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Analyse der transkriptionellen Regulation des humanen endogenen Retrovirus HTDV/HERV-K
No full textDas humane Genom besteht zu etwa 8% aus retroviralen Sequenzen. Davon sind ca. 1-2% dem humanen endogenen Retrovirus K (HERV-K) zuzuordnen. Das Virus ist mit ca. 30-50 Proviren und ca. 10.000 sLTRs im humanen Genom vertreten. HERV-K besitzt intakte ORFs für alle retroviralen Proteine und zusätzlich ein ORF für das akzessorische Protein Rec. Obwohl eine basale Transkription in verschiedenen Geweben nachgewiesen werden konnte, ist eine Expression von HERV-K Proteinen und Viruspartikeln nur in Keimzelltumoren nachgewiesen worden. In dieser Arbeit wurde die transkriptionelle Regulation des gewebespezifischen Promotors von HERV-K näher charakterisiert. Hierbei wurden regulatorisch wichtige Sequenzen mit Hilfe des Luziferase-Assays eingegrenzt. Transkriptionell sensitive Regionen wurden daraufhin im EMSA auf die Bindung von Transkriptionsfaktoren untersucht. Durch transiente Transfektionen von Luziferasereporterkonstrukten in verschiedenen Zelllinien stellte sich heraus, dass HERV-K LTRs in Keimzelltumorzellen aktiv sind, dass es aber auch inaktive LTRs gibt, die im Zuge der Evolution durch Punktmutationen ihre transkriptionelle Aktivität verloren haben. Aktive und inaktive LTRs unterscheiden sich durch Punktmutationen, die sich in verschiedenen Sequenzabschnitten häufen. Chimäre Konstrukte aus aktiven und inaktiven Sequenzabschnitten und gezielte Mutationen und Deletionen in aktiven HERV-K LTRs enthüllten verschiedene DNA-Bereiche, die transkriptionelle Sensitivität aufweisen. Die LTR-Bereiche bps 572-578, bps 757-798 und bps 809-823 waren im Aktivitäts-Assay besonders empfindlich gegenüber Mutation. In diesen Bereichen liegen zum einen GC/GT-Boxen, also Konsensussequenzen für die Transkriptionsfaktoren Sp1 und Sp3, zum anderen Konsensussequenzen für ein Inr und ein DPE. Das Binden von Sp1 und Sp3 auf den GC/GT-Boxen der bps 757-798 konnte durch eine EMSA-Analyse bewiesen werden. Der transkriptionell sensitive Bereich der bps 572-578 weist eine weitere putative GC/GT-Box auf. Die TATA-Box bei bp 532 zeigte sich unempfindlich gegenüber Mutation. Eine Beteiligung dieser Konsensussequenz am basalen Promotor wurde deshalb ausgeschlossen. Unterstützt durch die Ergebnisse einer 5’-RACE, ein Verfahren, dass den Transkriptionsstart eines bestimmten Gens bestimmen kann, konnte gezeigt werden, dass der basale HERV-K Promotor aus Konsensusequenzen für die Transkriptionsfaktoren Sp1 und Sp3 , einem Inr und einem DPE besteht. Sp1 und Sp3 sind ubiquitäre Transkriptionsfaktoren, die durch ihr Mengenverhältnis in einem bestimmten Gewebe oder durch posttranslationale Modifikationen unterschiedliche Auswirkungen auf die Transkription haben können. Diese Eigenschaften könnten zur Gewebespezifität von HERV-K beitragen. Die Core Promotor Komponenten Inr und DPE sind für die korrekte Positionierung von TFIID und damit der PolII verantwortlich. Des weiteren wurde eine starke Beteiligung des Testis-spezifischen Transkriptionsfaktors SRY an der Transkription von HERV-K belegt. Im Luziferase-Assay konnte ein SRY-Expressionsplasmid die Transkription eines aktiven LTRs in GH- wie auch in HeLa-Zellen steigern. Ein Indiz dafür, dass es sich um einen für die Transkription von HERV-K essentiellen Faktor handelt. Verschiedene SRY-Konsensussequenzen auf der HERV-K LTR machen eine Wirkung in cis wahrscheinlich, doch könnte auch in trans ein für die Transkription wichtiger Transkriptionsfaktor in seiner Expression verstärkt werden. Ebenfalls konnte bewiesen werden, dass epigenetische Mechanismen eine starke Rolle bei der Transkription von HERV-K spielen. Die Methylierung eines LTR-haltigen Luziferase-Reportervektors führte zum fast vollständigen Verlust der transkriptionellen Aktivität. Es bleibt abzuwarten, in welcher Weise die Schlüsselkomponenten Sp1, Sp3, SRY und Hypermethylierung des HERV-K Genoms zur Gewebespezifität von HERV-K beitragen
SRY-PCR.
