Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA.

OBJECTIVES: Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. METHODS: Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. RESULTS: The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10(-4), OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10(-7), OR 0.71; case-only pmeta=1.9×10(-4), OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. CONCLUSIONS: These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications.

Ethynylbenzenoid metabolites of Antrodia camphorata: synthesis and inhibition of TNF expression.

An improved synthesis of the anti-inflammatory natural product antrocamphin A (2), involving a key Castro-Stephens reaction, is presented, along with the first total synthesis of its congener antrocamphin B (3). Approaches towards the more complex co-metabolite antrodioxolanone (4) were unsuccessful, but a samarium diiodide-mediated pinacol coupling of antrocamphin B did provide the chiral epimers (51). Antrocamphin A (2) inhibits Tumour Necrosis Factor (TNF) reporter gene expression, but its development as an anti-inflammatory agent may be limited by cytotoxicity.

Diverse biological activities of the vascular non-inflammatory molecules - the Vanin pantetheinases.

The Vanin genes are a family that encode pantetheinases involved in recycling Coenzyme A, catalysing the breakdown of intermediate pantetheine to vitamin B5 for reuse in CoA biosynthesis. The role of pantetheinase in this most fundamental of cellular processes, was substantially characterised by the 1970s. The next 20 years saw little further interest in pantetheinase until various genetic studies implicated the Vanin locus in a range of normal and disease phenotypes, and a consequent interest in the other product of pantetheinase activity, cysteamine. This report seeks to bring together the early biochemical studies with recent biological data implicating cysteamine as a regulator of the oxidative state of a cell. Numerous studies now report a role for Vanin in inflammation, oxidative stress, cell migration and numerous diseases including cardiovascular disease.

TNF-inducible expression of lymphotoxin-ß in hepatic cells: an essential role for NF-κB and Ets1 transcription factors.

As TNF is one of the earliest signals that can be detected in the leukocyte-derived inflammatory cascade which drives subsequent cytokine production, we are interested in determining whether TNF is one of the initiating factors controlling liver remodeling and regeneration following chronic liver damage. One of the early responses is the expression of lymphotoxin-ß by hepatic progenitor oval cells. The aim of this study was to determine whether hepatic expression of LT-ß was controlled by TNF and to understand the basis of this regulation. We previously showed that LT-ß expression is transcriptionally controlled via the TNF-induced, inflammatory NF-κB pathway in T lymphocytes. Here we show that TNF is able to upregulate LT-ß expression in hepatic cells at the transcriptional level by the binding of NF-κB p50/p65 heterodimers and Ets1 to their respective sites in the LT-ß promoter.

Genetic variation in PARL influences mitochondrial content.

Given their involvement in processes necessary for life, mitochondrial damage and subsequent dysfunction can lead to a wide range of human diseases. Previous studies of both animal models and humans have suggested that presenilins-associated rhomboid-like protein (PARL) is a key regulator of mitochondrial integrity and function, and plays a role in cellular apoptosis. As a surrogate measure of mitochondrial integrity, we previously measured mitochondrial content in a Caucasian population consisting of large extended pedigrees, with results highlighting a substantial genetic component to this trait. To assess the influence of variation in the PARL gene on mitochondrial content, we re-sequenced 6.5 kb of the gene, identifying 16 SNPs and genotyped these in 1,086 Caucasian individuals, distributed across 170 families. Statistical genetic analysis revealed that one promoter variant, T-191C, exhibited significant effects (after correction for multiple testing) on mitochondrial content levels. Comparison of the transcription factor binding characteristics of the T-191C promoter SNP by EMSA indicates preferential binding of nuclear factors to the T allele, suggesting functional variation in PARL expression. These results suggest that genetic variation within PARL influences mitochondrial abundance and integrity.

Efforts toward elucidating Thalidomide's molecular target: an expedient synthesis of the first Thalidomide biotin analogue.

Herein we describe the synthesis of the first Thalidomide-biotin analogue in order to initiate investigations into the unknown molecular mode of action of Thalidomide. In this manner we describe the attachment of biotin tether through the Huisgen 1,3-dipolar cycloaddition or "click" synthetic methodology.

