A common atopy-associated variant in the Th2 cytokine locus control region impacts transcriptional regulation and alters SMAD3 and SP1 binding.

BACKGROUND: Type 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair gene RAD50, containing several RAD50 DNase1-hypersensitive sites (RHS), have been robustly associated with atopic traits in genome-wide association studies (GWAS). Functional variants in IL13 have been intensely studied, whereas no causative variants for the IL13-independent RAD50 signal have been identified yet. This study aimed to characterize the functional impact of the atopy-associated polymorphism rs2240032 located in the human RHS7 on cis-regulatory activity and differential binding of transcription factors. METHODS: Differential transcription factor binding was analyzed by electrophoretic mobility shift assays (EMSAs) with Jurkat T-cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry (LC-MS/MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and HeLa cells. RESULTS: The variant rs2240032 impacts transcriptional activity and allele-specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine-tuning of transcription regulation within this region. CONCLUSION: The human RHS7 critically contributes to the regulation of gene transcription, and the common atopy-associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding.

Peripheral monocytes of obese women display increased chemokine receptor expression and migration capacity.

CONTEXT: The activation of peripheral immune cells and the infiltration of immune cells into adipose tissue in obesity are implicated in the development of type 2 diabetes mellitus. OBJECTIVE: The aim of the study was to compare peripheral immune cells from obese and normal-weight women with regard to composition of immune cell subpopulations, surface expression of the chemokine receptors (CCRs) CCR2, CCR3, CCR5, and CXCR3 (chemokine (C-X-C motif) receptor 3) and cell-intrinsic migration capacity. DESIGN: This was a case-control study. SETTING: The study was conducted at a university clinical study center. PATIENTS: Obese females and normal-weight females were included for fluorescence-activated cell sorting analysis and migration assays. MAIN OUTCOME MEASURES: Peripheral blood mononuclear cells were prepared from fasting blood samples and used for fluorescence-activated cell sorting analysis and migration assays. RESULTS: An increase in the percentages of CD14(+)CD16(+) monocytes was observed in obese subjects compared with controls. The CCR profile of monocytes differed significantly in the obese state; in particular, CCR2 levels were increased. In addition, a higher chemotactic activity of monocytes from obese subjects was observed in a migration assay, which was associated with both insulin resistance and CCR2 expression. CONCLUSION: Our results suggest that the enhanced intrinsic migratory capacity of peripheral monocytes in obese women may be due to the increased CCR expression, further supporting a link between peripheral immune cell dysfunction and obesity.

LIGHT (TNFSF14) inhibits adipose differentiation without affecting adipocyte metabolism.

OBJECTIVE: The member of the tumor necrosis factor family LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells; TNFSF14 (tumor necrosis factor super family protein 14) is primarily expressed in lymphocytes, in which it induces the expression of pro-inflammatory cytokines and alterations of lipid homeostasis. Recently, the protein was shown to be upregulated in obesity and to induce cytokine secretion from adipocytes. RESEARCH METHODS AND PROCEDURES: Using an automated complementary DNA (cDNA) screen, LIGHT was identified to inhibit adipose differentiation. As cellular models for adipogenesis mouse 3T3-L1, human SGBS (Simpson-Golabi-Behmel syndrome) and primary human preadipocytes differentiated in vitro were used as well as primary human adipocytes to study adipocyte functions. Analysis of lipid deposition by Oil Red O staining, mRNA expression by quantitative reverse transcriptase-PCR, nuclear factor (NF)-κB activation as well as protein secretion by enzyme linked immunosorbent assay and Luminex technology was performed. RESULTS: LIGHT was found to inhibit lipid accumulation in the three models of preadipocytes in a dose-dependent manner without cytotoxic effects. This inhibition of differentiation was probably because of interference at early steps of adipogenesis, as early exposure during differentiation showed the strongest effect, as assessed by decreased peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα) mRNA expression. In contrast to TNFα, basal and insulin-stimulated glucose uptake and lipolysis of terminally differentiated mature adipocytes were not altered in the presence of LIGHT. At a concentration sufficient to inhibit differentiation, secretion of proinflammatory cytokines was not significantly induced and NF-κB activity was only modestly induced compared with TNFα. CONCLUSION: LIGHT is a novel inhibitor of human adipocyte differentiation without adversely influencing central metabolic pathways in adipocytes.

