Laboratoire de détermination du sexe

Daniel Pauli

Chargé(e) d'enseignement

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Régulation de la transcription par l’ARN polymérase II au niveau de la phase d’élongation

La transcription des gènes par l’ARN polymérase II peut être divisée en plusieurs phases, notamment la préinitiation, l’initiation ou amorçage, l’élongation et la terminaison. Si les deux premières phases sont évidemment très importantes pour le contrôle de l’expression de beaucoup de gènes, ce n’est que récemment que l’on s’est rendu compte que la transition entre initiation et élongation est importante pour la modulation de l’expression de près de 30% des gènes transcrits par l’ARN polymerase II. En effet, dans beaucoup de cas cette polymérase  ralenti et s’arrête même complètement (on parle de pause) après avoir synthétisé les premiers nucléotides d’un ARN messager. Le passage à une enzyme plus efficace pour poursuivre l’élongation de l’ARN nécessite des modifications post-traductionnelles de la plus grande sous-unité de l’ARN polymérase II. Cette sous-unité contient dans sa partie C-terminale de nombreuses copies d’un hexapeptide qui peut être phosphorylé au niveau de sérines en position 2 et 5 de ces répétitions. La phosphorylation des sérines 2 est particulièrement importante pour rendre la polymérase plus efficace. Cette phosphorylation est effectuée par un facteur appelé p-TEFb qui est constitué d’une kinase, Cdk9, et d’un co-facteur appelé cycline qui peut être soit la cycline T soit la cycline K.

Mon laboratoire combine des approches génétiques et moléculaires chez Drosophila melanogaster pour analyser in vivo la fonction des complexes p-TEFb. Nous avons généré des transgènes qui permettent une expression contrôlée d’une kinase Cdk9 inactive qui entre en compétition avec la kinase normale. Cette approche de mutation dominante négative nous a permis de décrire les effets d’une réduction de l’activité p-TEFb dans différents tissus de la drosophile. Cela permet de décrire l’importance de p-TEFb lors de la prolifération et de la différenciation cellulaire. Ce travail est une étape dans l’identification de gènes qui sont particulièrement fortement contrôlés au niveau de la transition entre l’initiation et l’élongation (relachement de polymerase en pause). Nous avons aussi créé des mutations et des différents transgènes pour les cyclines. Nous avons pû montrer que la cycline T et la cycline K sont toutes deux indispensables et que les deux types de complexes p-TEFb ont des activités différentes.

La figure montre une étape intermédiaire dans la formation d’un ovule normal (à gauche) ou exprimant la mutation dominante négative de la kinase Cdk9 (à droite). Les noyaux sont visibles par la coloration de l’ADN en bleu. Les structures circulaires en rouge sont une coloration pour une protéine localisée dans les canaux qui relient les cellules germinales entre elles. Dans la chambre ovocytaire normale, il y a 16 cellules germinales reliées par 15 canaux. Dans la chambre à oeuf mutante on observe 32 cellules germinales reliées par 31 canaux. Cette expérience montre que la réduction d’activité du complexe p-TEFb dans les cellules germinales abouti à une prolifération cellulaire excessive.
  • Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference. PLoS Genet 2021 Nov;17(11):e1009843. 10.1371/journal.pgen.1009843. PGENETICS-D-21-00807.

    résumé

    Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. Here, we investigate the iab-8 ncRNA from the Drosophila Bithorax Complex and show that this RNA is able to repress the transcription of genes located at its 3' end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, we find that its repressive activity is limited to the CNS, where, in wild-type embryos, it acts on the Hox gene, abd-A, located immediately downstream of it. The CNS specificity is achieved through a 3' extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3' extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote.

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  • Drosophila melanogaster positive transcriptional elongation factors regulate metabolic and sex-biased expression in adults. BMC Genomics 2017 May;18(1):384. 10.1186/s12864-017-3755-x. 10.1186/s12864-017-3755-x.

    résumé

    Transcriptional elongation is a generic function, but is also regulated to allow rapid transcription responses. Following relatively long initiation and promoter clearance, RNA polymerase II can pause and then rapidly elongate following recruitment of positive elongation factors. Multiple elongation complexes exist, but the role of specific components in adult Drosophila is underexplored.

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  • Transcriptional activation by GAGA factor is through its direct interaction with dmTAF3. Dev. Biol. 2008 May;317(2):660-70. S0012-1606(08)00102-4. 10.1016/j.ydbio.2008.02.008.

    résumé

    The GAGA factor (GAF), encoded by the Trithorax like gene (Trl) is a multifunctional protein involved in gene activation, Polycomb-dependent repression, chromatin remodeling and is a component of chromatin domain boundaries. Although first isolated as transcriptional activator of the Drosophila homeotic gene Ultrabithorax (Ubx), the molecular basis of this GAF activity is unknown. Here we show that dmTAF3 (also known as BIP2 and dTAF(II)155), a component of TFIID, interacts directly with GAF. We generated mutations in dmTAF3 and show that, in Trl mutant background, they affect transcription of Ubx leading to enhancement of Ubx phenotype. These results reveal that the gene activation pathway involving GAF is through its direct interaction with dmTAF3.