New thalidomide analogues derived through Sonogashira or Suzuki reactions and their TNF expression inhibition profiles.

A library of new thalidomide C4/5 analogues containing either a phenyl or alkyne tether were synthesized using Sonogashira or Suzuki cross coupling reactions from their aryl halogenated precursors. All thalidomide analogues were tested for their ability to inhibit the expression of the proinflammatory cytokine Tumor Necrosis Factor (TNF). More explicitly the use of a novel reporter system utilizing the promoter region of the TNF gene in a human T-cell line provided a rapid and effective measure of NFkappaB transcriptional activity. Several compounds either containing either an aryl-isobutyl or aryl-isopropoxy group were the most effective in inhibiting TNF expression, and were several times more active than thalidomide itself. Five of the more active derivatives indicated an apoptotic response while one of these compounds, containing an aldehyde tether, showed possible influence of cell cycling effects.

Notch signaling induces a transcriptionally permissive state at the Complement C3d Receptor 2 (CR2) promoter in a pre-B cell model.

During mammalian lymphoid development, Notch signaling is necessary at multiple stages of T lymphopoiesis, including lineage commitment, and later stages of T cell effector differentiation. In contrast, outside of a defined role in the development of splenic marginal zone B cells, there is conflicting evidence regarding whether Notch signaling plays functional roles in other B cell sub-populations. Complement receptor 2 (CR2) modulates BCR-signaling and is tightly regulated throughout differentiation. During B lymphopoiesis, CR2 is detected on immature and mature B cells with high surface expression on marginal zone B cells. Here, we have explored the possibility that Notch regulates human CR2 transcriptional activity using in vitro models including a co-culture system, co-transfection gene reporters and chromatin accessibility assays. We provide evidence that Notch signaling regulates CR2 promoter activity in a mature B cell line, as well as the induction of endogenous CR2 mRNA in a non-expressing pre-B cell line. The dynamics of endogenous gene activation suggests additional unidentified factors are required to mediate surface CR2 expression on immature and mature B lineage cells.

Whole-exome sequencing in multiplex preeclampsia families identifies novel candidate susceptibility genes.

OBJECTIVE: Preeclampsia is a common and serious heritable disorder of human pregnancy. Although there have been notable successes in identification of maternal susceptibility genes a large proportion of the heritability of preeclampsia remains unaccounted for. It is has been postulated that rare variation may account for some of this missing heritability. In this study, we performed whole-exome sequencing (WES) in multiplex families to identify rare exonic risk variants. METHODS: We conducted WES in 244 individuals from 34 Australian/New Zealand multiplex preeclampsia families. Variants were tested for association with preeclampsia using a threshold model and logistic regression. RESULTS: We found significant association for two moderately rare missense variants, rs145743393 (Padj = 0.0032, minor allele frequency = 0.016) in the chromosome 1 open reading frame 35 (C1orf35) gene, and rs34270076 (Padj = 0.0128, minor allele frequency = 0.024) in the pyroglutamylated RFamide peptide receptor (QRFPR) gene. To replicate these associations we performed imputation in our Australian genome wide association scan for preeclampsia and found no significant exonic variants in either C1orf35 or QRFPR. However, 11 variants demonstrating nominal significance (P < 0.05) in the genomic region between QRFPR and annexin A5 (ANXA5) were identified. We further leveraged publicly available genome-wide available summary data from the UK Biobank to investigate association of these two variants with the underlying clinical phenotypes of preeclampsia and detected nominal association of the QRFPR variant (rs34270076, P = 0.03) with protein levels in females. CONCLUSION: The study represents the first to use WES in multiplex families for preeclampsia and identifies two novel genes (QRFPR and C1orf35) not previously associated with preeclampsia and find nominal association of rs34270076 with protein levels, a key clinical feature of preeclampsia. We find further support for ANXA5 previously associated with pregnancy complications, including preeclampsia.

