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Polycomb group (PcG) proteins are able to maintain the memory of silent transcriptional states of homeotic genes throughout development. In Drosophila, they form multimeric complexes that bind to specific DNA regulatory elements named PcG response elements (PREs). To date, few PREs have been identified and the chromosomal distribution of PcG proteins during development is unknown. We used chromatin immunoprecipitation (ChIP) with genomic tiling path microarrays to analyze the binding profile of the PcG proteins Polycomb (PC) and Polyhomeotic (PH) across 10 Mb of euchromatin. We also analyzed the distribution of GAGA factor (GAF), a sequence-specific DNA binding protein that is found at most previously identified PREs. Our data show that PC and PH often bind to clustered regions within large loci that encode transcription factors which play multiple roles in developmental patterning and in the regulation of cell proliferation. GAF co-localizes with PC and PH to a limited extent, suggesting that GAF is not a necessary component of chromatin at PREs. Finally, the chromosome-association profile of PC and PH changes during development, suggesting that the function of these proteins in the regulation of some of their target genes might be more dynamic than previously anticipated.

Kif23 kinesin is an essential actor of cytokinesis in animals. It exists as two major isoforms, known as MKLP1 and CHO1, the longest of which, CHO1, contains two HXRXXS/T NDR/LATS kinase consensus sites. We demonstrate that these two sites are readily phosphorylated by NDR and LATS kinases in vitro, and this requires the presence of an upstream -5 histidine residue. We further show that these sites are phosphorylated in vivo and provide evidence revealing that LATS1,2 participate in the phosphorylation of the most C-terminal S814 site, present on both isoforms. This S814 phosphosite was previously reported to constitute a 14-3-3 binding site, which plays a role in Kif23 clustering during cytokinesis. Surprisingly, we found that phosphorylation of the upstream S716 NDR/LATS consensus site, present only in the longest Kif23 isoform, is required for efficient phosphorylation at S814, thus revealing sequential phosphorylation at these two sites, and differential regulation of Kif23-14-3-3 interaction for the two Kif23 isoforms. Finally, we provide evidence that Kif23 is largely unphosphorylated on S814 in post-abscission midbodies, making this Kif23 post-translational modification a potential marker to probe these structures.

Background One essential step in the massive analysis of transcriptomic profiles is the calculation of the correlation coefficient, a value used to select pairs of genes with similar or inverse transcriptional profiles across a large fraction of the biological conditions examined. Until now, the choice between the two available methods for calculating the coefficient has been dictated mainly by technological considerations. Specifically, in analyses based on double-channel techniques, researchers have been required to use covariation correlation, i.e. the correlation between gene expression changes measured between several pairs of biological conditions, expressed for example as fold-change. In contrast, in analyses of single-channel techniques scientists have been restricted to the use of coexpression correlation, i.e. correlation between gene expression levels. To our knowledge, nobody has ever examined the possible benefits of using covariation instead of coexpression in massive analyses of single channel microarray results. Results We describe here how single-channel techniques can be treated like double-channel techniques and used to generate both gene expression changes and covariation measures. We also present a new method that allows the calculation of both positive and negative correlation coefficients between genes. First, we perform systematic comparisons between two given biological conditions and classify, for each comparison, genes as increased (I), decreased (D), or not changed (N). As a result, the original series of n gene expression level measures assigned to each gene is replaced by an ordered string of n(n-1)/2 symbols, e.g. IDDNNIDID...DNNNNNNID, with the length of the string corresponding to the number of comparisons. In a second step, positive and negative covariation matrices (CVM) are constructed by calculating statistically significant positive or negative correlation scores for any pair of genes by comparing their strings of symbols. Conclusion This new method, applied to four different large data sets, has allowed us to construct distinct covariation matrices with similar properties. We have also developed a technique to translate these covariation networks into graphical 3D representations and found that the local assignation of the probe sets was conserved across the four chip set models used which encompass three different species (humans, mice, and rats). The application of adapted clustering methods succeeded in delineating six conserved functional regions that we characterized using Gene Ontology information.

