Caractérisation des facteurs HSF1 et HSF2 en tant que facteur maternel et régulateur de la réponse au stress

Au cours de ma thèse, je me suis intéressé aux facteurs de choc thermique HSFs (Heat Shock Factors) et aux fonctions qu'ils exercent au cours du développement. Mon projet de thèse comportait deux parties principales, la première concernant l'identification des gènes cibles du facteur maternel HSF1, et la seconde concernant les fonctions d'HSF1 et HSF2 au cours du développement précoce. Dans un premier temps, nous avons donc cherché à identifier les gènes cibles du facteur HSF1 par une analyse transcriptomique afin de mieux caractériser sa fonction maternelle dans l'ovocyte de souris. Ainsi, parmi les gènes régulés par HSF1, nous avons observé un enrichissement en gènes impliqués dans la cohésion des chromosomes homologues et des chromatides sœurs et nous avons mis en évidence la présence d'HSF1 sur le promoteur de 4 de ces gènes : Stag2, Stag3, Syce1 et Msh4. Ensuite, nous avons mis en évidence que la réduction de ces gènes dans les ovocytes Hsf1-/- entraine des défauts de progression à travers la prophase I aboutissant à une ségrégation précoce des chromosomes homologues lors de la métaphase I. L'ensemble de ces données montrent qu'HSF1 participe à la coordination de la dynamique des chromosomes au cours de la méiose chez la femelle. La deuxième partie de mon projet de thèse concernait les fonctions exercées par HSF1 et HSF2 au cours du développement précoce. En combinant l'utilisation de lignées transgéniques rapportrices (Hsp70.1 Luciférase) et de lignées mutantes pour Hsf1 ou Hsf2 (Hsf1-/-, Hsf2-/-), nous avons montré qu’HSF1 et HSF2 jouent un rôle dans l’activation zygotique du gène Hsp70.1 et qu’HSF2 intervient dans la réponse au choc thermique au stade blastocyste.During my PhD, I studied heat shock factors HSFs and their functions during development. My thesis project included two main parts. The first one was aimed to identify HSF1 dependent target genes, and the second part investigated the functions of HSF1 and HSF2 during early development. First, we sought to identify HSF1 target genes by a transcriptome analysis to further characterize its maternal function in mouse oocyte. Among the genes regulated by HSF1, we observed an enrichment of genes involved in the cohesion of homologous chromosomes and sister chromatids and we observed the presence of HSF1 on the promoter of 4 of these genes: Stag2, Stag3, Syce1 and Msh4. Then, we demonstrated that the lower expression of these genes in Hsf1-/- oocytes led to defects in prophase I progression and early segregation of homologous chromosomes at metaphase I. Taken together, these data show that HSF1 helps to coordinate the dynamics of chromosomes during female meiosis in mammals. The second part of my project was about the functions performed by HSF1 and HSF2 during early development. Using several mouse transgenic and knockout lines, we showed that HSF1 and HSF2 play a role in the activation of zygotic Hsp70.1 and that HSF2 takes part to the heat shock response at the blastocyst stage

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability.

International audienceA single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy

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 Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA

BACKGROUND: MBD5 and MBD6 are two uncharacterized mammalian proteins that contain a putative Methyl-Binding Domain (MBD). In the proteins MBD1, MBD2, MBD4, and MeCP2, this domain allows the specific recognition of DNA containing methylated cytosine; as a consequence, the proteins serve as interpreters of DNA methylation, an essential epigenetic mark. It is unknown whether MBD5 or MBD6 also bind methylated DNA; this question has interest for basic research, but also practical consequences for human health, as MBD5 deletions are the likely cause of certain cases of mental retardation. PRINCIPAL FINDINGS: Here we report the first functional characterization of MBD5 and MBD6. We have observed that the proteins colocalize with heterochromatin in cultured cells, and that this localization requires the integrity of their MBD. However, heterochromatic localization is maintained in cells with severely decreased levels of DNA methylation. In vitro, neither MBD5 nor MBD6 binds any of the methylated sequences DNA that were tested. CONCLUSIONS: Our data suggest that MBD5 and MBD6 are unlikely to be methyl-binding proteins, yet they may contribute to the formation or function of heterochromatin. One isoform of MBD5 is highly expressed in oocytes, which suggests a possible role in epigenetic reprogramming after fertilization

Fast-connecting open innovation practices: On the role of intermediaries to accelerate the absorptive capacity function