Quantitative differences in chromatin accessibility across regulatory regions can be directly compared in distinct cell-types.

Transcriptional activation in eukaryotes is often accompanied by alterations to chromatin structure at specific regulatory sites while other genomic regions may remain unchanged. In this study, we have examined the correlation between expression and chromatin accessibility of the human CR2 gene in a panel of cell lines (U937, REH, Ramos, and Raji) using the CHART-PCR assay with the accessibility agent micrococcal nuclease (MNase). To validate the use of this assay for comparing multiple cell-types, we first tested a series of genomic regions to determine if we could observe consistent, site-specific levels of MNase chromatin accessibility. Promoter regions of the ubiquitously expressed genes GAPDH and beta-actin were similar and showed high accessibility to MNase digestion in each of the cell lines, while on the other hand, promoter regions of developmentally restricted genes PAX-7 and SP-A2 showed consistently reduced chromatin accessibility. Since CHART-PCR detected site-specific differences in chromatin accessibility in a manner that could be compared between cell-types, we next examined chromatin accessibility over the CR2 core promoter in the panel of cell lines representing either CR2 expressing or CR2 non-expressing cell-types. Our data revealed significantly enhanced accessibility over the -289 to -101 and the -115 to -12 regions of the CR2 promoter in expressing B-cells (Ramos, Raji) compared to non-expressing cells (U937, REH). Thus, CHART-PCR assays detected a correlation between chromatin accessibility and expression of the human CR2 gene, while the accessibility of other genomic regions was site-specific, but not altered between cell-types.

Genetic association of preeclampsia to the inflammatory response gene SEPS1.

OBJECTIVE: The objective of the study was to test for a genetic association between the G-105A promoter polymorphism of the inflammatory mediator Selenoprotein S (SEPS1) and preeclampsia. STUDY DESIGN: A retrospective study in a large Norwegian case-control cohort compared maternal genotype and allele frequencies of the SEPS1 g.-105G>A polymorphism genotyped by SNPlex assay in preeclamptic (n = 1139) and control (n = 2269) women. Statistical significance was determined by chi2 and multivariate regression analyses. RESULTS: Women with preeclampsia were 1.34 times more likely to have the GA or AA genotype (P = .0039; 95% confidence interval [CI] 1.09 to 1.64) and 1.22 times more likely to carry the A allele (P = .023; odds ratio, 1.22; 95% CI, 1.02 to 1.46). CONCLUSION: The A allele of the SEPS1-105G>A polymorphism is a significant risk factor for preeclampsia in this population.

Integration site-specific transcriptional reporter gene analysis using Flp recombinase targeted cell lines.

While high-throughput genome-wide approaches are useful to identify important regulatory regions, traditional reporter gene methodologies still represent the ultimate steps in fine structure analysis of transcriptional control elements. However, there are still several inherent limitations in the currently available transient and stable transfection systems often leading to aberrant function of specific cis elements. In this study we overcome these problems and have developed a novel and widely applicable system that permits the comparison of transcriptional reporter gene activities following site-specific genomic integration. By using Flp recombinase-mediated integration, the system allows the integration and expression of a series of reporter gene constructs at exactly the same genomic location and orientation in all cells of any one culture. The resulting reporter gene lines carry a single reporter gene, which is incorporated within a measurably active chromatinized setting, thus more closely reflecting the endogenous gene environment.

JunB induced by constitutive CD30-extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling activates the CD30 promoter in anaplastic large cell lymphoma and reed-sternberg cells of Hodgkin lymphoma.

High expression of CD30 and JunB is characteristic of tumor cells in anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL). Possible interactions of CD30 and JunB were examined in this study. We found that the CD30 promoter in tumor cells of both nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK)-positive and NPM-ALK-negative ALCL and HL is regulated by a constitutively active CD30-extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK). Phosphorylation of ERK1/2 MAPK was confirmed in nuclei of tumor cells in both ALCL and HL. CD30-ERK1/2 MAPK signals induce JunB expression, which maintains high activity of the CD30 promoter. JunB induction seems to be largely independent of nuclear factor kappaB in ALCL and HL. These results show a common mechanism of CD30 overexpression in ALCL and HL, although the outcome of CD30 signaling differs between NPM-ALK-positive ALCL and NPM-ALK-negative ALCL, cutaneous ALCL, and HL as we recently reported.

