ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation

Abstract

Prdm14 is a sequence-specific transcriptional regulator of embryonic stem cell (ESC) pluripotency and primordial germ cell (PGC) formation. It exerts its function, at least in part, through repressing genes associated with epigenetic modification and cell differentiation. Here, we show that this repressive function is mediated through an ETO-family co-repressor Mtgr1, which tightly binds to the pre-SET/SET domains of Prdm14 and co-occupies its genomic targets in mouse ESCs. We generated two monobodies, synthetic binding proteins, targeting the Prdm14 SET domain and demonstrate their utility, respectively, in facilitating crystallization and structure determination of the Prdm14-Mtgr1 complex, or as genetically encoded inhibitor of the Prdm14-Mtgr1 interaction. Structure-guided point mutants and the monobody abrogated the Prdm14-Mtgr1 association and disrupted Prdm14's function in mESC gene expression and PGC formation in vitro. Altogether, our work uncovers the molecular mechanism underlying Prdm14-mediated repression and provides renewable reagents for studying and controlling Prdm14 functions.DOI: http://dx.doi.org/10.7554/eLife.10150.001

Monobodies and other synthetic binding proteins for expanding protein science

“…A Monobody binding to the pre‐SET/SET domain of a transcriptional regulator, human Prdm14 (different from the crystallization chaperone described earlier) inhibits its interaction with a co‐repressor Mtgr1 . When expressed in the nucleus of embryonic stem cell under an inducible promoter, it inhibited the progression to primordial germ cells.…”

“…In the recent structure of the extracellular region of an adhesion GPCR, GPR56/ADGRG1, a Monobody simultaneously interacts with two domains of GPR56 via two separate regions on its opposite ends, presenting yet another way to reduce the inter‐domain motions . A combination of a Monobody chaperone and linking of heterodimer into a single‐chain construct was used to determine the structure of an otherwise ill‐behaving Prdm14‐Mtgr1 complex . Furthermore, their small sizes may be important for crystallizing integral membrane proteins using the lipid cubic phase method, because of the limited size of cavities that can accommodate water‐exposed portions of the protein system, i.e., the water‐exposed portion of the target protein plus the chaperone .…”

“…Intracellular expression of Monobodies fused to epitope tags followed by affinity capture and mass spectroscopy‐based proteomic analysis has been used to validate the specificity of these molecules. The beauty of this approach is that one can use the same experimental system for both specificity validation and biological characterization . Whereas most of the Monobodies tested selectively capture their respective targets (e.g., only the targeted SH2 domain among ∼100 SH2 containing proteins), one Monobody developed for the SH2 domain of SHP2 exhibited substantial binding to a metabolic enzyme, an unanticipated off‐target .…”

Retinoic acid signaling in regulation of meiosis during embryonic development in mice

“…It could be rationalized that Wnt signaling might be required for making epiblast cells competent for responding to BMP signaling to initiate PGC fate specification. Additionally, PRDM14 interacts with MTGR1 (also called CBFA2T2) and together recruits HDACs (histone deacetylases) for inducing gene silencing (Nady et al, 2015;Tu et al, 2016). The PGC competent epiblast cells are initially directed toward somatic fate, but a three pronged repressive activity of Prdm1, Prdm14, and Tcfap2c actively suppresses the expression of somatic genes and promotes PGC specification (Ohinata et al, 2005;Weber et al, 2010;Yamaji et al, 2008).…”

“…PRDM14 protein has been shown to interact with the protein SUZ12, which is a component of PRC2 (polycomb repressive complex 2), and add a repressive mark in the form of H3K27me3 . Additionally, PRDM14 interacts with MTGR1 (also called CBFA2T2) and together recruits HDACs (histone deacetylases) for inducing gene silencing (Nady et al, 2015;Tu et al, 2016). Although signaling to induce PGC specification is active in the posterior epiblast, the anterior visceral endoderm secretes anti-BMP signaling factors that inhibit PGC induction in the anterior epiblast and thus restrict PGC induction to posterior epiblast.…”