Functional analysis of the Nanog tryptophan repeat

Abstract

The homeodomain transcription factor NANOG promotes self-renewal of embryonic stem cells (ESCs) and specification of primordial germ cells (PGCs). NANOG binds thousands of sites within the ESC genome together with OCT4, SOX2 and RNA polymerase II (RNAPII) regulating transcription of its target genes. Replacing tryptophans within the NANOG tryptophan repeat (WR) region for alanines (W10A) causes NANOG to lose its capacity to homodimerise and to interact with other proteins including RNAPII, SOX2 and RESF1. NANOGW10A mutant is also unable to sustain LIF-independent self-renewal. However, the requirements of the WR to interact with other proteins for NANOG function remains unclear. To characterise the cellular localisation of RESF1, endogenously tagged Resf1 mouse embryonic stem cell lines were produced. RESF1 is a widely expressed nuclear protein which interacts with NANOG in nuclei of ESCs. To quantify the requirements of Resf1 for ESC self-renewal and NANOG function, Resf1-/- ES cell lines were derived. In serum/LIF culture, Resf1 deletion promotes cell differentiation and decreases expression of Pou5f1, Nanog and Esrrb. Episomal expression of NANOG sustains LIF-independent self-renewal independently of Resf1. Resf1-/- ESCs are also capable to differentiate into epiblast stem cells. To investigate the role of Resf1 in PGC specification, Resf1-/- ESCs were differentiated into PGC-like cells (PGCLCs). Deletion of Resf1decreasedPGCLCspecification4-foldandpreventedefficientactivation of Blimp1, Prdm14 and Ap2γ. The effect of NANOG on OCT4, SOX2 and RNAPII chromatin binding was evaluated by inducing NANOG in Nanog-/- mouse ESCs. OCT4, SOX2 and RNAPII occupancy before and shortly after NANOG induction was measuredbyanalysingchromatinimmunoprecipitationfollowedbysequencing data sets. The k-mean clustering of Nanog binding sites revealed seven clusters with different response patterns of OCT4, SOX2 and RNAPII to NANOG. The sites where NANOG increases OCT4, SOX2 and RNAPII chromatin binding are mostly associate with the genes activated by NANOG, whereas sites where RNAPII binding decreases after NANOG induction associate with genes repressed by NANOG. To assess the requirements of the NANOG WR and the NANOG DNA binding for directing OCT4, SOX2 and RNAPII chromatin binding, NANOG variants with debilitating mutations in the WR or the homeodomain were induced in Nanog-/- ESCs and chromatin binding of NANOG, OCT4, SOX2 and RNAPII was assessed. The NANOGW10A has reduced chromatin binding at most of the NANOG binding sites. A reduced DNA binding capacity of recombinant NANOGW10A was also suggested by electrophoretic mobility shift assay. Moreover, NANOGW10A is unable to increase OCT4, SOX2 and RNAPII binding at the sites associated with gene activation. At the sites associated with gene repression, NANOGW10A induction leads to increased OCT4 binding. NANOG variant incapable to bind DNA (NANOGN51A) is unable to direct OCT4 and RNAPII binding. However, SOX2 binding temporarily decreases after NANOGN51A induction. In summary, Resf1 is not essential for ESC self-renewal and NANOG function but it is important for efficient specification of PGCLCs. NANOG directs the core transcription complex regulating self-renewal via the WR. This thesis contributes to understanding of NANOG function in ESC self-renewal and describes the function of previously uncharacterised RESF1 in ESC self-renewal and germ cell specification.