The Ume6 transcription factor in yeast is known to both repress and activate expression of diverse genes during growth and meiotic development. To obtain a more complete profile of the functions regulated by this protein, microarray analysis was used to examine transcription in wild-type and ume6Δ diploids during vegetative growth in glucose and acetate. Two different genetic backgrounds (W303 and SK1) were examined to identify a core set of strain-independent Ume6-regulated genes. Among genes whose expression is controlled by Ume6 in both backgrounds, 82 contain homologies to the Ume6-binding site (URS1) and are expected to be directly regulated by Ume6. The vast majority of those whose functions are known participate in carbon/nitrogen metabolism and/or meiosis. Approximately half of the Ume6 direct targets are induced during meiosis, with most falling into the early meiotic expression class (cluster 4), and a smaller subset in the middle and later classes (clusters 5-7). Based on these data, we propose that Ume6 serves a unique role in diploid cells, coupling metabolic responses to nutritional cues with the initiation and progression of meiosis. Finally, expression patterns in the two genetic backgrounds suggest that SK1 is better adapted to respiration and W303 to fermentation, which may in part account for the more efficient and synchronous sporulation of SK1.

Background The usefulness of the data from Affymetrix microarray analysis depends largely on the reliability of the files describing the correspondence between probe sets, genes and transcripts. Particularly, when a gene is targeted by several probe sets, these files should give information about the similarity of each alternative probe set pair. Transcriptional networks integrate the multiple correlations that exist between all probe sets and supply much more information than a simple correlation coefficient calculated for two series of signals. In this study, we used the PSAWN (Probe Set Assignment With Networks) programme we developed to investigate whether similarity of alternative probe sets resulted in some specific properties. Findings PSAWNpy delivered a full textual description of each probe set and information on the number and properties of secondary targets. PSAWNml calculated the similarity of each alternative probe set pair and allowed finding relationships between similarity and localisation of probes in common transcripts or exons. Similar alternative probe sets had very low negative correlation, high positive correlation and similar neighbourhood overlap. Using these properties, we devised a test that allowed grouping similar probe sets in a given network. By considering several networks, additional information concerning the similarity reproducibility was obtained, which allowed defining the actual similarity of alternative probe set pairs. In particular, we calculated the common localisation of probes in exons and in known transcripts and we showed that similarity was correctly correlated with them. The information collected on all pairs of alternative probe sets in the most popular 3’ IVT Affymetrix chips is available in tabular form at http://bns.crbm.cnrs.fr/download.html . Conclusions These processed data can be used to obtain a finer interpretation when comparing microarray data between biological conditions. They are particularly well adapted for searching 3’ alternative poly-adenylation events and can be also useful for studying the structure of transcriptional networks. The PSAWNpy, (in Python) and PSAWNml (in Matlab) programmes are freely available and can be downloaded at http://code.google.com/p/arraymatic . Tutorials and reference manuals are available at BMC Research Notes online (Additional file 1) or from http://bns.crbm.cnrs.fr/softwares.html .

