Ounissa Aït-Ahmed

Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms—in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3′ untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.

The yemanuclein-α: a new Drosophila DNA binding protein specific for the oocyte nucleus

Abstract The Drosophila yG 4.5 gene (now called yemanuclein-α gene), which maps at 98F, is a member of the yema gene cluster isolated in a search for differentially expressed maternal genes. The yemanuclein-α transcript (formerly yT 4.5) is specifically expressed in the female germ cells at early oogenic stages and displays a graded distribution along the antero-posterior axis of the oocyte. These provocative features are reminiscent of that of K10, bicoid and Bicaudal-D gene transcripts and lead us to hypothesize that the yemanuclein-α gene plays a key role in egg organization. We show in the present work that the yemanuclein-α is a nuclear protein highly specific for the oocyte nucleus. The sequence analysis of the 5696 bp EcoRI fragment containing the yemanuclein-α gene, and of 5 overlapping cDNAs, reveals a 3006 nucleotides long open reading frame (ORF) flanked by long untranslated 5′ and 3′ sequences. This ORF predicts a 109,215 kDa protein which is basic (pHi: 8.57), and serine rich (12.08%). It contains a 40 amino acid acidic domain in the first third of the protein with a potential α-helix organization; this domain has some similarity with the nucleolin acidic domain. Parts of the yemanuclein-α sequence are likely to form secondary structures known to interact with DNA. We demonstrate the DNA binding activity of the yemanuclein-α by affinity chromatography experiments. Our data indicate that the yemanuclein-α shares some of the features which are characteristic of genuine transcriptional activators.

Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding.

Background information. mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3′ untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU‐rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN‐BP (EDEN‐binding protein), which in turn stimulates deadenylation.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3′UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)240 insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)240.4, the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)240.4 expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3

Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication‐independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA‐mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3‐dependent intellectual disability.

Isolation of developmentally regulated genes in Drosophila melanogaster

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia‐like B104 repetitive sequence described by Scherr et al. [29]. We also isolated 2 maternally‐expressed genes, not previously identified in either classical genetic or similarly molecular‐based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.

Studies on protein kinases in two human rectocolic cell lines by polyacrylamide gel electrophoresis.Effect of the vasoactive intestinal peptide

Abstract Electrophoresis and subsequent assay of the enzyme directly onto the gel has allowed a rapid and quantitative characterization of the cyclic AMP-dependent and -independent histone kinases, protamine, phosvitin and casein kinases in HT 29 and HRT 18 cells. The technique has been applied to soluble extracts from cytoplasmic and nuclear fraction prepared in the presence and absence of neutral detergent. A more precise identification of these enzymes has been possible by analysing enzyme fractions obtained after ion-exchange chromatography of the above extracts. The protein kinase equipment of both cell lines was found to be identical (11 major components) but with different relative proportions of several enzymes. In cytoplasmic extracts: VIP activates only the type I, cytosolic, (band 4) and the type II, membrane-bound, (bands 6 and 8) cyclic AMP-dependent histone kinases. These enzymes account, respectively, for 34 and 55% of the total histone kinases in HT 29 and HRT 18 cells. The cyclic AMP-independent histone kinases (band 1,2,5 and 7) also phosphorylate protamine; band 5 was found 3o be much higher (4-fold) in HT 29 cells. In addition, two casein/phosvitin kinases have been identified in both cell lines with phosphorylating activity similar to the total histone kinases. In the nuclear extract two cyclic AMP-independent histone kinases have been found with electrophoretic mobility differing from the cytoplasmic enzymes. Also, two phosvitin/casein kinases specifically nuclear, due to their chromatographical and electrophoretical behaviour, have been characterized.

Expression in the central nervous system of a subset of the yema maternally acting genes during Drosophila embryogenesis. Post-embryonic expression extends to imaginal discs and spermatocytes

The yema gene region of Drosophila melanogaster is a cluster of maternally acting genes isolated in differential screens. At least ten transcripts are encoded by the yema gene region; most of them are produced by independent transcription units (eight different transcription units). Using RNA dot-blot analysis and in situ hybridization to tissue sections, we have realized a comprehensive survey of the temporal and spatial expression of the yema transcripts. All these transcripts are maternally expressed. Five of them display a strict maternal expression. They are found exclusively in the female germ line (nurse cells and oocyte). These transcripts are still present in the embryo as maternal information. However, a subset of the yema genes also shows an embryonic and a post-embryonic expression. Interestingly, this expression is essentially restricted to the central nervous system (CNS) throughout the fly development, to the larval and pupal imaginal discs and to a subset of cells in the male gonad, the spermatocytes. Strikingly, these expression sites mainly contain proliferating and/or differentiating cells.

Drosophila Yemanuclein associates with the cohesin and synaptonemal complexes

ABSTRACT Meiosis is characterized by two chromosome segregation rounds (meiosis I and II), which follow a single round of DNA replication, resulting in haploid genome formation. Chromosome reduction occurs at meiosis I. It relies on key structures, such as chiasmata, which are formed by repair of double-strand breaks (DSBs) between the homologous chromatids. In turn, to allow for segregation of homologs, chiasmata rely on the maintenance of sister chromatid cohesion. In most species, chiasma formation requires the prior synapsis of homologous chromosome axes, which is mediated by the synaptonemal complex, a tripartite proteinaceous structure specific to prophase I of meiosis. Yemanuclein (Yem) is a maternal factor that is crucial for sexual reproduction. It is required in the zygote for chromatin assembly of the male pronucleus, where it acts as a histone H3.3 chaperone in complex with Hira. We report here that Yem associates with the synaptonemal complex and the cohesin complex. A genetic interaction between yem1 (V478E) and the Spo11 homolog mei-W68, modified a yem1 dominant effect on crossover distribution, suggesting that Yem has an early role in meiotic recombination. This is further supported by the impact of yem mutations on DSB kinetics. A Hira mutation gave a similar effect, presumably through disruption of Hira–Yem complex.