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Mise au point d'un dosage d'activité kinase de la protéine DYRK1A et Régulation épigénétique de l'expression du gène codant le facteur de transcription ISL1

Abstract : Down syndrome is the most common aneuploidy, it originates from the presence of an extra 21st chromosome. The establishment of genotype/phenotype correlations in patients with Down's syndrome made it possible to highlight the DYRK1A kinase, encoded by the DYRK1A gene localized in the region DCR-1 on 21st chromosome, as a good candidate in the onset of mental retardation. Understanding the role and regulation of DYRK1A is thus necessary and for that, to get a reliable kinase activity assay is essential. First, we focused on the establishment of a new method of DYRK1A kinase activity assay using High Pressure Liquid Chromatography (HPLC). This method proved to be highly sensitive and affordable. Second, we sought to confirm previous data on in vitro activity of DYRK1A by using this new method. We also characterized the behavior of known inhibitors of DYRK1A (harmine) and the results obtained are in agreement with the literature. In collaboration with Dr. Dodd's team, we screened various molecules as potential inhibitors of DYRK1A. Finally, the activity assay was tested ex vivo, in mice brain extracts. Our results indicate that this new method of kinase activity assay is specific, reproducible and fast. This method can be potentially applied to other kinases, phosphatases and more broadly to other enzyme catalyzing a reaction of protein modification. GnRH plays a critical role by regulating LH and FSH secretion and synthesis via specific receptors (GnRHR) expressed at the surface of gonadotrope cells. Tissue-specific expression of Gnrhr is arbitrated by a combinatorial code of transcription factors that involves SF1, LHX3 and ISL1. Unlike for Gnrhr, we showed that Isl1 regulatory sequences upstream of the transcription start site (TSS) were not sufficient to direct pituitary-specific expression, suggesting the existence of additional regulatory mechanisms. Indeed regulatory regions (or promoters) as well as gene bodies, are altered by epigenetic modifications. Results of chromatin immunoprecipitation reveal that in cell lines expressing Isl1, namely gonadotrope cells, Isl1 was linked to histone H3 tri-methylated on Lys4 (H3K4Me3) at the TSS, an histone mark correlated with gene activity. In contrast, in cell lines where Isl1 was silent, Isl1 was bound by histone H3 tri-methylated on Lys27, a histone mark linked to gene repression. Similar correlation between histone modifications and gene activities where observed at the Gnrhr TSS. Our study further suggests that DNA methylation upstream of the CpG Island of Isl1 was inversely correlated with gene activity. Together, these data suggest that epigenetic modifications predominantly direct Isl1 tissue-specific expression whereas Gnrhr expression is primarily dependent on the presence of master tissue-specific transcription factors. Key words: ISL1, GnRH receptor, anterior pituitary, epigenetics regulation, histone modifications, DNA methylation, LIM-homeodomain proteins, gonadotrope cells.
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https://tel.archives-ouvertes.fr/tel-00777732
Contributor : Laure Tabouy <>
Submitted on : Friday, January 18, 2013 - 3:58:40 PM
Last modification on : Tuesday, August 18, 2020 - 2:40:04 PM
Long-term archiving on: : Friday, April 19, 2013 - 4:01:03 AM

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  • HAL Id : tel-00777732, version 1

Citation

Laure Tabouy. Mise au point d'un dosage d'activité kinase de la protéine DYRK1A et Régulation épigénétique de l'expression du gène codant le facteur de transcription ISL1. Neurobiologie. Université Paris-Diderot - Paris VII, 2012. Français. ⟨tel-00777732⟩

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