Small RGK GTPases, Rad, Gem, Rem1, and Rem2, are potent inhibitors of high-voltage-activated (HVA) Ca 2+ channels expressed in heterologous expression systems. However, the role of this regulation has never been clearly demonstrated in the nervous system. Using transcriptional analysis, we show that peripheral nerve injury specifically upregulates Gem in mice dorsal root ganglia. Following nerve injury, protein expression was increased in ganglia and peripheral nerve, mostly under its phosphorylated form. This was confirmed in situ and in vitro in dorsal root ganglia sensory neurons. Knockdown of endogenous Gem, using specific small-interfering RNA (siRNA), increased the HVA Ca 2+ current only in the large-somatic-sized neurons. Combining pharmacological analysis of the HVA Ca 2+ currents together with Gem siRNA-transfection of larger sensory neurons, we demonstrate that only the P/Q-type Ca 2+ channels were enhanced. In vitro analysis of Gem affinity to various Ca V βx-Ca V 2.x complexes and immunocytochemical studies of Gem and Ca V β expression in sensory neurons suggest that the specific inhibition of the P/Q channels relies on both the regionalized upregulation of Gem and the higher sensitivity of the endogenous Ca V 2.1-Ca V β4 pair in a subset of sensory neurons including the proprioceptors. Finally, pharmacological inhibition of P/Q-type Ca 2+ current reduces neurite branching of regenerating axotomized neurons. Taken together, the present results indicate that a Gem-dependent P/Q-type Ca 2+ current inhibition may contribute to general homeostatic mechanisms following a peripheral nerve injury.

Small RGK GTPases, Rad, Gem, Rem1, and Rem2, are potent inhibitors of high-voltage-activated (HVA) Ca^sup 2+^ channels expressed in heterologous expression systems. However, the role of this regulation has never been clearly demonstrated in the nervous system. Using transcriptional analysis, we show that peripheral nerve injury specifically upregulates Gem in mice dorsal root ganglia. Following nerve injury, protein expression was increased in ganglia and peripheral nerve, mostly under its phosphorylated form. This was confirmed in situ and in vitro in dorsal root ganglia sensory neurons. Knockdown of endogenous Gem, using specific small-interfering RNA (siRNA), increased the HVA Ca^sup 2+^ current only in the large-somatic-sized neurons. Combining pharmacological analysis of the HVA Ca^sup 2+^ currents together with Gem siRNA-transfection of larger sensory neurons, we demonstrate that only the P/Q-type Ca^sup 2+^ channels were enhanced. In vitro analysis of Gem affinity to various Ca^sub V^[beta]x-Ca^sub V^2.x complexes and immunocytochemical studies of Gem and Ca^sub V^[beta] expression in sensory neurons suggest that the specific inhibition of the P/Q channels relies on both the regionalized upregulation of Gem and the higher sensitivity of the endogenous Ca^sub V^2.1-Ca^sub V^[beta]4 pair in a subset of sensory neurons including the proprioceptors. Finally, pharmacological inhibition of P/Q-type Ca^sup 2+^ current reduces neurite branching of regenerating axotomized neurons. Taken together, the present results indicate that a Gem-dependent P/Q-type Ca^sup 2+^ current inhibition may contribute to general homeostatic mechanisms following a peripheral nerve injury.

Small RGK GTPases, Rad, Gem, Rem1, and Rem2, are potent inhibitors of high-voltage-activated (HVA) Ca super(2+) channels expressed in heterologous expression systems. However, the role of this regulation has never been clearly demonstrated in the nervous system. Using transcriptional analysis, we show that peripheral nerve injury specifically upregulates Gem in mice dorsal root ganglia. Following nerve injury, protein expression was increased in ganglia and peripheral nerve, mostly under its phosphorylated form. This was confirmed in situ and in vitro in dorsal root ganglia sensory neurons. Knockdown of endogenous Gem, using specific small-interfering RNA (siRNA), increased the HVA Ca super(2+) current only in the large-somatic-sized neurons. Combining pharmacological analysis of the HVA Ca super(2+) currents together with Gem siRNA-transfection of larger sensory neurons, we demonstrate that only the P/Q-type Ca super(2+) channels were enhanced. In vitro analysis of Gem affinity to various Ca sub(V) beta x-Ca sub(V)2.x complexes and immunocytochemical studies of Gem and Ca sub(V) beta expression in sensory neurons suggest that the specific inhibition of the P/Q channels relies on both the regionalized upregulation of Gem and the higher sensitivity of the endogenous Ca sub(V)2.1-Ca sub(V) beta 4 pair in a subset of sensory neurons including the proprioceptors. Finally, pharmacological inhibition of P/Q-type Ca super(2+) current reduces neurite branching of regenerating axotomized neurons. Taken together, the present results indicate that a Gem-dependent P/Q-type Ca super(2+) current inhibition may contribute to general homeostatic mechanisms following a peripheral nerve injury.

