Mad2l2 in primordial germ cell development and pluripotency

Mad2l2 in primordial germ cell development and pluripotency

Mehdi Pirouz

Doctoral thesis

Date of Examination: 2013-02-22

Date of issue: 2013-11-18

Advisor: Kessel, Michael Prof. Dr.

Referee: Engel, Wolfgang Prof. Dr.

Referee: Eichele, Gregor Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0001-BC58-8

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Abstract

English

The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while the cells are arrested in the G2 phase of their cell cycle. The significance and underlying molecular mechanism of these events were so far unknown. In this study, a role of the Mad2l2 (Mad2B, Rev7) gene product in development of PGCs was investigated. Mad2l2 is a HORMA domain protein, which is involved in proteinprotein interaction. Mad2l2 is essential for PGC, but not for somatic development. PGCs were specified normally in Mad2l2-/- embryos, but their chromatin did not proceed from the H3K9me2 to H3K27me3 state. Mad2l2-/- PGCs failed to arrest in the G2 phase, and were eliminated by apoptosis. Co-immunoprecipitation analysis showed that Mad2l2 could interact with the histone methyltransferases G9a and GLP, and thus lead to a downregulation of H3K9me2. It also interacts physically with Cdk1, which together with Cyclin B1 is the main driving force for mitotic entrance and progression. The inhibitory binding of Mad2l2 to Cdk1 could arrest the cell cycle in the G2 phase, and therefore allow the other histone methyltransferase, Ezh2, to upregulate H3K27me3. In conclusion, Mad2l2 is essential for epigenetic reprogramming in mouse PGCs. Mad2l2-/- ESC lines cultured in conventional condition (LIF/Serum) showed evidence for a spontaneous differentiation into epithelial-like cells with the molecular characteristics of primitive endoderm. They had a cell cycle similar to that of differentiated cells, failed to incorporate into chimeras, and deviated to primitive endoderm at the expense of pluripotency. They also manifested an epigenetic configuration distinct from normal ESCs, namely elevated H3K9me2 and H3K27me3 levels. Differentiating cells disappeared once the cultures were shifted to the chemically defined medium supplemented with two inhibitors of MEK and Wnt pathways (LIF/2i). Co-immunoprecipitation of Mad2l2 in ESCs followed by mass spectrometry identified CETN2 as its interacting partner. This interaction might be involved in regulation of Nanog expression and thus in sustaining self-renewal of ESCs. Altogether, these data point to essential role of Mad2l2 in pluripotent ESCs.

Keywords: Mad2l2, Primordial Germ Cell, Pluripotency, Cell Cycle, Epigenetic