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

Test fonctionnel de mesure des activités enzymatiques de réparation de l'ADN par excision resynthèse sur support miniaturisé : mise au point et applications

DNA repair is a very important cellular mechanism involved in various genetic diseases and in carcinogenesis. DNA repair systems exhibit complementarities and interactions recently highlighted. However, this complexity is not taken into account by functional assays available nowadays.Therefore we have designed a miniaturized in vitro assay allowing specific, functional and parallelized measurement of DNA repair activities. This test is an adaptation of an excision resynthesis assay to the microarray format.We first setup and optimized the microarray fabrication, the data analysis and normalization, and the repair reaction. We then validated the assay and demonstrated that DNA repair activities measured were specific. Finally we demonstrated the potential of the assay in two different domains. We first phenotyped different cell types: the results showed the similarity of human fibroblasts repair profiles, and also the different repair abilities existing between fibroblast, keratinocyte, and peripheral blood mononuclear cells. Moreover we studied adaptation of cells to gamma irradiation. We demonstrated that DNA repair activities are involved in the radio-adaptation of cells .Potential applications of this assay could be important in fundamental research as in applied one such as molecular screening, and in diagnosis.La réparation de l'ADN est un processus cellulaire très important comme le montre son implication dans de nombreuses maladies génétiques et la carcinogenèse. Les mécanismes des différents systèmes de réparation présentent des interactions et des complémentarités. Les tests fonctionnels utilisés jusqu'alors pour étudier les activités de réparation ne prennent pas en compte toute cette complexité.Nous avons développé un test in vitro offrant une mesure parallélisée, fonctionnelle et spécifique d'activités enzymatiques de réparation de l'ADN. Pour ce faire, nous avons adapté un test d'excision resynthèse au format biopuce.Nous avons mis au point les différentes étapes du test : la fabrication de la biopuce, la normalisation et l'analyse des données, les conditions de réactions biologiques. Nous avons ensuite validé le test en démontrant que nous mesurions des activités enzymatiques de réparation. Enfin deux expériences applicatives de ce test ont été réalisées. Nous avons tout d'abord mis en évidence la similitude des profils de réparation de trois souches de fibroblastes humains issus de cultures primaires, mais aussi les différences des capacités de réparation qu'il existe entre les fibroblastes, kératinocytes, et cellules mononuclées du sang périphérique. Par la suite, nous avons démontré que la réparation de l'ADN est impliquée dans la réponse adaptative des cellules au rayonnement ionisant. Les applications futures de ce test sont importantes, tant en recherche fondamentale qu'appliquée pour le criblage de molécules, ou encore dans le domaine diagnostic

Test fonctionnel de mesure des activités enzymatiques de réparation de l'ADN par excision resynthèse sur support miniaturisé : mise au point et applications

DNA repair is a very important cellular mechanism involved in various genetic diseases and in carcinogenesis. DNA repair systems exhibit complementarities and interactions recently highlighted. However, this complexity is not taken into account by functional assays available nowadays.Therefore we have designed a miniaturized in vitro assay allowing specific, functional and parallelized measurement of DNA repair activities. This test is an adaptation of an excision resynthesis assay to the microarray format.We first setup and optimized the microarray fabrication, the data analysis and normalization, and the repair reaction. We then validated the assay and demonstrated that DNA repair activities measured were specific. Finally we demonstrated the potential of the assay in two different domains. We first phenotyped different cell types: the results showed the similarity of human fibroblasts repair profiles, and also the different repair abilities existing between fibroblast, keratinocyte, and peripheral blood mononuclear cells. Moreover we studied adaptation of cells to gamma irradiation. We demonstrated that DNA repair activities are involved in the radio-adaptation of cells .Potential applications of this assay could be important in fundamental research as in applied one such as molecular screening, and in diagnosis.La réparation de l'ADN est un processus cellulaire très important comme le montre son implication dans de nombreuses maladies génétiques et la carcinogenèse. Les mécanismes des différents systèmes de réparation présentent des interactions et des complémentarités. Les tests fonctionnels utilisés jusqu'alors pour étudier les activités de réparation ne prennent pas en compte toute cette complexité.Nous avons développé un test in vitro offrant une mesure parallélisée, fonctionnelle et spécifique d'activités enzymatiques de réparation de l'ADN. Pour ce faire, nous avons adapté un test d'excision resynthèse au format biopuce.Nous avons mis au point les différentes étapes du test : la fabrication de la biopuce, la normalisation et l'analyse des données, les conditions de réactions biologiques. Nous avons ensuite validé le test en démontrant que nous mesurions des activités enzymatiques de réparation. Enfin deux expériences applicatives de ce test ont été réalisées. Nous avons tout d'abord mis en évidence la similitude des profils de réparation de trois souches de fibroblastes humains issus de cultures primaires, mais aussi les différences des capacités de réparation qu'il existe entre les fibroblastes, kératinocytes, et cellules mononuclées du sang périphérique. Par la suite, nous avons démontré que la réparation de l'ADN est impliquée dans la réponse adaptative des cellules au rayonnement ionisant. Les applications futures de ce test sont importantes, tant en recherche fondamentale qu'appliquée pour le criblage de molécules, ou encore dans le domaine diagnostic

High-resolution 4C reveals rapid p53-dependent chromatin reorganization of the CDKN1A locus in response to stress

