Yuhong Tang

Genome-wide DNA methylation patterns in CD4+ T cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus is a chronic-relapsing autoimmune disease of incompletely understood etiology. Recent evidence strongly supports an epigenetic contribution to the pathogenesis of lupus. To understand the extent and nature of dysregulated DNA methylation in lupus T cells, we performed a genome-wide DNA methylation study in CD4+ T cells in lupus patients compared to normal healthy controls. Cytosine methylation was quantified in 27,578 CG sites located within the promoter regions of 14,495 genes. We identified 236 hypomethylated and 105 hypermethylated CG sites in lupus CD4+ T cells compared to normal controls, consistent with widespread DNA methylation changes in lupus T cells. Of interest, hypomethylated genes in lupus T cells include CD9, which is known to provide potent T-cell co-stimulation signals. Other genes with known involvement in autoimmunity such as MMP9 and PDGFRA were also hypomethylated. The BST2 gene, an interferon-inducible membrane-bound protein that helps restrict the release of retroviral particles was also hypomethylated in lupus patients. Genes involved in folate biosynthesis, which plays a role in DNA methylation, were overrepresented among hypermethylated genes. In addition, the transcription factor RUNX3 was hypermethylated in patients, suggesting an impact on T-cell maturation. Protein-protein interaction maps identified a transcription factor, HNF4a, as a regulatory hub affecting a number of differentially methylated genes. Apoptosis was also an overrepresented ontology in these interaction maps. Further, our data suggest that the methylation status of RAB22A, STX1B2, LGALS3BP, DNASE1L1 and PREX1 correlates with disease activity in lupus patients.

Variants within MECP2, a key transcription regulator, are associated with increased susceptibility to lupus and differential gene expression in patients with systemic lupus erythematosus

OBJECTIVE Both genetic and epigenetic factors play an important role in the pathogenesis of lupus. The aim of this study was to examine methyl-CpG-binding protein 2 gene (MECP2) polymorphisms in a large cohort of patients with lupus and control subjects, and to determine the functional consequences of the lupus-associated MECP2 haplotype. METHODS We genotyped 18 single-nucleotide polymorphisms within MECP2, located on chromosome Xq28, in a large cohort of patients with lupus and control subjects of European descent. We studied the functional effects of the lupus-associated MECP2 haplotype by determining gene expression profiles in B cell lines in female lupus patients with and those without the lupus-associated MECP2 risk haplotype. RESULTS We confirmed, replicated, and extended the genetic association between lupus and genetic markers within MECP2 in a large independent cohort of lupus patients and control subjects of European descent (odds ratio 1.35, P = 6.65 x 10(-11)). MECP2 is a dichotomous transcription regulator that either activates or represses gene expression. We identified 128 genes that are differentially expressed in lupus patients with the disease-associated MECP2 haplotype; most ( approximately 81%) were up-regulated. Genes that were up-regulated had significantly more CpG islands in their promoter regions compared with genes that were down-regulated. Gene ontology analysis using the differentially expressed genes revealed significant association with epigenetic regulatory mechanisms, suggesting that these genes are targets for MECP2 regulation in B cells. Furthermore, at least 13 of the 104 up-regulated genes are regulated by interferon. The disease-risk MECP2 haplotype was associated with increased expression of the MECP2 transcription coactivator CREB1 and decreased expression of the corepressor histone deacetylase 1. CONCLUSION Polymorphism in the MECP2 locus is associated with lupus and, at least in part, contributes to the interferon signature observed in lupus patients.

Evidence for chronic, peripheral activation of neutrophils in polyarticular juvenile rheumatoid arthritis

Although strong epidemiologic evidence suggests an important role for adaptive immunity in the pathogenesis of polyarticular juvenile rheumatoid arthritis (JRA), there remain many aspects of the disease that suggest equally important contributions of the innate immune system. We used gene expression arrays and computer modeling to examine the function in neutrophils of 25 children with polyarticular JRA. Computer analysis identified 712 genes that were differentially expressed between patients and healthy controls. Computer-assisted analysis of the differentially expressed genes demonstrated functional connections linked to both interleukin (IL)-8- and interferon-γ (IFN-γ)-regulated processes. Of special note is that the gene expression fingerprint of children with active JRA remained essentially unchanged even after they had responded to therapy. This result differed markedly from our previously reported work, in which gene expression profiles in buffy coats of children with polyarticular JRA reverted to normal after disease control was achieved pharmacologically. These findings suggest that JRA neutrophils remain in an activated state even during disease quiescence. Computer modeling of array data further demonstrated disruption of gene regulatory networks in clusters of genes modulated by IFN-γ and IL-8. These cytokines have previously been shown to independently regulate the frequency (IFN-γ) and amplitude (IL-8) of the oscillations of key metabolites in neutrophils, including nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and superoxide ion. Using real-time, high-speed, single-cell photoimaging, we observed that 6/6 JRA patients displayed a characteristic defect in 12% to 23% of the neutrophils tested. Reagents known to induce only frequency fluctuations of NAD(P)H and superoxide ion induced both frequency and amplitude fluctuations in JRA neutrophils. This is a novel finding that was observed in children with both active (n = 4) and inactive (n = 2) JRA. A subpopulation of polyarticular JRA neutrophils are in a chronic, activated state, a state that persists when the disease is well controlled pharmacologically. Furthermore, polyarticular JRA neutrophils exhibit an intrinsic defect in the regulation of metabolic oscillations and superoxide ion production. Our data are consistent with the hypothesis that neutrophils play an essential role in the pathogenesis of polyarticular JRA.