No full textPCR-product of the 519 bp of the segment of the SRY-gene (left column in each section) confirmed the existence of the male specific Y sex chromosome in the examined case with hypospadias. One female and one male control Holstein cattle validated the result. The verification of the 563 bp amplicon of the GON4L gene (right column in each section) indicates the capability of the isolated DNA of the used samples.</p
FGF9 and WNT4 act as antagonistic signals to regulate mammalian sex determination
Get PDFThe genes encoding members of the wingless-related MMTV integration site (WNT) and fibroblast growth factor (FGF) families coordinate growth, morphogenesis, and differentiation in many fields of cells during development. In the mouse, Fgf9 and Wnt4 are expressed in gonads of both sexes prior to sex determination. Loss of Fgf9 leads to XY sex reversal, whereas loss of Wnt4 results in partial testis development in XX gonads. However, the relationship between these signals and the male sex-determining gene, Sry, was unknown. We show through gain- and loss-of-function experiments that fibroblast growth factor 9 (FGF9) and WNT4 act as opposing signals to regulate sex determination. In the mouse XY gonad, Sry normally initiates a feed-forward loop between Sox9 and Fgf9, which up-regulates Fgf9 and represses Wnt4 to establish the testis pathway. Surprisingly, loss of Wnt4 in XX gonads is sufficient to up-regulate Fgf9 and Sox9 in the absence of Sry. These data suggest that the fate of the gonad is controlled by antagonism between Fgf9 and Wnt4. The role of the male sex-determining switch— Sry in the case of mammals—is to tip the balance between these underlying patterning signals. In principle, sex determination in other vertebrates may operate through any switch that introduces an imbalance between these two signaling pathways
Conditional strategies to study gene function during gonadal development in mammals.
No full textSexual development in mammals involves a complex cascade of genetic events. These begin with a cell fate decision, whether to make Sertoli or follicle cells, that gives rise to the development of a male or female gonad, which is controlled by the testis-determining gene Sry. Following the expression of Sry, genes involved in the male pathway act to reinforce and maintain testis-specific cell fate decisions, as well as to repress the female pathway. Sox9 becomes rapidly upregulated after the onset of Sry expression, and is expressed in Sertoli cells throughout life. From mutation studies, SOX9 is known to be essential for male development in humans and to initiate Sertoli cell differentiation in mice. However, the function of SOX9 after sex determination and the reason for its maintenance in Sertoli cells remains unknown. In order to understand the function of Sox9 in the fetal and adult mouse testis, new tools have been generated to control gene activity in a conditional manner. This thesis mainly describes strategies to control either deletion of misexpression of Sox9. To make the tools useful at different stages, the tamoxifen-inducible Cre/loxP system was employed. This involves the establishment of two elements: a "Cre-driver" and a ' Sox9-responder". Cre-driver transgenes were made under the control of several gonadal-specific regulatory elements, as well as a strong, ubiquitous promoter. Responder mice allow Cre activated conditional misexpression or deletion of Sox9. Analyses on gonad morphologies and gene expression levels were compared between animals that have altered Sox9 expression and those that have not. The results reveal that Sox9 is necessary and sufficient for the expression of Sfl in the Sertoli cells, and suggest that Sox9 is antagonistic to the ovarian- specific gene Foxl2. The newly established Cre-drivers can also be applied in functional studies involving other genes implicated in sexual development
A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits
