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Thioredoxins and gene regulation in the Drosophila germline
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

Spermatogenesen är i många organismer en av de mest dramatiska förvandlingar som en cell kan genomgå – en vanlig, rund, diploid cell omvandlas till en nålformad, haploid cell med ett tätt packat cellmaskineri. I bananfluga, Drosophila melanogaster, innebär denna process flera karaktäristiska stadier. Ett av dessa är det primära spermatocyt-stadiet, som infaller innan cellen påbörjar meios-delning. Stadiet karaktäriseras av en uppluckrad kromatinstruktur i cellens kärna och ovanligt höga transkriptions- och translationshastigheter, för att producera allt det mRNA och de proteiner som behövs senare under spermatidomvandlingen. Två av de proteiner som uttrycks i höga nivåer i primära spermatocyter är ThioredoxinT (TrxT) och Painting of fourth (POF).

Thioredoxiner är små proteiner som har som funktion att reducera disulfidbryggor i andra proteiner, en mekanism som används i många olika fysiologiska sammanhang. I denna avhandling visar jag att TrxT-genen kodar för ett testikelspecifikt thioredoxin som binder specifikt till Y-kromosom-loopar i primära spermatocyter. TrxT-genen ligger precis bredvid deadhead (dhd), en gen som kodar för ett hon-specifikt thioredoxin som är lokaliserat till cellkärnorna i flugans äggstockar. Ett tredje thioredoxin i Drosophila är det allmänt uttryckta Thioredoxin-2 (Trx-2). Jag har upptäckt att flugor som saknar Trx-2 är livsdugliga, men att de lever kortare än vildtypsflugor, medan flugor med extra mycket Trx-2 har en ökad tålighet mot oxidativ stress. Slående nog är en total avsaknad av alla tre thioredoxiner inte förenat med letalitet, tvärtemot vad man skulle kunnat vänta sig. Alla tre thioredoxiner finns i de olika Drosophilider som undersökts och den ovanliga genorganisation som delas av TrxT och dhd är generellt konserverad.

Gen-namnet Painting of fourth härstammar från upptäckten att POF binder till (”målar”) Drosophilas kromosom 4. Jag visar i min avhandling att POFs bindning till den fjärde kromosomen är bevarad i olika Drosophila-arter och att POF kolokaliserar med både ett protein och en histon-modifiering, som är förknippade med doskompensation, i arter där POF också binder till hanens X-kromosom. POF uttrycks överallt i både honor och hanar, men i väldigt höga nivåer i hanens testiklar. Jag visar här att POF finns i cellkärnan hos primära spermatocyter, men också i kärnan på mognande spermatider, och att avsaknad av POF in hanens könsceller orsakar en global nedreglering av gener som ligger på kromosom 4. Kombinationen av mina POF- resultat tyder på att POF har en viktig funktion i det första kända fallet av genreglering av en hel autosomal kromosom.

Abstract [en]

The process of spermatogenesis is in many organisms one of the most dramatic cellular transformations - a normal round diploid cell is ultimately transformed into a needle shaped haploid cell with tightly packaged cell machinery. In Drosophila melanogaster this process involves several characteristic stages, one of these being the primary spermato-cyte stage, which is the stage prior to meiosis. This stage is characterized by a loose chromatin structure in the nucleus and exceptionally high rates of transcription and translation to produce essentially all the mRNAs and proteins that are needed later during spermatid formation. Two proteins that are expressed in high levels in primary spermatocytes are ThioredoxinT (TrxT) and Painting of fourth (POF).

Thioredoxins are small thiol proteins that reduce disulfides in other proteins, a mechanism that is utilized in many different contexts. In this thesis I show that the TrxT gene encodes a testis-specific thioredoxin that specifically associates to Y-chromosome loops in primary spermatocytes. TrxT is located right next to deadhead (dhd), a gene that encodes a female-specific thioredoxin that specifically locates to nuclei in the ovaries. A third thioredoxin in Drosophila is the ubiquitously expressed Thioredoxin-2 (Trx-2). I have found that flies lacking Trx-2 are viable but have shorter life spans than wild type flies, while over-expression of Trx-2 mediates an increased resistance to oxidative stress. Interestingly, a lack of all three thioredoxins does not result in lethality, contrary to what could be expected. All three thioredoxins are conserved among Drosophilids and the striking genomic organization of TrxT and dhd is generally conserved.