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  • Isolation of Su(var)3-7 mutations by homologous recombination in Drosophila melanogaster. Genetics 2002 Jul;161(3):1125-36. PMC1462191.

    résumé

    The Su(var)3-7 gene, a haplo-suppressor and triplo-enhancer of position-effect variegation (PEV), encodes a zinc finger heterochromatin-associated protein. To understand the role of this protein in heterochromatin and genomic silencing, mutations were generated by homologous recombination. The donor fragment contained a yellow(+) gene and 7.6 kb of the Su(var)3-7 gene inserted between two FRTs. The Su(var)3-7 sequence contained three stop codons flanking an I-SceI cut site located in the 5' half of the gene. Using two different screening approaches, we obtained an allelic series composed of three mutant alleles. The three mutations are dominant suppressors of PEV. One behaves as a null mutation and results in a maternal-effect recessive lethal phenotype that can be rescued by a zygotic paternal wild-type gene. A P transposon zygotically expressing a Su(var)3-7 full-length cDNA also rescues the mutant phenotype. One hypomorphic allele is viable and the pleiotropic phenotype showed by adult flies indicates that rapidly and late dividing cells seem the most affected by reduced amounts of Su(var)3-7 protein. All three mutants were characterized at the molecular level. Each expresses a portion of the Su(var)3-7 protein that is unable to enter the nucleus and bind chromatin.

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  • Tissue-specific expression of the heat shock protein HSP27 during Drosophila melanogaster development. J. Cell Biol. 1990 Sep;111(3):817-28. PMC2116260.

    résumé

    The alpha-crystallin-related heat shock (stress) protein hsp27 is expressed in absence of heat shock during Drosophila melanogaster development. Here, we describe the tissue distribution of this protein using an immunoaffinity-purified antibody. In embryos, hsp27 translated from maternal RNA is uniformly distributed, except in the yolk. During the first, second, and early third larval stages, hsp27 expression is restricted to the brain and the gonads. These tissues are characterized by a high level of proliferating cells. In late third instar larvae and early pupae, in addition to the central nervous system and the gonads, all the imaginal discs synthesize hsp27. The disc expression seems restricted to the beginning of their differentiation since it disappears during the second half of the pupal stage: no more hsp27 is observed in the disc-derived adult organs. In adults, hsp27 is still present in some regions of the central nervous system, and is also expressed in the male and female germ lines where it accumulates in mature sperm and oocytes. The transcript and the protein accumulate in oocytes since the onset of vitellogenesis with a uniform distribution similar to that found in embryos. The adult germ lines transcribe hsp27 gene while no transcript is detected in the late pupal and adult brain. These results suggest multiple roles of hsp27 during Drosophila development which may be related to both the proliferative and differentiated states of the tissues.

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  • Expression of the small heat shock genes during Drosophila development: comparison of the accumulation of hsp23 and hsp27 mRNAs and polypeptides. Genome 1989 ;31(2):671-6.

    résumé

    Seven heat shock genes are clustered within 15 kilobases of DNA at the Drosophila melanogaster chromosomal site 67B. They show a complex pattern of expression in the absence of external stress during normal development of this organism. In this paper, we quantitatively compare the abundance of the messenger RNAs for these seven genes at all major stages of Drosophila development and then focus on hsp23 and hsp27 for which available antibodies allow the comparison between the accumulation of the mRNAs and that of their corresponding polypeptides. Transcripts for both genes are maximally abundant in white prepupae. We observe that the amount of hsp23 message decreases more rapidly than that of hsp27 mRNA throughout the pupal period. The maximal abundance of the proteins occurs at the middle of the pupal stage, when their corresponding RNAs have almost completely disappeared. The peaks of expression of the proteins are also broader than those of their transcripts, indicating that the half-lives of the polypeptides are longer. These observations suggest that complex mechanisms regulate the expression of the small heat shock genes during Drosophila development.

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  • An unusual split Drosophila heat shock gene expressed during embryogenesis, pupation and in testis. J. Mol. Biol. 1988 Mar;200(1):47-53. 0022-2836(88)90332-4.

    résumé

    Gene 2, one of the seven heat shock genes from locus 67B of Drosophila melanogaster, is transcribed into two polyadenylated RNAs having different developmental profiles of expression. The smaller transcript, of about 560 nucleotides, is expressed from mid-embryogenesis to the first two larval stages and again at the beginning of pupation. The larger transcript, 780 nucleotides, contains an additional 5' exon, accounting for its larger size. It is detected in pupae and adults, is male-specific and is localized in the testes. Heat shock does not affect the abundance of these two transcripts but induces the accumulation of a third RNA species of about 2000 nucleotides. This heat-shock RNA has the same cap site as the embryonic transcript, while its 3' portion entirely includes the neighbouring hsp22 gene. It appears, therefore, that in this case, heat shock alters the normal transcription termination process. By contrast to most heat shock genes, gene 2 contains several micro introns. One long open reading frame common to the three transcripts encodes a putative polypeptide of 111 amino acid residues. No homology was found with the other small heat shock genes of locus 67B.

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  • A Drosophila heat shock gene from locus 67B is expressed during embryogenesis and pupation. J. Mol. Biol. 1987 Nov;198(2):235-40. 0022-2836(87)90309-3.

    résumé

    We present a detailed characterization of the structure and expression of gene 3 from the 67B locus of Drosophila melanogaster. Northern blot analysis reveals a major poly(A)+ transcript during two stages of development: mid-embryogenesis and beginning of pupation. After heat shock, the abundance of this mRNA is increased and small amounts of larger RNAs representing alternate terminations of the major transcript appear. In Schneider 3 tissue culture cells, beside the major transcript, we also observe small amounts of the larger RNAs after a heat shock. The sequencing of cDNA and genomic clones shows an intronless transcription unit with one long open reading frame. The deduced polypeptide has 169 amino acids. It shares a strong homology with the four small heat shock proteins in the region also conserved in the mammalian alpha-crystalline B2 chain. In gene 3, this homology is restricted to the first 50 residues along the conserved 83 amino acid stretch. Two heat shock regulatory elements are localized upstream from the gene.

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