog TNIP2, in case-control populations of diverse ethnic origins. TNIP1, TNIP2, and TAX1BP1 were fine-mapped in a total of 8,372 SLE cases and 7,492 healthy controls from European-ancestry, African American, Hispanic, East Asian, and African American Gullah populations. Levels of TNIP1 messenger RNA (mRNA) and ABIN1 protein in Epstein-Barr virus-transformed human B cell lines were analyzed by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively.ResultsWe found significant associations between SLE and genetic variants within TNIP1, but not in TNIP2 or TAX1BP1. After resequencing and imputation, we identified 2 independent risk haplotypes within TNIP1 in individuals of European ancestry that were also present in African American and Hispanic populations. Levels of TNIP1 mRNA and ABIN1 protein were reduced among subjects with these haplotypes, suggesting that they harbor hypomorphic functional variants that influence susceptibility to SLE by restricting ABIN1 expression.ConclusionOur results confirm the association signals between SLE and TNIP1 variants in multiple populations and provide new insight into the mechanism by which TNIP1 variants may contribute to SLE pathogenesis.

Objective: Genetic variants spanning UBE2L3 are associated with increased expression of the UBE2L3‐encoded E2 ubiquitin‐conjugating enzyme H7 (UbcH7), which facilitates activation of proinflammatory NF‐κB signaling and susceptibility to autoimmune diseases. We undertook this study to delineate how genetic variants carried on the UBE2L3/YDJC autoimmune risk haplotype function to drive hypermorphic UBE2L3 expression. Methods: We used bioinformatic analyses, electrophoretic mobility shift assays, and luciferase reporter assays to identify and functionally characterize allele‐specific effects of risk variants positioned in chromatin accessible regions of immune cells. Chromatin conformation capture with quantitative polymerase chain reaction (3C‐qPCR), chromatin immunoprecipitation (ChIP)–qPCR, and small interfering RNA (siRNA) knockdown assays were performed on patient‐derived Epstein‐Barr virus–transformed B cells homozygous for the UBE2L3/YDJC nonrisk or risk haplotype to determine if the risk haplotype increases UBE2L3 expression by altering the regulatory chromatin architecture in the region. Results: Of the 7 prioritized variants, 5 demonstrated allele‐specific increases in nuclear protein binding affinity and regulatory activity. High‐throughput sequencing of chromosome conformation capture coupled with ChIP (HiChIP) and 3C‐qPCR uncovered a long‐range interaction between the UBE2L3 promoter (rs140490, rs140491, rs11089620) and the downstream YDJC promoter (rs3747093) that was strengthened in the presence of the UBE2L3/YDJC risk haplotype, and correlated with the loss of CCCTC‐binding factor (CTCF) and gain of YY1 binding at the risk alleles. Depleting YY1 by siRNA disrupted the long‐range interaction between the 2 promoters and reduced UBE2L3 expression. Conclusion: The UBE2L3/YDJC autoimmune risk haplotype increases UBE2L3 expression through strengthening a YY1‐mediated interaction between the UBE2L3 and YDJC promoters. [ABSTRACT FROM AUTHOR]

Abstract Background Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. Results The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. Conclusion The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

