Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.

BACKGROUND: Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. RESULTS: We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. CONCLUSIONS: Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility

Prognostic role of HPV integration status and molecular profile in advanced anal carcinoma: An ancillary study to the epitopes-HPV02 trial.

peer reviewedSquamous Cell Carcinoma of the Anal canal (SCCA) is a rare disease associated with a Human Papillomavirus (HPV) infection in most cases, predominantly the HPV16 genotype. About 15% of SCCA are diagnosed in metastatic stage and some will relapse after initial chemoradiotherapy (CRT). Treatment of patients by Docetaxel, Cisplatin and 5-fluorouracil (DCF) has been recently shown to improve their complete remission and progression-free survival. The aim of this retrospective study was to explore the impact of HPV infection, HPV DNA integration, TERT promoter mutational status and somatic mutations of oncogenes on both progression-free (PFS) and overall survivals (OS) of patients treated by DCF. Samples obtained from 49 patients included in the Epitopes-HPV02 clinical trial, diagnosed with metastatic or non-resectable local recurrent SCCA treated by DCF, were used for analyses. Median PFS and OS were not associated with HPV status. Patients with episomal HPV had an improved PFS compared with SCCA patients with integrated HPV genome (p=0.07). TERT promoter mutations were rarely observed and did not specifically distribute in a subset of SCCA and did not impact DCF efficacy. Among the 42 genes investigated, few gene alterations were observed, and were in majority amplifications (68.4%), but none were significantly correlated to PFS. As no biomarker is significantly associated with patients' survival, it prompts us to include every patient failing CRT or with metastatic disease in DCF strategy

Molecular biomarkers in HPV-associated (pre-)cancers

Les Papillomavirus Humains (HPV) sont des petits virus infectant les épithéliums cutanés et/ou muqueux. Si dans la majorité des cas les infections par HPV sont asymptomatiques, certaines d’entre elles sont associées à l’apparition de condylomes ou de lésions précancéreuses et de cancers au niveau du col de l’utérus, du pénis, de la vulve, du vagin, du canal anal et des voies aérodigestives supérieures. L’oncogénicité des HPV est principalement due aux oncoprotéines virales E6 et E7 qui interagissent avec plus d’une centaine de partenaires protéiques cellulaires. Ainsi, de nombreuses voies de signalisation et la machinerie cellulaire sont dérégulés. Le type HPV16 est retrouvé dans la majorité des cancers associés à HPV, suivi de 12 autres, dit à Haut Risque (HR). Chaque type HPV est composé de plusieurs lignées et sous-lignées ayant des impacts différents sur le risque pour le patient de développer des lésions (au moins dans le cas d’HPV16). Il est donc nécessaire d’établir un protocole simple et efficace permettant le séquençage des génomes complets de ces HPV-HR pour pouvoir mieux comprendre les mécanismes d’oncogenèse associés à chaque type. Cela permet aussi d’étudier des variants spécifiques à chaque type et d’établir des liens avec les données cliniques. Les objectifs de cette thèse ont été de développer et de mettre au point une méthode de séquençage des 13 HPV-HR de façon simple, sans avoir à générer de sondes ou d’amorces spécifiques à chacun des types. En parallèle, un NGS de génome complet a été réalisé sur les HPV16 présents dans des frottis de col de l’utérus de patientes éliminant leur infection ou développant des lésions de haut grade. A l’aide de comparaisons statistiques, de prédictions in silico d’immunogénicité et de structures protéiques ainsi que d’analyses in vitro, nous avons tenté d’établir un lien entre la découverte de certaines mutations et leur impact phénotypique. Le protocole de séquençage a été réalisé à l’aide des sondes présentes dans la trousse de détection HPV Hybrid Capture 2 (HC2). Les résultats ont été validés en comparant le type détecté par NGS avec celui détecté en LiPA, un test de génotypage, et avec celui détecté en qPCR spécifique. Les mêmes types ont été retrouvés dans la majorité des cas, démontrant la preuve de principe de cette méthode. Dans le cas de l’étude des variants d’HPV16, quelques mutations ont été retrouvées significativement différentes entre les HPV qui ont été éliminés et ceux ayant favorisé l’apparition de lésion, mais c’est la mutation H78Y d’E6, se trouvant au sein du site d’interaction avec IRF3, que nous avons étudiée. L’expression en RT-qPCR d’E6 était semblable entre la version mutée et la version de référence du gène. Ce n’était pas le cas avec l’expression d’IFN-β, produit grâce à une stimulation par IRF3, qui était plus élevée avec la version mutée d’E6 mais sans atteindre la significativité. Etudier l’impact et le rôle des différentes mutations retrouvées au sein de variants spécifiques des 13 HPV-HR induisant des cancers est essentiel afin de prédire plus efficacement l’évolution d’une infection et a fortiori d’assurer une prise en charge médicale optimale.Human Papillomaviruses (HPV) are small virus infecting cutaneous and squamous epithelia. Most of the time, HPV infection is asymptomatic, but some HPV types are associated with warts or precancerous and cancerous lesions at the uterine cervix, penis, vulva, vagina, anal canal and head and neck. HPV oncogenicity is essentially due to viral oncoproteins E6 and E7 which interact with more than 100 protein partners from the host cell. Plenty of signalization pathways and cellular machinery are impacted by those interactions. HPV16 type is found in the majority of HPV-associated cancers, followed by 12 other High-Risk (HR) HPV. Each type is subdivided in lineages and sub-lineages, having different impact on cancer development risks (at least for HPV16 sub-lineages). It is therefore essential to establish an easy and reliable method to sequence HR-HPV whole genomes in order to understand oncogenesis mechanisms associated with each type. It will also allow the study of type specific variants and the establishment of links between those variants and cancers. This PhD thesis had 2 purposes: on the one hand, the development of a sequencing protocol to easily realize whole-genome sequencing of 13 HR-HPV, without any probe design. On the other hand, HPV16 whole-genome analysis was realized by NGS between groups of patients clearing their cervical infection or developing high-grade lesions. Thanks to statistical comparison, in silico immunogenicity prediction and protein structure predictions as well as in vitro analysis, we aimed to establish a link between certain mutations and a phenotypical impact. Concerning the development of the sequencing method, it was realized with probes included in the Hybrid Capture 2 (HC2) HPV screening test. Results were validated by comparing type detected by NGS, LiPA (genotyping technic) and specific qPCR. The same types were retrieved in most of the cases, showing the proof-of-principle of this new technic of library capture. In the case of the HPV16 whole-genome analysis, some mutations were significatively different between the 2 groups, but we choose to study the H78Y mutation within the E6 gene because of its position in the interaction site with IRF3. E6 expression by RT-qPCR was assessed for mutated and reference E6 that showed the same expression level. It was not the case for IFN-β expression, normally expressed under IRF3 activation, which was higher with the mutated version of E6 compared to the reference, without reaching significance, though. Studying the impact and the role of mutations found within specific variants of 13 HPV-HR associated with cancers is essential to predict efficiently the evolution of the infection and, by using this finer patient classification, for an optimal medical care

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development

Background: Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood.Results: We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes.Conclusions: Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility

Observed snow depth trends in the European Alps: 1971 to 2019

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