Characterization of factors involved in the X inactivation counting and choice process

Projectomschrijving

Cellen van vrouwelijke zoogdieren bevatten twee X-chromosomen, maar er wordt slechts een exemplaar gebruikt, het andere chromosoom wordt kort na de bevruchting uitgeschakeld. Het is bekend dat tijdens dit X-chromosoom inactivatie proces drie genen, Xist, Tsix en Xite, een cruciale rol spelen. Op welke manier bepaald wordt welk van de twee X-chromosomen geïnactiveerd wordt is echter niet bekend. Onderzoek in muizen heeft het vermoeden gewekt dat er nog een onbekende factor een rol speelt in het X-chromosoom inactivatie proces. Omdat fouten in het X-chromosoom inactivatie-proces kunnen leiden tot ernstige genetische aandoeningen is het van belang de factoren betrokken bij dit proces te leren kennen.

Producten

Titel: X-changing information on X inactivation.
Auteur: Barakat TS, Jonkers I, Monkhorst K, Gribnau J.
Titel: RNF12 is an X-Encoded dose-dependent activator of X chromosome inactivation.
Auteur: Jonkers I, Barakat TS, Achame EM, Monkhorst K, Kenter A, Rentmeester E, Grosveld F, Grootegoed JA, Gribnau J.

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Eindverslag

My research group studies different aspects of the X-chromosome inactivation process. We have initiated studies aimed to understand the mechanism involved in counting the number of X-chromosomes in a nucleus and the choice which X will be inactivated. Existing models explained X-chromosome inactivation as a mutually exclusive process, in which either one of the two X-chromosomes is inactivated. In contrast, our studies revealed that counting and choice is a stochastic process, in which each X-chromosome has an independent probability to be inactivated. The last few years we have tried to identify factors that count the number of X chromosomes in the nucleus, involved in the initiation of X-chromosome inactivation. Our studies revealed RNF12 to be an XCI-activator; two copies of this X-linked gene are sufficient for the initiation of X inactivation (Cell 2009). This is the first protein shown to be involved in the XCI counting process, and analysis of knockout female ES cell lines indicated the presence of one or more additional activators that cooperate with RNF12 in counting the number of X-chromosomes in the nucleus, and in initiation of X-chromosome inactivation (PLoS Genet.). Future research directions will involve the identification of the targets of E3 ubiquitin ligase RNF12, the role of Rnf12 in X-chromosome inactivation in vivo, and the identification of other XCI-activators.

Samenvatting van de aanvraag

In mammals, dosage compensation of X-encoded genes is achieved by random inactivation of one of the two X chromosomes in female cells. Three non-coding genes, Xist, Tsix and Xite, located in a small region on the X chromosome play a crucial role in the inactivation process. Xist accumulates on the inactive X chromosome and initiates silencing in cis. Tsix and Xite play an important role in down regulation of Xist during X chromosome inactivation (XCI). XCI starts with a counting and choice process, in which the number of X chromosomes is determined and the future active and inactive X chromosomes are elected. This process is directed by a stochastic mechanism, in which each X chromosome has a certain probability to be inactivated, independent of other X chromosome(s) within the nucleus. The molecular mechanisms driving the counting and choice process remain elusive so far. We have recently shown that XCI is still initiated on the wild type X chromosome in female mouse cells were the other X chromosome carries a heterozygous deletion of both Xist, Tsix and Xite. This result indicates the presence of a novel X-encoded activator of the XCI process. Here we propose to identify and characterize this XCI-activator, using different strategies. Preliminary evidence indicates that the XCI-activator acts through Xist, and that the nuclear concentration of the XCI-activator in combination with the Xist and Tsix promoter strengths determine the probability for an X chromosome to be inactivated. Skewed XCI in mice can therefore be explained as the consequence of two X chromosomes with different Xist and/or Tsix promoters. Skewing of XCI is also observed human, and is directly related to the onset and severity of several X-linked diseases. To analyze the role of Xist and Tsix promoter SNPs in skewing of XCI in mouse and human we are planning to sequence these promoters in mouse strains that show skewed XCI when intercrossed, and in human embryos that show skewed XCI. In contrast to mice, were important players in XCI, including Xist and Tsix, have been well characterized, knowledge about XCI in human is very limited. We therefore propose to study XCI in human induced pluripotent stem (iPS) cells, generated from human fibroblasts.

Onderwerpen

Kenmerken

Projectnummer:
91208011
Looptijd: 100%
2009
2014
Onderdeel van programma:
Gerelateerde subsidieronde:
TOP 2008 R1
Projectleider en penvoerder:
Prof. dr. J. Gribnau
Verantwoordelijke organisatie:
Erasmus MC