Protection against hydrogen peroxide-induced cell death in cultured human retinal pigment epithelial cells by 17β-estradiol: A differential gene expression profile

It has been demonstrated that estrogen receptors are present in the retinal pigment epithelium (RPE)-choroids complex regardless of sex. This suggests that estrogen could play a functional role in the outer retina, especially the RPE. To gain further insights on the molecular mechanisms differentially activated by 17beta-estradiol (betaE2) in RPE cells, we investigated gene expression changes in response to betaE2 in cultured RPE cells using cDNA microarray technology. A total of 47 genes among 21,329 human genes are significantly altered in response to betaE2 treatment in RPE cells. Among these 47 altered genes, 34 are up-regulated and 13 are down-regulated by betaE2. The products of 34 genes have a known or suspected function. These functions belong to various categories, including caspases; extracellular matrix proteins; metabolism pathway components; GTP/GDP exchangers and G-protein GTPase activity modulators; transcription activators and repressors. Six genes which may contribute to the unique functions of the RPE cells have been validated by both quantitative real-time reverse transcription (RT)-PCR and semi-quantitative RT-PCR. In addition, we also demonstrated that betaE2 quenches H2O2-induced up-regulation of apoptosis-related protein, and protects RPE cell degeneration. These results indicate that estrogen regulates functions of RPE cells and is involved in the maintaining and survival of RPE cells during oxidative stress, and its deficiency during menopause period may be a factor contributing to the development of age-related macular degeneration in elderly women.

17β‐estradiol (βE2) protects human retinal Müller cell against oxidative stress in vitro: Evaluation of its effects on gene expression by cDNA microarray

17β‐estradiol (βE2) is an effective neuroprotectant against hydrogen peroxide (H2O2)‐induced retinal neuronal cell death and light‐induced photoreceptor degeneration. Müller cells are the principal macroglia responsible for supporting retinal neuronal survival, information processing and removing metabolic waste. However, the role of βE2 on human Müller cells is unclear. In this study, the effects of βE2 on human Müller cell survival and gene expression were examined. Our data revealed that βE2 is able to increase human Müller cell viability after exposure to H2O2 through inhibition of apoptosis. Microarray analysis revealed significant changes in the expression of 69 genes (total of 21,324 genes screened) in cultured human Müller cells 6 h after βE2 treatment. Four of the βE2‐responsive genes [thrombospondin 1 (TSP1), mitogen‐activated protein kinase kinase kinase 3 (MAP3K3), large conductance calcium‐activated potassium channel β2 subunit (KCNMB2), and SRY (sex‐determining region Y)‐box 11 (SOX11)] were validated by both real‐time qRT‐PCR and semi‐quantitative RT‐PCR. Interestingly, exposure of human Müller cells to βE2 increased pigment epithelium‐derived factor (PEDF) gene expression as measured by both RT‐PCR and real time qRT‐PCR. Our data demonstrate, for the first time, that βE2 protects cultured human Müller cells against H2O2‐induced cell death through the inhibition of apoptosis. This protective effect may operate through regulation of genes, such as TSP1, MAP3K3, SOX11, TSP1, and PEDF, and may in turn exert an important role in protecting retinal neurons.