The gene name Painting of fourth originates from the finding that POF stains the 4th chromosome of Drosophila in a banded pattern on salivary gland chromosomes. I show in this thesis that POF binding to the equivalent of the 4th chromosome is conserved in genus Drosophila and that POF co-localizes with both a protein and a histone modification associated with dosage compensation in species where POF also binds the male X. POF is expressed ubiquitously in both males and females, but at very high levels in male testes. I show that POF is present in nuclei of primary spermatocytes, but also in nuclei of maturing spermatids and that a lack of POF in the male germline causes a global down-regulation of chromosome 4 genes. These results combined suggest a function of POF in the first known case of chromosome-wide gene regulation of an autosome.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet) , 2007. , 166 p.
Keyword [en]
Drosophila, thioredoxin, sex-specific, Pof, chromosome 4, gene regulation
Keyword [sv]
kromosom 4, genreglering, Drosophila, thioredoxin, könsspecifik, Pof
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1008ISBN: 978-91-7264-255-3 (print)OAI: oai:DiVA.org:umu-1008DiVA: diva2:139961
Public defence
2007-03-09, Sal B, 1D, Tandläkarhögskolan, Umeå Universitet, 90187 Umeå, 10:00
Opponent
Supervisors
Available from: 2007-02-15 Created: 2007-02-15 Last updated: 2012-06-28Bibliographically approved
List of papers
1. The ThioredoxinT and deadhead gene pair encode testis- and ovary-specific thioredoxins in Drosophila melanogaster.
Open this publication in new window or tab >>The ThioredoxinT and deadhead gene pair encode testis- and ovary-specific thioredoxins in Drosophila melanogaster.
2003 (English)In: Chromosoma, ISSN 0009-5915, Vol. 112, no 3, 133-43 p.Article in journal (Other academic) Published
Abstract [en]

So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster. Here, with the cloning and characterization of TrxT we describe an additional thioredoxin with testis-specific expression. TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region. We show that this regulatory region is sufficient for correct expression of the two genes. This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region. Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively. Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes. The association is lost in the absence of the Y chromosome. Our results suggest that nuclear thioredoxins may have regulatory functions in the germline.

Keyword
Amino Acid Sequence, Animals, Bacterial Proteins, DNA; Complementary/genetics, Drosophila Proteins/*genetics/metabolism, Drosophila melanogaster/*genetics/metabolism, Female, Fluorescent Antibody Technique, Gene Components, Gene Expression, In Situ Hybridization, Luminescent Proteins, Male, Membrane Proteins, Molecular Sequence Data, Ovary/metabolism, Regulatory Sequences; Nucleic Acid/genetics, Sequence Alignment, Sequence Analysis; DNA, Testis/metabolism, Thioredoxin/*genetics/metabolism, Transgenes/genetics, Y Chromosome/metabolism
Identifiers
urn:nbn:se:umu:diva-17037 (URN)14579129 (PubMedID)
Available from: 2007-10-26 Created: 2007-10-26 Last updated: 2012-06-28Bibliographically approved
2. Thioredoxin-2 affects lifespan and oxidative stress in Drosophila
Open this publication in new window or tab >>Thioredoxin-2 affects lifespan and oxidative stress in Drosophila
2007 (English)In: Hereditas, ISSN 0018-0661, E-ISSN 1601-5223, Vol. 144, no 1, 25-32 p.Article in journal (Other academic) Published
Abstract [en]

Thioredoxins are proteins that have thiol-reducing activity and a characteristic conserved active site (WCGPC). They have

several documented functions, e.g. roles in defences against oxidative stress and as electron donors for ribonucleotidereductase.

In Drosophila melanogaster there are three ‘‘classical’’ thioredoxins with the conserved active site: deadhead,

ThioredoxinT and Thioredoxin-2. Here, we report the creation of null-mutations in the Thioredoxin-2 (Trx-2) gene.

