DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription.DOI: http://dx.doi.org/10.7554/eLife.00726.001

To monitor severe acute respiratory syndrome (SARS) infection, a coronavirus protein microarray that harbors proteins from SARS coronavirus (SARS-CoV) and five additional coronaviruses was constructed. These microarrays were used to screen Ϸ400 Canadian sera from the SARS outbreak, including samples from confirmed SARS-CoV cases, respiratory illness patients, and healthcare professionals. A computer algorithm that uses multiple classifiers to predict samples from SARS patients was developed and used to predict 206 sera from Chinese fever patients. The test assigned patients into two distinct groups: those with antibodies to SARSCoV and those without. The microarray also identified patients with sera reactive against other coronavirus proteins. Our results correlated well with an indirect immunofluorescence test and demonstrated that viral infection can be monitored for many months after infection. We show that protein microarrays can serve as a rapid, sensitive, and simple tool for large-scale identification of viral-specific antibodies in sera.infectious disease ͉ protein chip ͉ virus diagnostics

Altered DNA methylation status is associated with human diseases and cancer; however, the underlying molecular mechanisms remain elusive. We previously identified many human transcription factors, including Krüppel-like factor 4 (KLF4), as sequence-specific DNA methylation readers that preferentially recognize methylated CpG (mCpG), here we report the biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. We show that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. In-depth mechanistic studies reveal that recruitment of KLF4 to the methylated cis-regulatory elements of these genes result in chromatin remodeling and transcription activation. Our study demonstrates a new paradigm of DNA methylation-mediated gene activation and chromatin remodeling, and provides a general framework to dissect the biological functions of DNA methylation readers and effectors.DOI: http://dx.doi.org/10.7554/eLife.20068.001

A protein microarray representing 149 Yersinia pestis proteins was developed to profile antibody responses in EV76-immunized rabbits. Antibodies to 50 proteins were detected. There are 11 proteins besides F1 and V antigens to which the predominant antibody response occurred, and these proteins show promise for further evaluation as candidates for subunit vaccines and/or diagnostic antigens.Plague, one of the most dangerous diseases, is caused by Yersinia pestis. The increasing possibility of antibiotic-resistant Y. pestis strains means that a vaccine effective against bubonic and pneumonic plague is urgently needed (12,14,18). The current interest is in developing plague vaccines that consist of purified protein subunits, with improved protection and reduced side effects (25,31,34). The F1 or V single-subunit vaccine and the F1 plus V combination vaccine have been shown to provide effective protection against bubonic and pneumonic plague in animal models (1,31,34). There may still exist other Y. pestis antigens that provide protection. These novel vaccine candidates in conjunction with F1 and V can be developed as multicomponent subunit vaccines (21). It may improve protection against F1-and/or V-antigen mutant but virulent strains. Therefore, evaluation of Y. pestis proteins beside F1 and V for their efficacy in inducing specific antibody in the infected animals or human patients is urgently needed for plague vaccine development.Genomic sequences of Y. pestis CO92 (27), KIM (9), and 91001 (30) have been released in the past 3 years. Decoding of whole-genome sequence provides unprecedented opportunities for vaccine design. The microarray immobilized with multiple antigens, using a simple fluorescence analysis, allows high-throughput parallel detection and quantification of multiple specific antibodies in a miniaturized, low-sample-consumption format. In this study, a microarray representing 149 Y. pestis proteins was developed to profile the serum antibodies of rabbits that were immunized with live plague vaccine, providing an overall picture of the immunogenicity of the proteins tested.Construction of an antigen microarray representing 149 Y. pestis proteins. In our present work, 202 genes were selected for cloning and expression (additional information is available at http://bioinflab.org/journals/ruifu/supplementary_TableS1 .pdf/). The digested PCR products of specific genes were cloned into expression plasmid vector pET-32a (Novagen), and recombinant plasmids were transformed into BL21(DE3) cells. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, 172 genes were successfully expressed in Escherichia coli. The expressed proteins were subjected to purification in a 96-well format using Ni-NAT agarose (QIA-GEN) according to Brauns' method with a few modifications (5). For quality control, the purified proteins were printed onto silylated glass slides (CEL) and incubated with Cy5-labeled antibody specific for the six-His tag. Only the proteins giving a signal-to-background ratio of ...

Calcium channel subunits, including CACNA1C, have been associated with multiple psychiatric disorders. Specifically, genome wide association studies (GWAS) have repeatedly identified the single nucleotide polymorphism (SNP) rs1006737 in intron 3 of CACNA1C to be strongly associated with schizophrenia and bipolar disorder. Here, we show that rs1006737 marks a quantitative trait locus for CACNA1C transcript levels. We test 16 SNPs in high linkage disequilibrium with rs1007637 and find one, rs4765905, consistently showing allele-dependent regulatory function in reporter assays. We find allele-specific protein binding for 13 SNPs including rs4765905. Using protein microarrays, we identify several proteins binding ≥3 SNPs, but not control sequences, suggesting possible functional interactions and combinatorial haplotype effects. Finally, using circular chromatin conformation capture, we show interaction of the disease-associated region including the 16 SNPs with the CACNA1C promoter and other potential regulatory regions. Our results elucidate the pathogenic relevance of one of the best-supported risk loci for schizophrenia and bipolar disorder.

Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver with a poorly understood etiology. Detection of non-organ-specific and liver-related autoantibodies using immunoserological approaches has been widely used for diagnosis and prognosis. However, unambiguous and accurate detection of the disease requires the identification and characterization of disease-specific autoantigens. In the present study, we have profiled the autoantigen repertoire of patients with AIH versus those with other liver diseases, identifying and validating three novel and highly specific biomarkers for AIH. In Phase I we fabricated a human protein chip of 5,011 non-redundant proteins and used it to quickly identify 11 candidate autoantigens with relative small serum collection. In Phase II we fabricated an AIH-specific protein chip and obtained autoimmunogenic profiles of serum samples from 44 AIH patients, 50 healthy controls, and 184 additional patients suffering from hepatitis B, hepatitis C, systemic lupus erythematosus, primary Sjögren’s syndrome, rheumatoid arthritis, or primary biliary cirrhosis. Using this two-phase approach, we identified three new antigens, RPS20, Alba-like, and dUTPase, as highly AIH-specific biomarkers, with sensitivities of 47.5% (RPS20), 45.5% (Alba-like), and 22.7% (dUTPase). These potential biomarkers were further validated with additional AIH samples in a double-blind design. Finally, we demonstrated that these new biomarkers could be readily applied to ELISA-based assays for use in clinical diagnosis/prognosis.

Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites, and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems.

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