Get PDFWe tested the hypothesis that genes encoded on the sex chromosomes play a direct role in sexual differentiation of brain and behavior. We used mice in which the testis-determining gene (Sry) was moved from the Y chromosome to an autosome (by deletion of Sry from the Y and subsequent insertion of an Sry transgene onto an autosome), so that the determination of testis development occurred independently of the complement of X or Y chromosomes. We compared XX and XY mice with ovaries (females) and XX and XY mice with testes (males). These comparisons allowed us to assess the effect of sex chromosome complement (XX vs XY) independent of gonadal status (testes vs ovaries) on sexually dimorphic neural and behavioral phenotypes. The phenotypes included measures of male copulatory behavior, social exploration behavior, and sexually dimorphic neuroanatomical structures in the septum, hypothalamus, and lumbar spinal cord. Most of the sexually dimorphic phenotypes correlated with the presence of ovaries or testes and therefore reflect the hormonal output of the gonads. We found, however, that both male and female mice with XY sex chromosomes were more masculine than XX mice in the density of vasopressin-immunoreactive fibers in the lateral septum. Moreover, two male groups differing only in the form of their Sry gene showed differences in behavior. The results show that sex chromosome genes contribute directly to the development of a sex difference in the brain
A conserved NR5A1-responsive enhancer regulates SRY in testis-determination
No full textPublisher Copyright: © The Author(s) 2024.The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5’ regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process
SRY and mammalian sex determination
No full textThis chapter discusses the understanding of the biology of sry and sex determination particularly what is known of the biochemical basis of sry function and its relationship to the other genes in the sex determination pathway. Most importantly, it attempts to identify those areas, in which ignorance is greatest and address some of the issues that might concern the researchers in sex determination. Data discussed in this chapter, primarily from the studies of mice and humans; the symbol “sry” are used to refer exclusively to the murine gene and “sRY” to that of humans and other mammals. Two central tenets allow the geneticist to frame the question of the genetic basis of sex determination in mammals, in terms of which gene or genes on the Y chromosome are required for the initiation of testis development. The isolation of the predicted gene(s), known as the testis determining factor (TDF) in humans and testis-determining Y gene (Tdy) in mice, was the subject of an intense international research effort that culminated, with the identification of the human sry gene. Proof of the identity of sry and TDF came in the form of a chromosomally female mouse transgenic for the murine Sry gene: this mouse developed as a normal male, albeit sterile due to the presence of two X chromosomes and the absence of Y chromosomal genes required for spermatogenesis. Sry was thus, shown to be the only Y-linked gene (though by no means the only gene) required for testis determination in mammals
SRY binds to WDR5 promoter.
No full text<p>(A) EMSA analysis of HA tagged SRY with wild type and mutant probes. Arrows indicate SRY-probe complex and supershift. (B) ChIP analysis of HA tagged SRY on WDR5 promoter,WDR5 promoter proximal upstream region, or MyoD promoter. Mouse IgG serves as a negative control. Graphs show mean ± SD, n = 3.</p
Comparative analysis of anti-mouse SRY antibodies
No full textThe Y chromosome gene SRY is the initiator of male sexual differentiation in mammals, but the molecular and cellular mechanisms operating downstream of SRY remain undefined. A deeper understanding of these issues relies on the ability to visualize SRY protein endogenously under a number of experimental conditions. Here we compare the specificity and effectiveness of several available antibodies to mouse SRY. Two antibodies cross-reacted with other SOX proteins in immunofluorescence analyses of transfected cells, and one of these two was unable to detect SRY on Western blots. A third antibody was both avid and specific, and was able to detect endogenous SRY in developing Sertoli cells in mouse genital ridges. Our findings underline the need to distinguish between useful and spurious reagents for biochemical and immunolocalization studies involving mouse SRY protein
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