Requirements for selective recruitment of Ets proteins and activation of mb-1/Ig-alpha gene transcription by Pax-5 (BSAP).

Pax-5, a member of the paired domain family of transcription factors, is a key regulator of B lymphocyte-specific transcription and differentiation. A major target of Pax-5-mediated activation is the mb-1 gene, which encodes the essential transmembrane signaling protein Ig-alpha. Pax-5 recruits three members of the Ets family of transcription factors: Ets-1, Fli-1 and GABPalpha (with GABPbeta1), to assemble ternary complexes on the mb-1 promoter in vitro. Using the Pax-5:Ets-1:DNA crystal structure as a guide, we defined amino acid requirements for transcriptional activation of endogenous mb-1 genes using a novel cell-based assay. Mutations in the beta-hairpin/beta-turn of the DNA-binding domain of Pax-5 demonstrated its importance for DNA sequence recognition and activation of mb-1 transcription. Mutations of amino acids contacting Ets-1 in the crystal structure reduced or blocked mb-1 promoter activation. One of these mutations, Q22A, resulted in greatly reduced mb-1 gene transcript levels, concurrent with the loss of its ability to recruit Fli-1 to bind the promoter in vitro. In contrast, the mutation had no effect on recruitment of the related Ets protein GABPalpha (with GABPbeta1). These data further define requirements for Pax-5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors.

Finding potential cis-regulatory loci using allele-specific chromatin accessibility as weights in a kernel-based variance component test.

We present a novel approach to detect potential cis-acting regulatory loci that combines the functional potential, an empirical DNase-seq based estimate of the allele-specificity of DNase-I hypersensitivity sites, with kernel-based variance component association analyses against expression phenotypes. To test our method we used public ENCODE whole genome DNase-I sequencing data, from a single sample, to estimate the functional potentials of the subset of 10,552 noncoding heterozygous single-nucleotide polymorphisms (SNPs) that were also present in the Genetic Analysis Workshop 19 (GAW19) family-based data set. We then built two covariance kernels, one nonweighted and one weighted by the functional potentials, and conducted kernel-based variance component association analyses against the 20,527 transcript expression phenotypes in the GAW19 family-based data set. We found signals of potential cis-regulatory effects, that surpassed the Bonferroni significance threshold, for ten transcripts. Stepwise removal of the cis-located SNPs from the weighted kernel lead to the disappearance of the association signal from our top transcript hit. We found compelling evidence of allele-specific cis-regulation for four transcripts using both kernels, and our results agree with previous research that suggests the involvement of specific cis-located variants in the regulation of their neighboring gene.

Focused transcription from the human CR2/CD21 core promoter is regulated by synergistic activity of TATA and Initiator elements in mature B cells.

Complement receptor 2 (CR2/CD21) is predominantly expressed on the surface of mature B cells where it forms part of a coreceptor complex that functions, in part, to modulate B-cell receptor signal strength. CR2/CD21 expression is tightly regulated throughout B-cell development such that CR2/CD21 cannot be detected on pre-B or terminally differentiated plasma cells. CR2/CD21 expression is upregulated at B-cell maturation and can be induced by IL-4 and CD40 signaling pathways. We have previously characterized elements in the proximal promoter and first intron of CR2/CD21 that are involved in regulating basal and tissue-specific expression. We now extend these analyses to the CR2/CD21 core promoter. We show that in mature B cells, CR2/CD21 transcription proceeds from a focused TSS regulated by a non-consensus TATA box, an initiator element and a downstream promoter element. Furthermore, occupancy of the general transcriptional machinery in pre-B versus mature B-cell lines correlate with CR2/CD21 expression level and indicate that promoter accessibility must switch from inactive to active during the transitional B-cell window.

