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ABSTRACT: Heat shock factor 1 (HSF1), while recognized as the major regulator of the heat shock transcriptional response, also exerts important functions during mammalian embryonic development and gametogenesis. In particular, HSF1 is required for oocyte maturation, the adult phase of meiosis preceding fertilization. To identify HSF1 target genes implicated in this process, comparative transcriptomic analyses were performed with wild-type and HSF-deficient oocytes. This revealed a network of meiotic genes involved in cohesin and synaptonemal complex (SC) structures, DNA recombination, and the spindle assembly checkpoint (SAC). All of them were found to be regulated by HSF1 not only during adult but also in embryonic phases of female meiosis. Additional investigations showed that SC, recombination nodules, and DNA repair were affected in Hsf1(-/-) oocytes during prenatal meiotic prophase I. However, targeting Hsf1 deletion to postnatal oocytes (using Zp3 Cre; Hsf1(loxP/loxP)) did not fully rescue the chromosomal anomalies identified during meiotic maturation, which possibly caused a persistent SAC activation. This would explain the metaphase I arrest previously described in HSF1-deficient oocytes since SAC inhibition circumvented this block. This work provides new insights into meiotic gene regulation and points out potential links between cellular stress and the meiotic anomalies frequently observed in humans.
Molecular and cellular biology 06/2011; 31(16):3410-23. · 6.06 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2011; 6(2):e17109. · 4.09 Impact Factor
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ABSTRACT: Gene encoding heat shock protein (Hsps) are induced following a thermal stress thanks to the activation of heat shock transcription factor (HSF) which interacts with heat shock elements (HSE) located within the sequence of Hsp promoters. This cellular and protective response (heat shock response (HSR)) is well known and evolutionarily conserved. Nevertheless, HSR does not function in all the cells produced during the life of a multicellular organism, e.g., early mouse embryos. Taking advantage of mouse transgenic and knockout models, we investigated the roles of trans (HSF 1 and 2) and cis (HSE) regulatory elements in the control of Hsp70.1 (Hspa1b) through several developmental steps from oocytes to blastocysts. Our studies confirm that, even in absence of any stress, HSF1 regulates Hsp70.1 in oocytes and early embryos. Our data emphasize the role of maternal and paternal HSFs in the developmentally regulated expression of Hsp70.1 observed when the zygotic genome activation occurs. Furthermore, in this unstressed developmental condition, affinity and binding to HSEs might be more permissive than in the stress response. Finally, submitting blastocyst to different stress conditions, we show that HSF2 is differentially required for Hsp expression and cell survival. Taken together, our findings indicate that the role of heat shock trans and cis regulatory elements evolve along the successive steps of early embryonic development.
Cell Stress and Chaperones 10/2010; 16(3):275-85. · 3.01 Impact Factor
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ABSTRACT: 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.
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.
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.
PLoS ONE 01/2010; 5(8):e11982. · 4.09 Impact Factor
