A critical but divergent role of PRDM14 in human primordial germ cell fate revealed by inducible degrons

Abstract

PRDM14 is a crucial regulator of mouse primordial germ cells (mPGC), epigenetic reprogramming and pluripotency, but its role in the evolutionarily divergent regulatory network of human PGCs (hPGCs) remains unclear. Besides, a previous knockdown study indicated that PRDM14 might be dispensable for human germ cell fate. Here, we decided to use inducible degrons for a more rapid and comprehensive PRDM14 depletion.We show that PRDM14 loss results in significantly reduced specification efficiency and an aberrant transcriptome of human PGC-like cells (hPGCLCs) obtained in vitro from human embryonic stem cells (hESCs). Chromatin immunoprecipitation and transcriptomic analyses suggest that PRDM14 cooperates with TFAP2C and BLIMP1 to upregulate germ cell and pluripotency genes, while repressing WNT signalling and somatic markers. Notably, PRDM14 targets are not conserved between mouse and human, emphasising the divergent molecular mechanisms of PGC specification. The effectiveness of degrons for acute protein depletion is widely applicable in various developmental contexts. MainGametes develop from primordial germ cells (PGCs), the embryonic precursors, which are apparently specified in ~week 2-3 (Wk2-3) human embryos 1, 2 . While the requirement for BMP and WNT signalling for PGC specification is conserved between mouse and human 3-5 , the gene regulatory network for hPGC fate has diverged 6 . Mouse PGCs (mPGCs) are specified by three core transcription factors (TFs): Prdm1 (encoding BLIMP1), Prdm14 and Tfap2c (encoding AP2γ) 7, 8 , among which PRDM14 plays a central role; loss of Prdm14 abrogates mPGC specification 9 , while its overexpression is sufficient to induce mPGC fate in vitro 8 .During mPGC specification, PRDM14 induces upregulation of germline-specific genes, assists BLIMP1mediated repression of somatic transcripts and initiates global epigenetic reprogramming 7, 8, 10, 11 . PRDM14 also has a significant role in preimplantaion development 12 , as well as pluripotency induction and maintenance in both mouse and human [13][14][15][16] . Indeed, PRDM14 knockdown in hESCs led to a decrease in OCT4 levels and elevated expression of lineage markers 13, 17, 18 .Despite its critical function in mPGC specification, the role of PRDM14 in hPGC development remains uncertain, due to its low and potentially cytoplasmic expression in gonadal hPGCs 3 . Furthermore, a partial PRDM14 knockdown suggested it might not be important for hPGC specification in vitro 19 , within the TF network for hPGC specification that has diverged significantly from mouse 1, 6, 20 . In particular, SOX17 is a key determinant of hPGC fate, acting upstream of BLIMP1 and TFAP2C 3 , but it is dispensable for mPGC 2 development 21, 22 . Understanding whether PRDM14 has a role in hPGC specification is critical towards gaining insights on the molecular divergence between mouse and human PGCs.An inducible system for PRDM14 loss of function during hPGCLC specification from hESCs is critical, since PRDM14 is also vital for hESC pluripotency 13 . According...

Testing the role of SOX15 in human primordial germ cell fate

“…For ProteoTuner overexpression, SOX15 cDNA was cloned into the PB-EF1-myc-DD-IRES-Puro backbone using InFusion. This plasmid was stably integrated into N3tdT hESCs using the PiggyBac system 6 . Full plasmid sequences with annotation are listed in the Extended data 24 .…”

“…Genotyping gels for AID knock-ins are shown in Extended Figure 1 26 . After the AID tag was introduced, cells were subsequently transfected with TIR1 using the PiggyBac system 6 , and the selectable marker was excised using transient expression of Dre recombinase 27 .…”

“…In mice, PGC fate is specified by a core network of three transcription factors: BLIMP1, PRDM14, and AP2γ 2 . These genes are also important in hPGCs 6,7 , but their activities depend on SOX17, which is the crucial specifier of hPGC fate 5 . Indeed, pigs, which are not closely related to primates, rely on SOX17 for germline specification 8 , suggesting that the SOX17-driven mode of PGC specification is likely to represent a pathway conserved among non-rodent mammals 9 .…”

“…This happens rapidly, causing complete depletion within one hour. The presence of a Venus fluorescent reporter tag is compatible with AID, and this combination has previously been used successfully in hPGCLCs 6 . In accordance with the known role for SOX17 in the human germline, we found that its depletion prevented hPGCLC specification.…”

Unique and redundant roles of SOX2 and SOX17 in regulating the germ cell tumour fate

“…In the end, in PGC-like cells all three transcription factors form a self-regulatory network including feedback loops regulating their own expression 35,38 . Interestingly, PRDM14 was recently introduced as a fourth factor (next to SOX17, TFAP2C and PRDM1) maintaining human PGC pluripotency 43 . A different study further suggested that an aberrant overexpression of PRDM14 during PGC development could favour a malignant transformation of the cells 44 .…”