The honeybee is a model system to study learning and memory, and Ca^sup 2+^ signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca^sup 2+^ channel subunit. We identified two splice variants of the Apis Ca^sub V^[beta] Ca^sup 2+^ channel subunit (Am-Ca^sub V^[beta]) and demonstrated expression in muscle and neurons. Although AmCa^sub V^[beta] shares with vertebrate Ca^sub V^[beta] subunits the SH3 and GK domains, it beholds a unique N terminus that is alternatively spliced in the first exon to produce a long (a) and short (b) variant. When expressed with the Ca^sub V^2 channels both, AmCa^sub V^[beta]a and AmCa^sub V^[beta]b, increase current amplitude, shift the voltage-sensitivity of the channel, and slow channel inactivation as the vertebrate Ca^sub V^[beta]^sub 2a^ subunit does. However, as opposed to Ca^sub V^[beta]^sub 2a^, slow inactivation induced by Am-Ca^sub V^[beta]a was insensitive to palmitoylation but displayed a unique PI3K sensitivity. Inactivation produced by the b variant was PI3K-insensitive but staurosporine/H89-sensitive. Deletion of the first exon suppressed the sensitivity to PI3K inhibitors, staurosporine, or H89. Recording of Ba^sup 2+^ currents in Apis neurons or muscle cells evidenced a sensitivity to PI3K inhibitors and H89, suggesting that both AmCa^sub V^[beta] variants may be important to couple cell signaling to Ca^sup 2+^ entry in vivo. Functional interactions with phospho-inositide and identification of phosphorylation sites in AmCa^sub V^[beta]a and AmCa^sub V^[beta]b N termini, respectively, suggest that AmCa^sub V^[beta] splicing promoted two novel and alternative modes of regulation of channel activity with specific signaling pathways. This is the first description of a splicing-dependent kinase switch in the regulation of Ca^sup 2+^ channel activity by Ca^sub V^[beta] subunit.[PUBLICATION ABSTRACT]

The honeybee is a model system to study learning and memory, and Ca2+ signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca2+ channel subunit. We identified two splice variants of the Apis CaVβ Ca2+ channel subunit (Am-CaVβ) and demonstrated expression in muscle and neurons. Although AmCaVβ shares with vertebrate CaVβ subunits the SH3 and GK domains, it beholds a unique N terminus that is alternatively spliced in the first exon to produce a long (a) and short (b) variant. When expressed with the CaV2 channels both, AmCaVβa and AmCaVβb, increase current amplitude, shift the voltage-sensitivity of the channel, and slow channel inactivation as the vertebrate CaVβ2a subunit does. However, as opposed to CaVβ2a, slow inactivation induced by Am-CaVβa was insensitive to palmitoylation but displayed a unique PI3K sensitivity. Inactivation produced by the b variant was PI3K-insensitive but staurosporine/H89-sensitive. Deletion of the first exon suppressed the sensitivity to PI3K inhibitors, staurosporine, or H89. Recording of Ba2+ currents in Apis neurons or muscle cells evidenced a sensitivity to PI3K inhibitors and H89, suggesting that both AmCaVβ variants may be important to couple cell signaling to Ca2+ entry in vivo. Functional interactions with phospho-inositide and identification of phosphorylation sites in AmCaVβa and AmCaVβb N termini,respectively, suggest that AmCaVβ splicing promoted two novel and alternative modes of regulation of channel activity with specific signaling pathways. This is the first description of a splicing-dependent kinase switch in the regulation of Ca2+ channel activity by CaVβ subunit.