Laboratory of sex determination

Daniel Pauli

Lecturer

  • T: +41 22 379 64 39
  • office 3003a (Sciences III)

Transcriptionnal regulation of RNA polymerase II at the level of the elongation phase

Transcription by RNA polymerase II can be divided in several stages, in particular the preinitiation, the initiation, the elongation and the termination. If the first two  phases are clearly very important in the regulation of most genes, it was recently realized that  the transition between initiation and elongation could be critical for modulation of the expression of about 30% of the genes trancribed by RNA polymerase II. Indeed, in many cases, the polymerase slows down and even stops completely (pauses) after synthesis of a few messenger RNA nucleotides. The switch of the enzyme into a processive elongation mode requires post-translational modifications of the largest subunit of RNA polymerase II. This subunit contains in its C-terminal domain many hexaptide repeats, which can be phosphorylated on serine 2 and 5. Serine 2 phosphorylation is particularly important to make the polymerase more efficient. This phosphorylation is carried out by a factor called p-TEFb composed of a kinase, Cdk9, and a cyclin co-factor, either cyclin T or cyclin K.

My laboratory uses genetic and molecular approaches in Drosophila melanogaster to analyse in vivo the function of p-TEFb complexes. We generated several transgenes that allow a conditional expression of an inactive Cdk9 kinase that competes with the normal kinase. This dominant negative approach allowed us to describe the effects of reducing p-TEFb activity in different tissues. We have described the importance of p-TEFb for cell proliferation and differentiation. It is also a step in the identification of genes whose expression is critically regulated at the level of the transition between initiation and elongation (release of RNA polymerase II pausing). We have also generated mutations and various transgenes for the two cyclins. We have shown that cyclin T and cyclin K are both essential and that the two types of p-TEFb complexes have different activities.

immuno_staining_ovaries.jpg_s600

The figure shows one particular stage during the formation of a normal oocyte (left) or of an oocyte expressing the mutant dominant negative Cdk9 kinase (right). The nuclei are detected by staining of the DNA in blue. The circular structures in red are a staining of a protein localized in the channels that link the germ cells. In the normal egg chamber, there are 16 germ cells connected by 15 channels. In the mutant egg chamber, one can observe 32 germ cells connected by 31 channels. This experiment shows that a reduction of p-TEFb activity in germ cells leads to an excess of cell proliferation.
  • 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.

    abstract

    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.

    abstract

    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.

    abstract

    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.

    abstract

    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.

    abstract

    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.

    abstract

    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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