A regulatory program involving hundreds of genes is coordinated by p53 to prevent carcinogenesis in response to stress. Given the importance of chromatin loops in gene regulation, we investigated whether DNA interactions participate in the p53 stress response. To shed light on this issue, we measured the binding dynamics of cohesin in response to stress. We reveal that cohesin is remodeled at specific loci during the stress response and that its binding within genes negatively correlates with transcription. At p53 target genes, stressinduced eviction of cohesin from gene bodies is concomitant to spatial reorganization of loci through the disruption of functional chromatin loops. These findings demonstrate that chromatin loops can be remodeled upon stress and contribute to the p53-driven stress response. Additionally, we also propose a mechanism whereby transcription-coupled eviction of cohesin from CDKN1A might act as a molecular switch to control spatial interactions between regulatory elements

High-Resolution 4C Reveals Rapid p53-Dependent Chromatin Reorganization of the <i>CDKN1A</i> Locus in Response to Stress

<div><p>A regulatory program involving hundreds of genes is coordinated by p53 to prevent carcinogenesis in response to stress. Given the importance of chromatin loops in gene regulation, we investigated whether DNA interactions participate in the p53 stress response. To shed light on this issue, we measured the binding dynamics of cohesin in response to stress. We reveal that cohesin is remodeled at specific loci during the stress response and that its binding within genes negatively correlates with transcription. At p53 target genes, stress-induced eviction of cohesin from gene bodies is concomitant to spatial reorganization of loci through the disruption of functional chromatin loops. These findings demonstrate that chromatin loops can be remodeled upon stress and contribute to the p53-driven stress response. Additionally, we also propose a mechanism whereby transcription-coupled eviction of cohesin from <i>CDKN1A</i> might act as a molecular switch to control spatial interactions between regulatory elements.</p></div

Model of the <i>CDKN1A</i> chromatin loop molecular switch.

<p>Following activation of <i>CDKN1A</i> by p53 in response to stress, the passage of the transcriptional machinery evicts the cohesin and disrupts the chromatin loop. This releases the internal promoter from the influence of the repressor element and results in the induction of p21C.</p

The spatial organization of the <i>FDXR</i> locus is remodeled in response to stress.

<p>(<b>A</b>) 4C high-resolution interaction map of the <i>FDXR</i> intragenic cohesin site. HCT116 p53^+/+ cells were treated (Dauno) or not (NT) with daunorubicin. The 4C cohesin site viewpoint is highlighted in red; Rad21 ChIP-seq tracks are also shown. p53 binding sites are shown in green in the gene track. (<b>B</b>) Model of the dynamic spatial organization of the <i>FDXR</i> locus in response to stress.</p

The spatial organization of the <i>CDKN1A</i> locus is remodeled in response to stress.

<p>(<b>A</b>) 4C high-resolution interaction map of <i>CDKN1A</i>’s intragenic cohesin site. HCT116 p53^+/+ cells were treated (Dauno) or not (NT) with daunorubicin. The 4C cohesin site viewpoint is highlighted in red; Rad21 ChIP-seq tracks are also shown. P53 binding sites are indicated in green in the gene track. (<b>B</b>) 4C high-resolution interaction map of the <i>CDKN1A</i> locus. HCT116 p53^+/+ cells were treated (Dauno) or not (NT) with daunorubicin. The 4C tracks for the promoter, nucleosome-depleted region (NDR), and interacting region are presented with their respective viewpoints in red. Rad21 ChIP-seq tracks are also shown. P53 binding sites are shown in green in the gene track. (<b>C</b>) Model of the dynamic spatial organization of the <i>CDKN1A</i> locus in response to stress. (<b>†</b>) Indicates the same 4C peak between between (<b>A</b>) and (<b>B</b>).</p

The <i>CDKN1A</i> stress-dependent chromatin loop controls the interaction between a <i>CDKN1A</i> alternative internal promoter and a downstream repressor element.

<p>(<b>A</b>) <i>CDKN1A</i> gene with the DNA fragments spanning the <i>CDKN1A</i> nucleosome-depleted region (NDR) cloned for the luciferase assay. Base counting starts at position chr6:36,649,572 strand (+) hg19. (<b>B</b>) Luciferase reporter assay to measure the promoter activity of the <i>CDKN1A</i> NDR. Signal was normalized to the empty vector. (<b>C</b>) Luciferase reporter assay to test the directionality of the putative promoter. Signal was normalized to the empty vector. (<b>D</b>) Experiments to locate the TSS and the 3’ splicing junction of the mRNA transcribed from the internal promoter. The 5’ RACE products from two biological replicates obtained using two different sets of primers were sequenced and aligned to the <i>CDKN1A</i> gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163885#pone.0163885.s018" target="_blank">S9 Table</a>). The 3’ splicing was determined by performing a PCR on cDNA using a forward primer overlapping the 5’ RACE product and a reverse primer located on exon 2 of p21. The sequencing results of the PCR are shown along with the p21C splicing sequence. Experiments were performed in HCT116 p53^+/+ cells treated with daunorubicin. Red rectangles correspond to primers. (<b>E</b>) p21 and p21C mRNA levels assayed by RT-qPCR in HCT116 p53^+/+ cells exposed to 125, 250, or 1000 nM daunorubicin and collected over a 24 h time course. (<b>F</b>) p21C mRNA level assayed by RT-qPCR in HCT116 p53^+/+ and p53^-/- cells exposed to 250 nM daunorubicin for 8 h. (<b>G</b>) Luciferase reporter assay to test the repressor activity of the interacting region on the <i>CDKN1A</i> internal promoter. The luciferase signal is normalized to Control DNA1.</p