Daniela Ulgiati

Association of a common complement receptor 2 haplotype with increased risk of systemic lupus erythematosus

A genomic region on distal mouse chromosome 1 and its syntenic human counterpart 1q23–42 show strong evidence of harboring lupus susceptibility genes. We found evidence of linkage at 1q32.2 in a targeted genome scan of 1q21–43 in 126 lupus multiplex families containing 151 affected sibpairs (nonparametric linkage score 2.52, P = 0.006). A positional candidate gene at 1q32.2, complement receptor 2 (CR2), is also a candidate in the murine Sle1c lupus susceptibility locus. To explore its role in human disease, we analyzed 1,416 individuals from 258 Caucasian and 142 Chinese lupus simplex families and demonstrated that a common three-single-nucleotide polymorphism CR2 haplotype (rs3813946, rs1048971, rs17615) was associated with lupus susceptibility (P = 0.00001) with a 1.54-fold increased risk for the development of disease. Single-nucleotide polymorphism 1 (rs3813946), located in the 5′ untranslated region of the CR2 gene, altered transcriptional activity, suggesting a potential mechanism by which CR2 could contribute to the development of lupus. Our findings reveal that CR2 is a likely susceptibility gene for human lupus at 1q32.2, extending previous studies suggesting that CR2 participates in the pathogenesis of systemic lupus erythematosus.

Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case–control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls, P=0.016, OR=0.90 (0.82–0.98)). Two of these SNPs are in exon 10, directly 5′ of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

A Proteomics Approach for the Identification of DNA Binding Activities Observed in the Electrophoretic Mobility Shift Assay

Transcription factors lie at the center of gene regulation, and their identification is crucial to the understanding of transcription and gene expression. Traditionally, the isolation and identification of transcription factors has been a long and laborious task. We present here a novel method for the identification of DNA-binding proteins seen in electrophoretic mobility shift assay (EMSA) using the power of two-dimensional electrophoresis coupled with mass spectrometry. By coupling SDS-PAGE and isoelectric focusing to EMSA, the molecular mass and pI of a protein complex seen in EMSA were estimated. Candidate proteins were then identified on a two-dimensional array at the predetermined pI and molecular mass coordinates and identified by mass spectrometry. We show here the successful isolation of a functionally relevant transcription factor and validate the identity through EMSA supershift analysis.

TNF and Phorbol Esters Induce Lymphotoxin-β Expression through Distinct Pathways Involving Ets and NF-κB Family Members

Lymphotoxin-β (LT-β) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT-α on the cell surface to form the heterotrimeric LTα1,β2 complex, which binds the LT-β receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer’s patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT-β promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT-β promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-κB motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT-β promoter. TNF inducibility was localized to the NF-κB site positioned at −83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT-β in response to different immunological or inflammatory stimuli.

The impact of histone post-translational modifications on developmental gene regulation

Eukaryotic genomic DNA is orderly compacted to fit into the nucleus and to inhibit accessibility of specific sequences. DNA is manipulated in many different ways by bound RNA and proteins within the composite material known as chromatin. All of the biological processes that require access to genomic DNA (such as replication, recombination and transcription) therefore are dependent on the precise characteristics of chromatin in eukaryotes. This distinction underlies a fundamental property of eukaryotic versus prokaryotic gene regulation such that chromatin structure must be regulated to precisely repress or relieve repression of particular regions of the genome in an appropriate spatio-temporal manner. As well as playing a key role in structuring genomic DNA, histones are subject to site-specific modifications that can influence the organization of chromatin structure. This review examines the molecular processes regulating site-specific histone acetylation, methylation and phosphorylation with an emphasis on how these processes underpin differentiation-regulated transcription.

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.

The Role of Sp Family Members, Basic Krupple-Like Factor, and E Box Factors in the Basal and IFN-γ Regulated Expression of the Human Complement C4 Promoter

The fourth component of human complement (C4) is a serum protein that is expressed in the liver and other organs. The promoter region of the C4 gene has been analyzed in reporter gene assays in two cell lines that represent hepatic (HepG2) and monocytic (U937) lineages. Analysis indicated that regions important for basal transcription in HepG2 cells included Sp1 and E box sites within the first 100 bp upstream of the transcription initiation site but not the nuclear factor-1 site important in the control of the mouse C4 gene. Also, a region encompassing −468 to −310 was able to repress activity 2-fold. However, when a CACCC or GT box sequence at −140 was mutated the repressive activity of the upstream region resulted in almost no activity. The −140 region consists of a series of four closely positioned GT boxes that were shown to bind Sp1, Sp3, and basic Krupple-like factor in EMSA. This novel two-part regulatory element may be involved in the regulated expression of C4. However, IFN-γ a major activator of C4 expression did not signal through this two-part regulatory element. We were able to map the position of an IFN-γ responsive element in U937. IFN-γ was able to increase transcription by up to 20-fold with mutations in the E box sequence at −78 to −73, thus completely abolishing induction. We conclude that the E box binding factors, which appear to be distinct from upstream stimulatory factors 1 and 2, are totally responsible for IFN-γ induction of C4.

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.

The role of notch signaling in the development of a normal B-cell repertoire

The notch signaling pathway is evolutionarily conserved across the animal kingdom and regulates developmental ‘decisions’, such as cell fate commitment, differentiation, proliferation and apoptosis. In the mammalian immune system, notch signaling events have been extensively studied during T lymphopoiesis, and have a role both during early development, as well as differentiation into discreet effector cell compartments. In contrast, the impact of notch signaling in the B‐cell compartment is less obvious. It is clear that notch signaling is crucial to generate the marginal zone B‐cell population located within the spleen; however, the full effects of notch signaling during normal B‐cell development remain unresolved. Nevertheless, there is compelling evidence that notch signaling regulates multiple stages of B‐cell differentiation and in shaping the antibody repertoire; however, the molecular details have not been elucidated. This review explores the relationship between notch signaling and B‐cell development with attention to how these processes contribute to a normal B‐cell repertoire.

Cell- and stage-specific chromatin structure across the Complement receptor 2 (CR2/CD21) promoter coincide with CBF1 and C/EBP-β binding in B cells

Stringent developmental transcription requires multiple transcription factor (TF) binding sites, cell-specific expression of signaling molecules, TFs and co-regulators and appropriate chromatin structure. During B-lymphopoiesis, human Complement receptor 2 (CR2/CD21) is detected on immature and mature B cells but not on B cell precursors and plasma cells. We examined cell- and stage-specific human CR2 gene regulation using cell lines modeling B-lymphopoiesis. Chromatin accessibility assays revealed a region between -409 and -262 with enhanced accessibility in mature B cells and pre-B cells, compared to either non-lymphoid or plasma cell-types, however, accessibility near the transcription start site (TSS) was elevated only in CR2-expressing B cells. A correlation between histone acetylation and CR2 expression was observed, while histone H3K4 dimethylation was enriched near the TSS in both CR2-expressing B cells and non-expressing pre-B cells. Candidate sites within the CR2 promoter were identified which could regulate chromatin, including a matrix attachment region associated with CDP, SATB1/BRIGHT and CEBP-beta sites as well as two CBF1 sites. ChIP assays verified that both CBF1 and C/EBP-beta bind the CR2 promoter in B cells raising the possibility that these factors facilitate or respond to alterations in chromatin structure to control the timing and/or level of CR2 transcription.