International audienceFirms that engage in open innovation (OI) activities seek to leverage on external knowledge to innovate and use the open innovation intermediaries to conduct and structure their collaborative efforts. The access to external knowledge often requires an internal knowledge effort to be able to recognize, assimilate and use external knowledge which imply the organization of absorptive capacity (AC) function. Still, the absorption of isolated ideas in OI initiatives is a major issue. The purpose of this research is to investigate how the OI intermediaries ensure the AC for firms participating in the OI through the process of searching and integrating knowledge created through the innovation process. Through an exploratory case study of an intermediary platform that connects the technical experts throughout the world – IdexLab and its clients, we investigate IdexLab’s capacity to prepare and conduct the AC activity based on “novelty search” algorithms. We find that the OI platform can incorporate function to automatize the AC and facilitate further diffusion of ideas

Unraveling Complex Interplay between Heat Shock Factor 1 and 2 Splicing Isoforms

Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs). The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called a and b, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs) deleted for both Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1a is significantly more active than the b isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short b isoform of HSF2 exerts a negative role on HSF1b-dependent transactivation. To further assess the impact of HSF2b inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relativ

HSPA5 expression but not localization is modified in MT embryos.

<p>(<b>A</b>) RTqPCR was used to determine the level of Hspa5 transcripts in WT and MT samples: GV oocyte, ovulated MII oocytes, 1-cell or zygote (30 hours post hCG) and 2-cell (42–44 hours post hCG) embryos. Relative quantities of mRNA were normalized against the quantity of ribosomal S16 transcripts and Hspa5 transcript level in GV oocytes was arbitrarily given the value 1. (<b>B</b>) HSPA5 was immunodetected by Western blot using WT and MT samples as indicated. Experimental procedure was similar as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017109#pone-0017109-g002" target="_blank">Figure 2C</a>. The number written below each lane corresponds to the relative densitometric value (HSPA5/alphaTUB.). (<b>C</b>) WT embryos (pronuclear -PN5- and mitotic 1-cell stages) and MT mitotic zygotes were prepared for immunofluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017109#s4" target="_blank">materials and methods</a>. Representative images show that ER chaperones HSPA5 and HSP90B1 did not completely colocalize. HSP90B1 was found within the spindle structure but not HSPA5 as shown in inset (i). HSPA5 and HSP90B1 were both present in the granular region surrounding the spindle but higher magnification (inset ii) showed that even in this region where both ER chaperones were present, they were not fully colocalized. HSPA5 seemed to be more abundant in the cortex region of the zygote. HSPA5 staining was not visibly modified in MT embryos.</p

Development of embryos obtained by crossing <i>Zp3-cre; Hsp90b1^flox/flox</i> (MT) or control females with wild-type (WT) males.

<p>Development of embryos obtained by crossing <i>Zp3-cre; Hsp90b1^flox/flox</i> (MT) or control females with wild-type (WT) males.</p

Thinner zona pellucida in HSP90B1 deficient oocytes.

<p>(<b>A</b>) Representative images of wild-type (WT) and mutant (MT, obtained from Zp3-cre<i>; Hsp90b1^flox/flox</i> females) ovulated oocytes showing that zona pellucida is thinner in mutant oocytes. (<b>B</b>) Ratio between thickness of zona pellucida and oocyte diameter was calculated and indicated in arbitrary unit (A.U.). Mutant zona pellucida (n = 99) were significantly thinner than wild type ones (n = 36). p<0.001. (<b>C</b>) Histological sections of adult ovaries were prepared for immunofluorescence against ZP2 and representative examples of WT and MT oocytes from secondary follicles are shown. Arrows indicate the ZP2 positive structures in mutant oocytes.(<b>D</b>) Percentage of WT and MT oocytes containing ZP2 positive structures at late primary, secondary and tertiary follicular stages. Numbers indicated above the bar correspond to the number of follicles for each category.</p

Peri-spindle accumulation of filamentous actin in mutant embryos.

<p>(<b>A</b>) Representative pictures of WT and MT zygotes at the metaphase stage of the first mitosis stained with phalloidin (green: filamentous actin) and TO-PRO-3 (blue: DNA) shown with original contrast and enhanced contrast to better visualize actin accumulated around the spindle. (<b>B</b>) Representative examples of plot profiles (Image J) analyzing the actin staining (normal contrast) along the diameter section of the zygotes. (<b>C</b>) Graph showing the relative intensity of actin staining measured at the peri-spindle region and cortex in WT zygotes (n = 5) and MT zygotes (n = 13). Data presented as mean value (A.U.: arbitrary unit). p<0.01.</p