Genetic variants in the leukemia-associated Rho guanine nucleotide exchange factor (ARHGEF12) gene are not associated with T2DM and related parameters in Caucasians (KORA study).

OBJECTIVE: The aim of our study was to determine the variant pattern of the leukemia-associated Rho guanine nucleotide exchange factor (LARG, or ARHGEF12) gene and investigate whether LARG variants are associated with diabetes mellitus type 2 (T2DM), the metabolic syndrome (MetS), or related parameters such as insulin sensitivity in German Caucasians. DESIGN: We analyzed single nucleotide polymorphisms (SNPs) in the LARG gene in the 55-74-year-old individuals of the population-based German Caucasian Cooperative Health Research in the region of Augsberg (KORA) survey 4 (S4). METHODS: Sequencing of Tyr1306Cys, which was of functional relevance in Pima Indians, in 48 randomly selected individuals and genotyping of 11 additional LARG SNPs in 1653 subjects were performed. Four linkage disequilibrium (LD) blocks (r(2)> or =0.8) were established and each block was statistically analyzed for association with metabolic traits. The association with T2DM and the MetS was analyzed by logistic regression in 1462 subjects, and HOMA-IR (homeostasis model assessment of insulin resistance) as a measure of insulin sensitivity was analyzed by the Kruskal-Wallis test in 1346 fasting subjects. RESULTS: The polymorphism Tyr1306Cys, which was significantly associated with insulin sensitivity in Pima Indians, was not found in the KORA S4 population. Statistical analysis yielded no significant associations (P>0.05) between the analyzed LARG variants and T2DM, the MetS, or related parameters such as insulin sensitivity. CONCLUSIONS: Caucasian individuals and Pima Indians differ in their genetic variance pattern in the LARG gene region. There is no evidence in the Caucasian KORA study that variants of the LARG gene confer susceptibility for T2DM, insulin sensitivity, or the MetS.

Defective octamer-dependent transcription is responsible for silenced immunoglobulin transcription in Reed-Sternberg cells.

The absence of immunoglobulin (Ig) expression in B-cell-derived Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD) was initially suggested to be caused by crippling mutations in the Ig promoter or coding region. More recent investigations have, however, challenged this concept. This study addressed the role of mutations in the Ig promoter region in HRS cells. Nine cases of cHD and 3 B-cell-derived HD lines were analyzed for mutations in the TATA box and octamer motif of the Ig promoter. Mutations in the octamer motif were found in only 1 of the 9 cases and in 1 of the 3 HD cell lines (L1236). Furthermore, in all cases either a complete lack or strong reduction in the expression of the Oct2 transcription factor and the BOB.1/OBF.1 coactivator were found. The relevance of the rare promoter mutations was investigated by assaying the activity of Ig promoter reporter constructs transfected into the HD cell line L1236, which harbors a mutated octamer motif. These Ig reporter constructs were completely inactive in L1236 cells; however, their activity could be reconstituted by the cotransfection of a BOB.1/OBF.1 expression vector. The additional transfection with an Oct2 expression vector did not further enhance the Ig promoter activity. The conclusions drawn from these results are that crippling mutations in the Ig promoter and coding region are not the sole cause for the lack of Ig expression in HRS cells and that defects in the transcription machinery such as absence of BOB.1/OBF.1 are more important for this phenomenon.

Down-regulation of BOB.1/OBF.1 and Oct2 in classical Hodgkin disease but not in lymphocyte predominant Hodgkin disease correlates with immunoglobulin transcription.

