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Dissection of the epigenetic asymmetry in mouse zygotes

Kunzmann, Rico. Dissection of the epigenetic asymmetry in mouse zygotes. 2012, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9911

Abstract

PRC1 targeting to heterochromatin and its dependency with the Suv39h/Hp1β pathway in mouse zygotes.
Constitutive heterochromatin in mouse is detected around the centromeres of chromosomes. It consists of the repetitive AT-rich major satellite repeats. In mouse zygotes, maternal pericentric heterochromatin (PCH) displays the marks of the Suv39h pathway: tri-methylation of lysine 9 on histone tail H3 (H3K9me3), H4K20me3 and Hp1β (Santos et al., 2005). Paternal PCH on the other hand does not exhibit the Suv39h marks. It is enriched for proteins of the Polycomb repressive complex 1 (PRC1) (Puschendorf et al., 2008). This complex, has been shown to be important for gene repression (Lewis, 1978). In mouse zygotes, however, PRC1 also represses paternal transcription of major satellite repeats (Puschendorf et al., 2008).
The first major question addressed in this thesis is how PRC1 is targeted to PCH. We show that PRC1 targeting to paternal PCH is dependent on two protein modules of Cbx2, a core member of PRC1 in early mouse zygotes. The first module is the N-terminal chromodomain (CD), which preferentially binds H3K27me3 (Kaustov et al., 2011), a histone mark that appears on paternal PCH in the late zygotic stages. The second module is an AT-hook motif, which is located just C-terminal of the CD. Insertion of a point mutation into either the CD or the AT-hook results in reduced heterochromatin enrichment. Nevertheless, only the introduction of point mutations into both modules results in complete loss of enrichment. Finally, we show that among all five Cbx paralogs in mouse, only Cbx2 contains an AT-hook motif, which is highly conserved from fish to humans. Thus, this targeting mechanism of PRC1 is strictly Cbx2 dependent.
The second major question addressed in this thesis concerned the hierarchy of the two major epigenetic, repressive pathways at PCH in mouse zygotes. Zygotes that are deficient for the Suv39h2 histone methyltransferase (HMT), exhibit PRC1 enrichment at paternal and maternal PCH. This observations suggests a hierarchy between the two epigenetic, repressive pathways. We show that Hp1β, a downstream member of the Suv39h pathway, but not the Suv4-20 HMTs, prevents PRC1 members from binding to maternal PCH. In Hp1β maternally deficient zygotes (Hp1βm-z+) as in Suv39h2m-z+ zygotes, PRC1 members strongly localize to maternal PCH. Furthermore, we show that enhancing the affinity of the Cbx2 CD for H3K9me3 by a single amino acid (aa) exchange enables its co-localization with members of the Suv39h pathway on maternal PCH. We show that this aa residue confers the H3K27me3 specificity of Cbx2. Interestingly, this aa residue is conserved in Cbx2 among eumetazoa.
Taken together, we propose a simple targeting mechanism for PRC1 to heterochromatin, based on a CD and an AT-hook motif. Furthermore, we map the interdependency of the Suv39h pathway with PRC1 to the CDs of Hp1β and Cbx2.
Size Difference of Maternal and Paternal Pronuclei
Although the maternal and the paternal genomes are equally large, the paternal pronucleus (PN) is bigger than the maternal PN. This suggests that DNA in the maternal PN is more compacted. Interestingly, this size difference is not only observed in PN5 zygotes but throughout zygotic development, which suggests that it is maintained by proteins present in the zygote.
Here we show that the size difference between maternal PN and paternal PN is due to Hp1β. The maternal PN is smaller than the paternal PN because of Hp1β, putatively bound to maternal H3K9me3. In Hp1βm-z+ zygotes, this size difference is lost. Microinjection of recombinant Hp1β into Hp1βm-z+ zygotes reestablishes the size decrease of maternal PN, suggesting that it is a zygotic phenotype. Furthermore, the maternal and paternal PN sizes of wt zygotes can be decreased by the presence and the enzymatic activity of abundant exogenously provided proteins of the Suv39h pathway.
Impact of Epigenetic Repressors on Zygotic 5mC to 5hmC conversion
Within a few hours after fertilization, the paternal genome rapidly loses its global 5mC DNA methylation (Mayer et al., 2000a; Oswald et al., 2000; Santos et al., 2002). The maternal genome remains DNA methylated in the zygote. Recently, it was shown that paternal 5mC is converted to 5hmC by the Tet3 proteins (Gu et al., 2011).
Zygotes, which were maternally deficient for either PcG proteins or members of three H3K9 HMT pathways, were analyzed for their maternal and paternal 5mC and 5hmC content. The most evident effect on the 5mC to 5hmC conversion in zygotes was observed in maternally deficient G9a and Hp1β zygotes. In both lines, enhanced conversion of maternal 5mC was observed. This suggests that G9a and even more so Hp1β protect maternal 5mC from conversion to 5hmC.
Advisors:Peters, Antoine
Committee Members:Reuter, Gunter
Item Type:Thesis
Thesis no:9911
Bibsysno:Link to catalogue
Number of Pages:164 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:49
Deposited On:05 Jul 2012 09:31

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