Oocyte–Targeted Deletion Reveals That Hsp90b1 Is Needed for the Completion of First Mitosis in Mouse Zygotes

Hsp90b1 is an endoplasmic reticulum (ER) chaperone (also named Grp94, ERp99, gp96,Targ2, Tra-1, Tra1, Hspc4) (MGI:98817) contributing with Hspa5 (also named Grp78, BIP) (MGI:95835) to protein folding in ER compartment. Besides its high protein expression in mouse oocytes, little is known about Hsp90b1 during the transition from oocyte-to-embryo. Because the constitutive knockout of Hsp90b1 is responsible for peri-implantation embryonic lethality, it was not yet known whether Hsp90b1 is a functionally important maternal factor.To circumvent embryonic lethality, we established an oocyte-specific conditional knockout line taking advantage of the more recently created floxed Hsp90b1 line (Hsp90b1(flox), MGI:3700023) in combination with the transgenic mouse line expressing the cre recombinase under the control of zona pellucida 3 (ZP3) promoter (Zp3-cre, MGI:2176187). Altered expression of Hsp90b1 in growing oocytes provoked a limited, albeit significant reduction of the zona pellucida thickness but no obvious anomalies in follicular growth, meiotic maturation or fertilization. Interestingly, mutant zygotes obtained from oocytes lacking Hsp90b1 were unable to reach the 2-cell stage. They exhibited either a G2/M block or, more frequently an abnormal mitotic spindle leading to developmental arrest. Despite the fact that Hspa5 displayed a similar profile of expression as Hsp90b1, we found that HSPA5 and HSP90B1 did not fully colocalize in zygotes suggesting distinct function for the two chaperones. Consequently, even if HSPA5 was overexpressed in Hsp90b1 mutant embryos, it did not compensate for HSP90B1 deficiency. Finally, further characterization of ER compartment and cytoskeleton revealed a defective organization of the cytoplasmic region surrounding the mutant zygotic spindle.Our findings demonstrate that the maternal contribution of Hsp90b1 is critical for the development of murine zygotes. All together our data indicate that Hsp90b1 is involved in unique and specific aspects of the first mitosis, which brings together the maternal and paternal genomes on a single spindle

The Identification of Protein Kinase C Iota as a Regulator of the Mammalian Heat Shock Response Using Functional Genomic Screens

BACKGROUND: The heat shock response is widely used as a surrogate of the general protein quality control system within the cell. This system plays a significant role in aging and many protein folding diseases as well as the responses to other physical and chemical stressors. METHODS/PRINCIPAL FINDINGS: In this study, a broad-based functional genomics approach was taken to identify potential regulators of the mammalian heat shock response. In the primary screen, a total of 13724 full-length genes in mammalian expression vectors were individually co-transfected into human embryonic kidney cells together with a human HSP70B promoter driving firefly luciferase. A subset of the full-length genes that showed significant activation in the primary screen were then evaluated for their ability to hyper-activate the HSP70B under heat shock conditions. Based on the results from the secondary assay and gene expression microarray analyses, eight genes were chosen for validation using siRNA knockdown. Of the eight genes, only PRKCI showed a statistically significant reduction in the heat shock response in two independent siRNA duplexes compared to scrambled controls. Knockdown of the PRKCI mRNA was confirmed using quantitative RT-PCR. Additional studies did not show a direct physical interaction between PRKCI and HSF1. CONCLUSIONS/SIGNIFICANCE: The results suggest that PRKCI is an indirect co-regulator of HSF1 activity and the heat shock response. Given the underlying role of HSF1 in many human diseases and the response to environmental stressors, PRKCI represents a potentially new candidate for gene-environment interactions and therapeutic intervention

Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Whole-Genome Analysis Reveals That Active Heat Shock Factor Binding Sites Are Mostly Associated with Non-Heat Shock Genes in Drosophila melanogaster