Characterization of two Trx-2 mutants indicated that Trx-2 affects the lifespan of D. melanogaster, and is involved in the

organism’s oxidative stress protection system. We found that the mutants have a shorter lifespan than wild-type flies, and

thioredoxin double mutant flies showed lower tolerance to oxidative stress than wild-type flies, while flies carrying multiple

copies of a Trx-2 rescue construct showed higher tolerance. These findings suggest that Trx-2 has modest or redundant

functions in Drosophila physiology under unstressed conditions, but could be important during times of environmental

stress.

Place, publisher, year, edition, pages
Oxford: Blackwell, 2007
Keyword
Analysis of Variance, Animals, Animals; Genetically Modified, Blotting; Western, Crosses; Genetic, Drosophila/*genetics, Female, Gene Deletion, Genes; Insect, Homozygote, Longevity/*genetics, Male, Membrane Proteins/*genetics/*physiology, Oxidative Stress/*genetics, Thioredoxin/*genetics
Identifiers
urn:nbn:se:umu:diva-17027 (URN)10.1111/j.2007.0018-0661.01990.x (DOI)17567437 (PubMedID)
Available from: 2007-10-26 Created: 2007-10-26 Last updated: 2017-12-14Bibliographically approved
3. Organization and regulation of sex-specific thioredoxin encoding genes in genus Drosophila
Open this publication in new window or tab >>Organization and regulation of sex-specific thioredoxin encoding genes in genus Drosophila
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2129 (URN)
Available from: 2007-02-15 Created: 2007-02-15 Last updated: 2010-04-12Bibliographically approved
4. Painting of fourth in genus Drosophila suggests autosome-specific gene regulation
Open this publication in new window or tab >>Painting of fourth in genus Drosophila suggests autosome-specific gene regulation
2004 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 26, 9728-9733 p.Article in journal (Refereed) Published
Abstract [en]

Painting of fourth (POF) is a chromosome-specific protein in Drosophila and represents the first example of an autosome-specific protein. POF binds to chromosome 4 in Drosophila melanogaster, initiating at the proximal region, followed by a spreading dependent on chromosome 4-specific sequences or structures. Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila, a complex including the male-specific lethal proteins, "paints" the male X chromosome, mediating its hypertranscription, explained to some extent by the acetylation of lysine 16 on histone H4. Here, we show that Pof is essential for viability in both sexes and for female fertility. POF binding to an autosome, the F element, is conserved in genus Drosophila, indicating functional conservation of the autosome specificity. In three of nine studied species, POF binds to the male X chromosome. When bound to the male X, it also colocalizes with the dosage compensation protein male-specific lethal 3, suggesting a relationship to dosage compensation. The chromosome specificity is determined at the species level and not by the amino acid sequence. We argue that POF is involved in a chromosome-specific regulatory function.

Place, publisher, year, edition, pages
Washington: National Academy of Sciences, 2004
Keyword
Animals, Cell Line, Chromosomal Proteins; Non-Histone/*genetics, Chromosomes/*genetics/metabolism, Conserved Sequence/genetics, DNA Transposable Elements/genetics, Drosophila/classification/cytology/*genetics, Drosophila Proteins/*genetics, Evolution; Molecular, Exons/genetics, Female, Fertility/genetics, Gene Expression Regulation, Genes; Essential/genetics, Introns/genetics, Male, Molecular Sequence Data, Mutation/genetics, Organ Specificity, Ovary/cytology/metabolism, Response Elements/genetics, Species Specificity, Substrate Specificity, X Chromosome/genetics/metabolism
Identifiers
urn:nbn:se:umu:diva-17035 (URN)10.1073/pnas.0400978101 (DOI)15210994 (PubMedID)
Available from: 2008-01-12 Created: 2008-01-12 Last updated: 2017-12-14Bibliographically approved
5. Painting of fourth in Drosophila spermatogenesis
Open this publication in new window or tab >>Painting of fourth in Drosophila spermatogenesis
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2131 (URN)
Available from: 2007-02-15 Created: 2007-02-15 Last updated: 2010-05-11Bibliographically approved

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CiteExportLink to record
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Citation style
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