Statistical monitoring of weak spots for improvement of normalization and ratio estimates in microarrays

BackgroundSeveral aspects of microarray data analysis are dependent on identification of genes expressed at or near the limits of detection. For example, regression-based normalization methods rely on the premise that most genes in compared samples are expressed at similar levels and therefore require accurate identification of nonexpressed genes (additive noise) so that they can be excluded from the normalization procedure. Moreover, key regulatory genes can maintain stringent control of a given response at low expression levels. If arbitrary cutoffs are used for distinguishing expressed from nonexpressed genes, some of these key regulatory genes may be unnecessarily excluded from the analysis. Unfortunately, no accurate method for differentiating additive noise from genes expressed at low levels is currently available.ResultsWe developed a multistep procedure for analysis of mRNA expression data that robustly identifies the additive noise in a microarray experiment. This analysis is predicated on the fact that additive noise signals can be accurately identified by both distribution and statistical analysis.ConclusionsIdentification of additive noise in this manner allows exclusion of noncorrelated weak signals from regression-based normalization of compared profiles thus maximizing the accuracy of these methods. Moreover, genes expressed at very low levels can be clearly identified due to the fact that their expression distribution is stable and distinguishable from the random pattern of additive noise.

Temporal dynamics of gene expression in the lung in a baboon model of E. coli sepsis

BackgroundBacterial invasion during sepsis induces disregulated systemic responses that could lead to fatal lung failure. The purpose of this study was to relate the temporal dynamics of gene expression to the pathophysiological changes in the lung during the first and second stages of E. coli sepsis in baboons.ResultsUsing human oligonucleotide microarrays, we have explored the temporal changes of gene expression in the lung of baboons challenged with sublethal doses of E. coli. Temporal expression pattern and biological significance of the differentially expressed genes were explored using clustering and pathway analysis software. Expression of selected genes was validated by real-time PCR. Cytokine levels in tissue and plasma were assayed by multiplex ELISA. Changes in lung ultrastructure were visualized by electron microscopy. We found that genes involved in primary inflammation, innate immune response, and apoptosis peaked at 2 hrs. Inflammatory and immune response genes that function in the stimulation of monocytes, natural killer and T-cells, and in the modulation of cell adhesion peaked at 8 hrs, while genes involved in wound healing and functional recovery were upregulated at 24 hrs.ConclusionThe analysis of gene expression modulation in response to sepsis provides the baseline information that is crucial for the understanding of the pathophysiology of systemic inflammation and may facilitate the development of future approaches for sepsis therapy.

A dynamic model of gene expression in monocytes reveals differences in immediate/early response genes between adult and neonatal cells

Neonatal monocytes display immaturity of numerous functions compared with adult cells. Gene expression arrays provide a promising tool for elucidating mechanisms underlying neonatal immune function. We used a well-established microarray to analyze differences between LPS-stimulated human cord blood and adult monocytes to create dynamic models for interactions to elucidate observed deficiencies in neonatal immune responses.We identified 168 genes that were differentially expressed between adult and cord monocytes after 45 min incubation with LPS. Of these genes, 95% (159 of 167) were over-expressed in adult relative to cord monocytes. Differentially expressed genes could be sorted into nine groups according to their kinetics of activation. Functional modelling suggested differences between adult and cord blood in the regulation of apoptosis, a finding confirmed using annexin binding assays. We conclude that kinetic studies of gene expression reveal potentially important differences in gene expression dynamics that may provide insight into neonatal innate immunity.

5α-Androstane-3α,17β-diol activates pathway that resembles the epidermal growth factor responsive pathways in stimulating human prostate cancer LNCaP cell proliferation

5α-Androstane-3α,17β-diol (3α-diol) is considered to have no androgenic effects in androgen target organs unless it is oxidized to 5α-dihydrotestosterone (5α-DHT). We used microarray and bioinformatics to identify and compare 3α-diol and 5α-DHT responsive gene in human prostate LNCaP cells. Through a procedure called ‘hypervariable determination’, a similar set of 30 responsive genes involving signal transduction, transcription regulation, and cell proliferation were selected in 5α-DHT-, 3α-diol-, and epidermal growth factor (EGF)-treated samples. F-means cluster and networking procedures showed that the responsive pattern of these genes was more closely related between 3α-diol and EGF than between 5α-DHT and 3α-diol treatments. We conclude that 3α-diol is capable of stimulating prostate cell proliferation by eliciting EGF-like pathway in conjunction with androgen receptor pathway.

Mobile classification in microarray experiments.

In a homogeneous group of samples, there are genes whose expression variations can be attributed to factors other than experimental errors. These factors can include natural biological oscillations or metabolic processes. These genes are rarely classified as ‘interesting’ based on their variability profile. However, their dynamic behaviour can tease out important clues about naturally occurring biological processes in the organism under study and can be used for group classification. Dynamical discriminate function analysis was developed on the concept that stable classification parameters (roots) can be derived from highly variable gene‐expression data. Stability of these combinations implies a strongly compensatory relationship that may divulge functional interconnections.