Milan de Putter, Halla Rós Eyjólfsdóttir, Kristján Hólm Grétarsson, Deo Prakash Pandey and Erna Magnúsdóttir
Primordial germ cells (PGCs) are the founders of the germ cell line and are essential for transferring genetic and epigenetic information across generations. Understanding PGC specification and germ cell line development could therefore be key to addressing reproductive issues such as sub- or infertility.
The RHOX factors are poorly characterized homeobox transcription factors expressed in the germ cell line. In this research, we used mouse embryonic stem cells (ESCs) engineered for either endogenous expression or doxycycline-inducible overexpression of HA- or 3xTY1-tagged RHOX5 and RHOX6. These cells can be induced into epiblast-like cells (EpiLCs) and subsequently into PGC-like cells to study the behaviour of these two RHOX factors in three distinct stages mirroring the in vivo developmental process.
Immunofluorescence microscopy showed heterogeneous expression of RHOX5 and RHOX6 in irregular subpopulations of ESCs and at increasing frequency 24h, 48h, and 72h post-differentiation into EpiLCs. Rapid Immunoprecipitation and Mass Spectrometry of Endogenous proteins (RIME) using crosslinked chromatin immunoprecipitation (ChIP) of overexpressed RHOX5-HA and RHOX6-HA in ESCs identified approx. 700 high-confidence enriched proteins using label-free quantitation (fold change>4 over empty vector control, adj. p-value <0.05). The most enriched proteins, with notable candidates for direct interactions involved in chromatin modification, stem cell maintenance, and pluripotency networks, exhibited between 8 to 12 times fold enrichment over the control. Furthermore, ongoing ChIP-seq experiments aim to uncover RHOX factor binding sites, providing insights into their direct genomic targets. Together, these results could suggest novel roles for the RHOX factors through characterization of their molecular mechanisms and interactions.
