70 research outputs found
Discriminating nucleosomes containing histone H2A.Z or H2A based on genetic and epigenetic information
Detection of prokaryotic promoters from the genomic distribution of hexanucleotide pairs
BACKGROUND: In bacteria, sigma factors and other transcriptional regulatory proteins recognize DNA patterns upstream of their target genes and interact with RNA polymerase to control transcription. As a consequence of evolution, DNA sequences recognized by transcription factors are thought to be enriched in intergenic regions (IRs) and depleted from coding regions of prokaryotic genomes. RESULTS: In this work, we report that genomic distribution of transcription factors binding sites is biased towards IRs, and that this bias is conserved amongst bacterial species. We further take advantage of this observation to develop an algorithm that can efficiently identify promoter boxes by a distribution-dependent approach rather than a direct sequence comparison approach. This strategy, which can easily be combined with other methodologies, allowed the identification of promoter sequences in ten species and can be used with any annotated bacterial genome, with results that rival with current methodologies. Experimental validations of predicted promoters also support our approach. CONCLUSION: Considering that complete genomic sequences of over 1000 bacteria will soon be available and that little transcriptional information is available for most of them, our algorithm constitutes a promising tool for the prediction of promoter sequences. Importantly, our methodology could also be adapted to identify DNA sequences recognized by other regulatory proteins
PCRTiler: automated design of tiled and specific PCR primer pairs
Efficiency and specificity of PCR amplification is dependent on several parameters, such as amplicon length, as well as hybridization specificity and melting temperature of primer oligonucleotides. Primer design is thus of critical importance for the success of PCR experiments, but can be a time-consuming and repetitive task, for example when large genomic regions are to be scanned for the presence of a protein of interest by chromatin immunoprecipitation experiments. We present here a webserver that allows the automated design of tiled primer pairs for any number of genomic loci. PCRTiler splits the target DNA sequences into smaller regions, and identifies candidate primers for each sub-region by running the well-known program Primer3 followed by the elimination of primers with a high cross-hybridization potential via BLAST. Tiling density and primer characteristics are specified by the user via a simple and user-friendly interface. The webserver can be accessed at http://pcrtiler.alaingervais.org:8080/PCRTiler. Additionally, users may download a standalone Java-based implementation of this software. Experimental validation of PCRTiler has demonstrated that it produces correct results. We have tiled a region of the human genome, in which 96 of 123 primer pairs worked in the first attempt, and 105 of 123 (85%) could be made to work by optimizing the conditions of the PCR assay
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A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination
Abstract RPA-coated single-stranded DNA (RPA–ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA–ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR
Variant Histone H2A.Z Is Globally Localized to the Promoters of Inactive Yeast Genes and Regulates Nucleosome Positioning
H2A.Z is an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability. The mechanism(s) by which H2A.Z regulates these various biological functions remains poorly defined, in part due to the lack of knowledge regarding its physical location along chromosomes and the bearing it has in regulating chromatin structure. Here we mapped H2A.Z across the yeast genome at an approximately 300-bp resolution, using chromatin immunoprecipitation combined with tiling microarrays. We have identified 4,862 small regions—typically one or two nucleosomes wide—decorated with H2A.Z. Those “Z loci” are predominantly found within specific nucleosomes in the promoter of inactive genes all across the genome. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning at the GAL1 promoter. Within HZAD domains, the regions where H2A.Z shows an antisilencing function, H2A.Z is localized in a wider pattern, suggesting that the variant histone regulates a silencing and transcriptional activation via different mechanisms. Our data suggest that the incorporation of H2A.Z into specific promoter-bound nucleosomes configures chromatin structure to poise genes for transcriptional activation. The relevance of these findings to higher eukaryotes is discussed
Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements†
The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein–DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process
The Euchromatic and Heterochromatic Landscapes Are Shaped by Antagonizing Effects of Transcription on H2A.Z Deposition
A role for variant histone H2A.Z in gene expression is now well established but little is known about the mechanisms by which it operates. Using a combination of ChIP–chip, knockdown and expression profiling experiments, we show that upon gene induction, human H2A.Z associates with gene promoters and helps in recruiting the transcriptional machinery. Surprisingly, we also found that H2A.Z is randomly incorporated in the genome at low levels and that active transcription antagonizes this incorporation in transcribed regions. After cessation of transcription, random H2A.Z quickly reappears on genes, demonstrating that this incorporation utilizes an active mechanism. Within facultative heterochromatin, we observe a hyper accumulation of the variant histone, which might be due to the lack of transcription in these regions. These results show how chromatin structure and transcription can antagonize each other, therefore shaping chromatin and controlling gene expression
Low levels of 3,3′-diindolylmethane activate estrogen receptor α and induce proliferation of breast cancer cells in the absence of estradiol
Étude des interactions régulatrices au niveau de l'extrémité droite du génome de l'actinophage
Afin d'étudier l'expression génétique chez les actinomycètes, un système impliquant les bactériophages a été choisi. Dans un premier temps, l'actinophage JHJ-3 a été isolé et caractérisé biologiquement. Ce phage a été utilisé pour étudier le(s) gène(s) qui contrôle(nt) la lysogénie dans des souches du genre Saccharopolyspora et dans un deuxième temps, le réplicon de JHJ-3 a été utilisé afin d'étudier certains éléments de réplication d'actinomycètes et de s'en servir pour la construction de vecteurs de clonage. Premièrement, JHJ-3 a été isolé de la souche de Saccharopolyspora hirsuta 367 UC8106, puis a été caractérisé d'un point de vue biologique par rapport à JHJ-1, qui est un mutant virulent de JHJ-3. Les deux bactériophages infectent plusieurs souches du genre Saccharopolyspora. JHJ-3 a été défini comme un phage tempéré pour sa capacité de former des plages turbides sur un hôte sensible, sa capacité de lysogéniser plusieurs souches du genre Saccharopolyspora et finalement pour son incapacité à superinfecter une souche lysogénisée par JHJ-3. Parallèlement, JHJ-1 a été utilisé pour étudier les systèmes de restriction/modification du genre Saccharopolyspora; cette étude a permis de démontrer que certaines souches de Saccharopolyspora ont moins de systèmes de restriction et sont préférentiellement utilisables pour des transformations. Le gène du répresseur (jrpI) de JHJ-3 a été choisi comme gène modèle pour l'étude du contrôle de la lysogénie. Celui-ci a été localisé, cloné et séquencé. Des études fonctionnelles nous ont permis de conclure que jrpl coderait pour un répresseur de transcription pour sa capacité d'inhiber celle-ci à partir de promoteurs clonés du phage JHJ-1. De plus, jrpl serait impliqué dans l'établissement de l'état lysogène chez Sac. hirsuta. Comme deuxième modèle d'étude, le réplicon de JHJ-3 a été utilisé. Celui-ci a aussi été cloné, réduit à 767 pb et puis séquencé. Lorsque cloné dans un plasmide, le réplicon de JHJ-3 est capable de se répliquer dans au moins trois genres d'actinomycètes (Streptomyces, Saccharopolyspora et Amycolatopsis) à un nombre de copies moyen par chromosome bactérien. A cause de ces propriétés intéressantes, le réplicon de JHJ-3 a aussi été utilisé pour la construction de plusieurs cosmides tels que pOJ305, pOJCOS305 et pOJ31
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