In contrast to the tumor cells (L&H cells) of lymphocyte predominant Hodgkin disease (LPHD), Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD) are unable to transcribe immunoglobulin, despite the presence of rearranged immunoglobulin genes. Although initial studies have suggested crippling immunoglobulin gene mutations to be the cause of absent immunoglobulin expression in cHD, recent work of our group has demonstrated an impaired activation of the immunoglobulin promoter as a superior mechanism. As immunoglobulin transcription is mainly regulated by the B-cell transcription factors Oct2 and BOB.1/OBF.1, we analyzed 35 cases of LPHD, 32 cases of cHD, and 2 Hodgkin disease cell lines for the expression of these transcription factors and also in parallel for immunoglobulin expression. Our results demonstrate an absence of Oct2 and/or BOB.1/OBF.1 in cHD and a striking overexpression of Oct2 in LPHD. Immunoglobulin expression was lacking in cHD but present in LPHD. Furthermore, the reintroduction of BOB.1/OBF.1 and Oct2 into cultured HRS cells restored the activity of cotransduced immunoglobulin promoter constructs. Our findings dismiss the concept that the different immunoglobulin expression in cHD and LPHD is due to disrupting mutations of immunoglobulin V genes in cHD but is most likely due to a down-regulation of Oct2 and/or BOB.1/OBF.1. This study further revealed Oct2 as a new and valuable marker for the identification of L&H cells and their distinction from HRS cells. The impairment of immunoglobulin transcription with a down-regulated synthesis of Oct2 and BOB.1/OBF.1 is the first established general recurrent defect found in HRS cells.

Hodgkin and reed-sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription.

Single cell studies aimed at clarifying the nature and clonality of Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin's disease (HD) have so far produced conflicting results. Using an improved single cell procedure, the HRS cells of 25 patients with nodular sclerosing HD lacking B- and T-cell antigens, with and without Epstein-Barr virus infection, were analyzed for the presence of immunoglobulin (Ig) gene rearrangements. One patient with HD developed follicular lymphoma 2 years later. Both lymphomas originated from a common precursor identified as a germinal center B cell. The data show that all but one of the investigated cases harbored rearranged Ig genes, which were clonal in all instances and carried a high load of somatic mutations. The Ig coding capacity was preserved in 18 of the 24 cases (75%) with rearrangements. However, expression of Ig messenger RNA was not detectable in the HRS cells with the exception of Ig kappa light chain expression in some tumor cells of 1 case. The lack of Ig gene transcription in HRS cells was confirmed by analyzing the HD cell lines L428 and KM-H2 in transient transfection experiments. An Ig promoter/enhancer reporter construct showed virtually no activity in these cells compared to 5 control B-cell lines. We conclude that (1) classical HD is a B-cell lymphoma in most instances, (2) HRS cells are clonal without any exception, (3) they are derived from germinal center B-cells that (4) mostly lack crippling mutations but (5) have consistently lost their Ig gene transcription ability, due to functional defects in the Ig gene regulatory elements. (Blood. 2000;95:1443-1450)

The BOB.1 / OBF.1 co-activator is essential for octamer-dependent transcription in B cells.

The BOB.1 / OBF.1 / OCA-B protein (henceforth designated as BOB.1 / OBF.1) is a B cell-specific co-activator of the Oct1 and Oct2 transcription factors. It is involved in mediating the transcriptional activity of these proteins. Surprisingly, animals deficient for BOB.1 / OBF.1 showed normal expression of genes that contain an octamer motif in their regulatory regions. Here we have addressed the role of BOB.1 / OBF.1 for octamer-dependent transcription. We show that promoters exclusively dependent on functional octamer motifs are completely inactive in BOB.1 / OBF. 1-deficient B cells. The lack of activity is a direct consequence of lack of the co-activator. To show this, a hormone-regulated conditional allele of BOB.1 / OBF.1 was introduced into the BOB.1 / OBF.1-deficient B cells. This resulted in the hormone-dependent transcriptional activity of octamer-dependent reporters in these cells. The BOB.1 / OBF.1 requirement for octamer promoter function was also observed when an authentic immunoglobulin kappa-promoter was assayed. BOB.1 / OBF.1 dependence could not be overcome by including the strong enhancer element from the immunoglobulin heavy chain gene. Induction of pre-B cells with lipopolysaccharide led to increased Oct2 levels but did not significantly increase octamer-dependent transcription in BOB.1 / OBF.1-deficient B cells. Thus, these results demonstrate that BOB.1 / OBF.1 itself is a non-redundant protein in B cells and absolutely required for octamer-dependent transcriptional activity.

Application of differential cDNA screening techniques to the identification of unique gene expression in tumours and lymphocytes.

Development, differentiation and cell death in all organisms are mediated by tightly regulated programs of differential gene expression. Furthermore, changes in gene expression profiles are responsible for tumour formation and tumour progression, as well as for many other human diseases. Thus, the definition of distinct patterns of gene expression is not only essential for understanding complex biological processes but also leads to the identification of novel targets for therapy of various diseases.