During heat shock (HS) and other stresses, HS gene transcription in eukaryotes is up-regulated by the transcription factor heat shock factor (HSF). While the identities of the major HS genes have been known for more than 30 years, it has been suspected that HSF binds to numerous other genes and potentially regulates their transcription. In this study, we have used a chromatin immunoprecipitation and microarray (ChIP-chip) approach to identify 434 regions in the Drosophila genome that are bound by HSF. We have also performed a transcript analysis of heat shocked Kc167 cells and third instar larvae and compared them to HSF binding sites. The heat-induced transcription profiles were quite different between cells and larvae and surprisingly only about 10% of the genes associated with HSF binding sites show changed transcription. There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites. Analysis of the locations of the HSF binding sites revealed that 57% were contained within genes with approximately 2/3rds of these sites being in introns. We also found that the insulator protein, BEAF, has enriched binding prior to HS to promoters of genes that are bound by HSF upon HS but that are not transcriptionally induced during HS. When the genes associated with HSF binding sites in promoters were analyzed for gene ontology terms, categories such as stress response and transferase activity were enriched whereas analysis of genes having HSF binding sites in introns identified those categories plus ones related to developmental processes and reproduction. These results suggest that Drosophila HSF may be regulating many genes besides the known HS genes and that some of these genes may be regulated during non-stress conditions

Contribution à l'étude du réseau de régulation génique de la différenciation cardiaque chez la drosophile : approches génomiques

Un grand nombre de maladies cardiaques apparaissent à la suite de problèmes développementaux dus à des mutations dans des gènes très conservés durant l'évolution. Il est donc crucial d'identifier les acteurs intervenants dans la cardiogenèse. Durant ma thèse, j'ai étudié la différenciation cardiaque, en utilisant la Drosophile comme modèle, avec comme objectifs d'identifier des nouveaux intervenants grâce à l'acquisition de données globales par une approche génomique tissue spécifique. J'ai tout d'abord mis au point un protocole permettant d'acquérir des données génomique spécifiquement dans le tube cardiaque de la Drosophile lors de la différenciation des cardiomyocytes. Ce fut une étape primordiale pour le reste de ma thèse. Ce protocole me permet d'isoler spécifiquement les cellules du système cardiaque.A partir de là, j'ai réalisé une cinétique transcriptomique qui a permis de mettre en évidence environ 1000 gènes différenciellement exprimés au cours de la différenciation cardiaque. Ensuite, En collaboration avec Delphine Potier, une thésarde bio-informaticienne de l'équipe, des modules Cis-régulateur (CRM) pouvant conduire à l'expression dans le tube cardiaque ont été prédits, ainsi que des facteurs de transcription putatifs régulant ces CRM. Ces nouveaux acteurs du réseau de régulation génique cardiaque sont en cours de validation.Dans un second temps, je me suis intéressé à un facteur de transcription à homéoboîte clé de la cardiogenèse : Abdominal-A (AbdA). AbdA est essentiel pour la différenciation de la partie postérieure du tube cardiaque, le cœur proprement dit, et à l'acquisition de sa fonction. Cependant, ses cibles ainsi que son action tissue spécifique sont encore inconnu à ce jourAfin d'apporter un élément de réponse, j'ai analysé le transcriptome consécutif à un Gain de Fonction de AbdA spécifiquement dans le système cardiaque ce qui m'a permis de mettre en évidence plus de 1000 gènes dérégulés par AbdA lors de la différenciation cardiaque.Cardiac diseases generally arise from developmental disorders caused by mutations in genes that are highly conserved during evolution. It is therefore of prime interest to identify actors which participate to cardiogenesis. During my thesis, I have analyzed cardiac differentiation, using Drosophila as a model. My objective was to identify news actors of the cardiac Gene Regulatory Network (GRN) using a genomic approach and starting from tissue specific whole data acquisition.First, I have set a protocol to allow tissue specific genomic data acquisition during cardiac differentiation. It was a critical step for my thesis. This protocol allowed me to isolate specifically cardiac system cells.Using this protocol, I analyzed the transcriptome dynamics and determined that 1000 genes are dynamically expressed during cardiac differentiation.Next, in collaboration with Delphine Potier, a PhD student in bio-informatic in the team, cis-regulatory modules (CRM), which can drive expression in the cardiac tube, have been predicted, and also putative transcription factors regulating these CRM. These new cardiac GRN actors are currently tested in vivo.In a second time, I have analyzed the function of Abdominal-A (AbdA) a homeobox transcription factor which plays a key role during cardiogenesis. :. AbdA is crucial for the differentiation of the posterior part of the organ, called heart My aim was to identify cardiac specific AbdA targets

Tissue-specific cell sorting from Drosophila embryos: Application to gene expression analysis

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The fabulous destiny of the Drosophila heart

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