Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.

The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

IntroductionCommon variable immunodeficiency (CVID) is the most common primary immunodeficiency in adults. 1 Recurrent bacterial infections of the respiratory tract are the clinical hallmark present in nearly all patients. 2 In addition, up to 40% of the patients show gastrointestinal disease, concomitant lymphoproliferative disorders, autoimmune phenomena, or granulomatous inflammation. 2 The pathogenic understanding of antibody deficiency in humans has always been hampered by the great heterogeneity of the syndrome. 3 In 1966, Rosen and Janeway started to group antibody deficiencies by their mode of inheritance. 4 In 1973, Cooper included the clinical course and serum immunoglobulin levels, thereby separating hyper-IgM syndromes and selective IgA deficiency. 5 The remaining group of still very heterogeneous antibody deficiencies was termed CVID. Consecutive attempts to subclassify CVID by B-cell function in vitro 6,7 failed to reach diagnostic acceptance because of laborious and poorly standardized procedures and a lack of clinical relevance.In 2002, we and others suggested a flow cytometric classification of CVID according to the B-cell phenotype. 8,9 The abnormalities of circulating B cells in patients with CVID had already been recognized earlier, 10 but only with the ease and the broad availability of flow cytometry was a widespread and systematic analysis of these aberrations possible. The Freiburg classification divided patients into 3 groups by analyzing the expression of IgM, IgD, CD27 and CD21. 8 Group 1 was characterized by a severe reduction of switched memory B cells (IgD Ϫ IgM Ϫ CD27 ϩ less than 0.4% of lymphocytes), while group 2 representing 25% of the analyzed CVID patients exhibited nearly normal numbers of class-switched memory B cells, suggesting a post germinal center defect. The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Methods PatientsAll patients were diagnosed as having CVID based on the European Society for Immunodeficiencies/Pan-American Group for Immunodeficiency (ESID/PAGID) criteria, 11 including a marked decrease of IgG (at least 2 standard deviations [SDs] below the mean for age) and a marked decrease in at least one of the isotypes IgM or IgA, the onset of clinical significant immunodeficiency at greater than 2 years of age, and the exclusion of defined causes of hypogammaglobulinemia (see also www.esid.org). Not all patients have been evaluated for absent isohemagglutinins and/or poor response to vaccines. For the final evaluation of B-cell phenotyping, the following exclusion criteria were adopted: patients younger than 6 years of age at the time of flowcytometric evaluation, patients on immunosuppressive treatment, patients suffering currently from malignancies, and patients with less than 1% peripheral B cells. Altogether, 303 patients of origi...