The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. Their distinctive feature is a conserved 500 amino acid semaphorin domain, a ligand-receptor interaction module also present in plexins and scatter-factor receptors. We report the crystal structure of a secreted 65 kDa form of Semaphorin-3A (Sema3A), containing the full semaphorin domain. Unexpectedly, the semaphorin fold is a variation of the beta propeller topology. Analysis of the Sema3A structure and structure-based mutagenesis data identify the neuropilin binding site and suggest a potential plexin interaction site. Based on the structure, we present a model for the initiation of semaphorin signaling and discuss potential similarities with the signaling mechanisms of other beta propeller cell surface receptors, such as integrins and the LDL receptor.

IntroductionBalanced translocations that lead to expression of aberrant fusion proteins are a hallmark of acute leukemias. 1 Many of these fusion proteins function as aberrant transcription factors that are an initiating event in leukemogenesis. 2 Some translocations, eg those involving the MLL gene can lead to phenotypically diverse forms of leukemia, whereas the t(15;17) uniformly leads to acute promyelocytic leukemia. The reasons why some translocations are leukemia-type specific, whereas others are not, is unknown.Histone modifications such as methylation of histone H3 at lysine 4 and 9 and acetylation of histone H3 are closely linked to the transcriptional activation status. In t(15;17), the chimeric PML-RAR␣ fusion protein has been shown to recruit corepressors such as DAXX, histone deacetylase (HDAC) activity, DNA methyltransferase activity, and the SUV39H1 histone methyltransferase to RAR␤2, the most extensively studied target gene. [3][4][5][6][7] PML-RAR␣ also interacts with histone deacetylase 1. 8 PML-RAR␣ homodimerization has been shown to relax the relatively stringent RAR␣ DNA binding specificity. 9,10 This gain of function is supposed to lead to many additional genomic binding sites that are not well defined. As a consequence, virtually all direct genomic targets of PML-RAR␣ are currently unknown. In addition, on a global level, the mechanistic alterations occurring at PML-RAR␣ target genes remain to be clarified.On a phenotypic level, the PML-RAR␣ fusion protein blocks differentiation and apoptosis and enhances self-renewal. 11,12 In mouse models, PML-RAR␣ induces a disease similar to acute promyelocytic leukemia (APL). 13,14 Microarray analyses elucidated several leukemogenic mechanisms and pathways. [15][16][17][18][19] For example, PML-RAR␣ induces activation of the Wnt signaling pathway. 17 Also, PML-RAR␣ alters the apoptotic response and expression of differentiation genes. 15 However, these studies do not distinguish between direct and indirect effects on gene expression. This knowledge is crucial to understand the mechanistic implications of PML-RAR␣ and to elucidate the reasons for the unique phenotype that is associated with its activities.The possibilities to understand transcription factor functions has recently been significantly improved by genome-wide approaches that identify target genes in vivo using Chromatin immunoprecipitation (ChIP)-Chip approaches. 20,21 In addition, the ability to use this method to map epigenetic modifications such as histone acetylation at promoters known to bind the transcription factor allows for the identification of the functional consequences of transcription factor binding to its genomic targets. 22,23 Using ChIP-chip analyses, we identified 372 direct PML-RAR␣ genomic targets and show that PML-RAR␣ induces heterochromatin formation on virtually all of its identified target genes. Several of the identified genes are known tumor suppressors and for one of the novel genes (S100P), we show a potential role in the PML-RAR␣-associated block in differenti...

Most current microarray oligonucleotide probe design strategies are based on probe design factors (PDFs), which include probe hybridization free energy (PHFE), probe minimum folding energy (PMFE), dimer score, hairpin score, homology score and complexity score. The impact of these PDFs on probe performance was evaluated using four sets of microarray comparative genome hybridization (aCGH) data, which included two array manufacturing methods and the genomes of two species. Since most of the hybridizing DNA is equimolar in CGH data, such data are ideal for testing the general hybridization properties of almost all candidate oligonucleotides. In all our data sets, PDFs related to probe secondary structure (PMFE, hairpin score and dimer score) are the most significant factors linearly correlated with probe hybridization intensities. PHFE, homology and complexity score are correlating significantly with probe specificities, but in a non-linear fashion. We developed a new PDF, pseudo probe binding energy (PPBE), by iteratively fitting dinucleotide positional weights and dinucleotide stacking energies until the average residue sum of squares for the model was minimized. PPBE showed a better correlation with probe sensitivity and a better specificity than all other PDFs, although training data are required to construct a PPBE model prior to designing new oligonucleotide probes. The physical properties that are measured by PPBE are as yet unknown but include a platform-dependent component. A practical way to use these PDFs for probe design is to set cutoff thresholds to filter out bad quality probes. Programs and correlation parameters from this study are freely available to facilitate the design of DNA microarray oligonucleotide probes.

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