Identification of a thalidomide derivative that selectively targets tumorigenic liver progenitor cells and comparing its effects with lenalidomide and sorafenib.

BACKGROUND & AIMS: The availability of non-tumorigenic and tumorigenic liver progenitor cell (LPC) lines affords a method to screen putative anti-liver cancer agents to identify those that are selectively effective. To prove this principle we tested thalidomide and a range of its derivatives and compared them to lenalidomide and sorafenib, to assess their growth-inhibitory effects. METHODS: Cell growth, the mitotic and apoptotic index of cell cultures were measured using the Cellavista instrument (SynenTec) using commercially available reagents. RESULTS: Neither lenalidomide nor thalidomide (100 µM) affected tumorigenic LPCs but killed their non-tumorigenic counterparts. Sorafenib arrested growth in both cell types. All but two derivatives of thalidomide were ineffective; of the two effective derivatives, one (thalidomide C1) specifically affected the tumorigenic cell line (10 µM). Mitotic and apoptotic analyses revealed that thalidomide C1 induced apoptotic cell death and not mitotic arrest. CONCLUSIONS: This study shows that screens incorporating non-tumorigenic and tumorigenic liver cell lines are a sound approach to identify agents that are effective and selective. A high throughput instrument such as the Cellavista affords robust and reproducible objective measurements with a large number of replicates that are reliable. These experiments show that neither lenalidomide nor thalidomide are potentially useful for anti-liver cancer therapy as they kill non-tumorigenic liver cells and not their tumorigenic counterparts. Sorafenib in contrast, is highly effective, but not selective. One tested thalidomide derivative has potential as an anti-tumor drug since it induced growth arrest; and importantly, it selectively induced apoptotic cell death only in tumorigenic liver progenitor cells.

Polymorphism of the CD30 promoter microsatellite repressive element is associated with development of primary cutaneous lymphoproliferative disorders.

Lymphomatoid papulosis is a preneoplastic cutaneous lymphoproliferative disorder characterized by overexpression of CD30, a member of the tumor necrosis factor receptor superfamily. CD30 signaling is known to have an effect on the growth and survival of lymphoid cells. Therefore, we hypothesized that the development of lymphomatoid papulosis and progression to an associated neoplasm such as cutaneous and systemic anaplastic large cell lymphoma may reflect an underlying genetic defect. In this study, we determined that two allelic forms of the CD30 promoter microsatellite repressive element, designated 30M377 and 30M362, are associated with the development of lymphomatoid papulosis and CD30+ lymphomas in lymphomatoid papulosis patients, respectively. These findings suggest that allele-specific differences in the control of CD30 transcription may determine the pathogenesis of the spectrum of CD30+ cutaneous lymphoproliferative disorders.

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells.

Effects of copy number variable regions on local gene expression in white blood cells of Mexican Americans.

Only few systematic studies on the contribution of copy number variation to gene expression variation have been published to date. Here we identify effects of copy number variable regions (CNVRs) on nearby gene expression by investigating 909 CNVRs and expression levels of 12059 nearby genes in white blood cells from Mexican-American participants of the San Antonio Family Heart Study. We empirically evaluate our ability to detect the contribution of CNVs to proximal gene expression (presumably in cis) at various window sizes (up to a 10 Mb distance) between the gene and CNV. We found a ~1-Mb window size to be optimal for capturing cis effects of CNVs. Up to 10% of the CNVs in this study were found to be significantly associated with the expression of at least one gene within their vicinity. As expected, we find that CNVs that directly overlap gene sequences have the largest effects on gene expression (compared with non-overlapping CNVRs located nearby), with positive correlation (except for a few exceptions) between estimated genomic dosage and expression level. We find that genes whose expression level is significantly influenced by nearby CNVRs are enriched for immunity and autoimmunity related genes. These findings add to the currently limited catalog of CNVRs that are recognized as expression quantitative trait loci, and have implications for future study designs as well as for prioritizing candidate causal variants in genomic regions associated with disease.