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To unravel the functions of Brdt, we explored spermatogenesis in three mouse models, which enabled us to demonstrate that Brdt is one of the most ssvi factors involved in male genome programming. Ligand binding activity of BD1-mutBC. In summary we present here a comprehensive functional study of a member of the BET family, demonstrating for the first time in a physiological setting that the bromodomains can have specific roles and act in a stage-dependent manner.

Consideration of the histone code and of the corresponding reading factors has opened new ways to tackle the in vivo mechanistic issue. Chromatin was visualized by Hoechst staining.

Along this line of investigation, we showed that during spermatogenesis, the paternally and maternally methylated imprinting control regions ICRs carry different histone modifications during the stages that precede the global histone-to-protamine exchange.

Pour obtenir le grade de. Arrêté ministériel : 7 août Jonathan GAUCHER

The strain was validated by replacing a wild-type WT plasmid with a plasmid expressing Flag-tagged H3 or H4. Figure 3B shows that significant amounts of Brdt could be co-immunoprecipitated with both Cdk9 and cyclin T1 only from wild type testes.


S3B; data not shown. D Graphic representation of the substitution mutants affected in sporulation. Chez la souris, les spermatogonies X3 se divisent 5 fois et forment successivement les spermatogonies A2, A3 A4, Intermediaire In et B.

SUMMARY Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of self-renewing progenitor cells into a defined number of mitotic divisions followed by entry into meiosis and major chromatin reorganization in haploid spermatids.

This work opens the way to new strategies to specifically target their activity. Some of these H2A and H2B variants have been subjected to structural genetisue functional analyses, and all of these studies point to the nucleosomes containing these variants as being significantly less stable than those composed of canonical histones.

BD1 failed to bind monoacetylated H4 tails, but recognized four diacetylated H4 peptides out of six testedhighlighting the cooperative nature of ligand recognition Fig. Immunoprecipitation of Cdk9 and CyclinT1 was as follows. The positions of the GFP-Brdt fusion proteins in the input and immunoprecipated materials are indicated by red arrows.

Introduction One of the unknown biological processes remains the molecular basis of post-meiotic haploid genome reprogramming [Rousseaux et al. It is striking that these modifications have a similar relative temporal relationship during sporulation and mammalian spermatogenesis, suggesting that histone H4 phosphorylation and acetylation dynamics during the final differentiation stages to generate mature gametes has been conserved through evolution.

At the same time, in meiotic cells, Brdt silences genes normally active in progenitor pre-meiotic cells. Spores were dissected and then germinated on YPD plates. After washes in Phosphate buffer mM ph7. We undertook a systematic investigation of chromatin reorganization during gametogenesis, using the model eukaryote Saccharomyces cerevisiae to examine sporulation, which has strong similarities with higher eukaryotic spermatogenesis. However, almost nothing is known of the actual molecular machinery involved in histone replacement.


BRDT was recently identified as the first factor capable of binding to hyperacetylated histone H4. Unique chromatin remodeling and transcriptional regulation in spermatogenesis. There are also some hints on the possibility of histone degradation through the ubiquitin proteasome system.

Brdt requires its first bromodomain, BD1, to prime the transcription of genes in meiotic cells, which become fully active in early post-meiotic cells.

Chromatin acetylation was induced by treating cells overnight with the histone deacetylase inhibitor trichostatin A TSA. Because of the capacity of Brdt s BD1 to specifically act on hyperacetylated histones, we questioned whether the replacement of histones is maintained in the absence of BD1. These results indicate that at least partial ligandbinding activity by BD1 is required for compaction, whereas that by BD2 is dispensable Fig. Upon activation of its gene, in early spermatocytes, Brdt switches on a meiotic gene expression program including critical genes known to direct meiotic divisions.

Pour obtenir le grade de. Arrêté ministériel : 7 août Jonathan GAUCHER – PDF

The right panels show the expression of the same genes in testis from the three indicated genotypes, at 20 dpp. The differentially expressed genes fold change threshold of 1. However, these mice would be difficult to obtain, as Prm haploinsufficency results in male sterility [34], and therefore Prm conditional mutants would cour to be generated, and then crossed with mice lacking TPs.

Indeed, our investigations now indicate that BRDT plays a major role in controlling chromatin acetylation-dependent events in spermatids. More than substitution mutants have been created and validated by sequencing.

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