Objective: Genetic variants in the region of tumor necrosis factor–induced protein 3–interacting protein 1 (TNIP1) are associated with autoimmune disease and reduced TNIP1 gene expression. The aim of this study was to define the functional genetic mechanisms driving TNIP1 hypomorphic expression imparted by the systemic lupus erythematosus–associated TNIP1 H1 risk haplotype. Methods: Dual luciferase expression and electrophoretic mobility shift assays were used to evaluate the allelic effects of 11 risk variants on enhancer function and nuclear protein binding in immune cell line models (Epstein‐Barr virus [EBV]–transformed human B cells, Jurkat cells, and THP‐1 cells), left in a resting state or stimulated with phorbol 12‐myristate 13‐acetate/ionomycin. HiChIP was used to define the regulatory 3‐dimensional (3‐D) chromatin network of the TNIP1 haplotype by detecting in situ long‐range DNA contacts associated with H3K27ac‐marked chromatin in EBV B cells. Then, quantitative reverse transcription–polymerase chain reaction (qRT‐PCR) was used to determine the expression of genes within the 3‐D chromatin network. Results: Bioinformatics analyses of 50 single‐nucleotide polymorphisms on the TNIP1 H1 risk haplotype identified 11 non–protein‐coding variants with a high likelihood of influencing TNIP1 gene expression. Eight variants in EBV B cells, 5 in THP‐1 cells, and 2 in Jurkat cells exhibited various allelic effects on enhancer activation, resulting in a cumulative suppressive effect on TNIP1 expression (net effect of risk variants −7.14 fold, −6.80 fold, and −2.44 fold, respectively; n > 3). Specifically, in EBV B cells, only 2 variants (rs10057690 and rs13180950) exhibited allele‐specific loss of both enhancer activity and nuclear protein binding (each P < 0.01 relative to nonrisk alleles). In contrast, the rs10036748 risk allele reduced binding affinities of the transcriptional repressors basic helix‐loop‐helix family member 40/differentially expressed in chondrocytes 1 (bHLHe40/DEC1) (P < 0.05 relative to nonrisk alleles) and CREB‐1 (P not significant) in EBV B cells, resulting in a gain of enhancer activity (P < 0.05). HiChIP and qRT‐PCR analyses revealed that overall transcriptional repression of the TNIP1 haplotype extended to the neighboring genes DCTN4 and GMA2, both of which also showed decreased expression in the presence of the TNIP1 risk haplotype (P < 0.001 and P < 0.01, respectively, relative to the nonrisk haplotype); notably, it was found that these genes share a 3‐D chromatin network. Conclusion: Hypomorphic TNIP1 expression results from the combined concordant and opposing effects of multiple risk variants carried on the TNIP1 risk haplotype, with the strongest regulatory effect in B lymphoid lineage cells. Furthermore, the TNIP1 risk haplotype effect extends to neighboring genes within a shared chromatin network. [ABSTRACT FROM AUTHOR]

The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1. [ABSTRACT FROM AUTHOR]

Functional characterization of causal variants present on risk haplotypes identified through genome-wide association studies (GWAS) is a primary objective of human genetics. In this report, we evaluate the function of a pair of tandem polymorphic dinucleotides, 42 kb downstream of the promoter of TNFAIP3, (rs148314165, rs200820567, collectively referred to as TT>A) recently nominated as causal variants responsible for genetic association of systemic lupus erythematosus (SLE) with tumor necrosis factor alpha inducible protein 3 (TNFAIP3). TNFAIP3 encodes the ubiquitin-editing enzyme, A20, a key negative regulator of NF-κB signaling. A20 expression is reduced in subjects carrying the TT>A risk alleles; however, the underlying functional mechanism by which this occurs is unclear. We used a combination of electrophoretic mobility shift assays (EMSA), mass spectrometry (MS), reporter assays, chromatin immunoprecipitation-PCR (ChIP-PCR) and chromosome conformation capture (3C) EBV transformed lymphoblastoid cell lines (LCL) from individuals carrying risk and non-risk TNFAIP3 haplotypes to characterize the effect of TT>A on A20 expression. Our results demonstrate that the TT>A variants reside in an enhancer element that binds NF-κB and SATB1 enabling physical interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-κB to the TT>A risk alleles or knockdown of SATB1 expression by shRNA, inhibits the looping interaction resulting in reduced A20 expression. Together, these data reveal a novel mechanism of TNFAIP3 transcriptional regulation and establish the functional basis by which the TT>A risk variants attenuate A20 expression through inefficient delivery of NF-κB to the TNFAIP3 promoter. These results provide critical functional evidence supporting a direct causal role for TT>A in the genetic predisposition to SLE. [ABSTRACT FROM AUTHOR]

Background: Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. Methodology/Principal Findings: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. Conclusions/Significance: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples. [ABSTRACT FROM AUTHOR]