5 research outputs found
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NANOGP1 as a Model to Study the Consequences of Gene Duplications on Human Pluripotency and Development
Gene duplication events play an important role in genome evolution; they can also create developmental strategies that differ between species. However, the functional contribution of duplicated genes in early human development and pluripotency is poorly understood. To address this knowledge gap, I investigated NANOGP1, which is a duplicated pseudogene of a key pluripotency factor called NANOG. NANOGP1 was chosen as a model for studying gene duplication in human pluripotency for several reasons. Firstly, NANOGP1 is an evolutionarily conserved duplicate in Hominidae that appears to have an intact coding sequence. The pseudogene is currently annotated as non-protein-coding, although no functional assays have been performed to test this. Secondly, upon investigating the expression of pseudogenes in human naïve pluripotent stem cells (PSCs), I found that NANOGP1 is among the top 1% of the highest expressed pseudogenes. Because high expression levels of NANOG are crucial for maintaining human pluripotency, I hypothesised that a duplicated copy of this important developmental regulator could have similar properties and might contribute to the regulation of human pluripotency.
Gene expression profiling revealed that NANOG and NANOGP1 have overlapping but distinct expression patterns, both in human embryos and in PSC states. NANOGP1 is highly expressed in naïve pluripotent cells but is significantly downregulated in primed pluripotent cells, while NANOG expression levels do not differ to the same extent between the two pluripotent states. RNA splicing analysis predicted that NANOGP1 encodes a protein with an intact homeodomain and transactivation domain, but lacking part of the N-terminus. The divergent N-terminus is the main structural difference between NANOG and NANOGP1 and was therefore used in this study to distinguish between the two genes. Using CRISPR/Cas12a-mediated gene editing in naïve PSCs, I introduced an epitope tag at the start of the predicted protein sequence, and this enabled me to demonstrate for the first time that endogenous NANOGP1 encodes an expressed protein.
The ability to be translated into the stable protein raised the possibility that NANOGP1 could have a functional role. To test this, I performed a series of assays and established that at least two key functional properties are conserved between NANOG and NANOGP1: gene autorepression, and the ability to promote primed-to-naïve PSC reprogramming. Alongside this, however, downregulating NANOGP1 expression using inducible CRISPRi in naïve PSCs did not lead to a differentiation phenotype,
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which is in contrast to NANOG loss of function. Finally, using ChIP-seq, I showed that NANOGP1 shared a subset of chromatin binding sites with NANOG, and also, surprisingly, had a small number of unique, NANOG-independent sites particularly at the promoters of neural-associated genes.
Overall, I conclude that NANOGP1, a previously overlooked duplicated copy of NANOG, is an expressed, protein-coding transcription factor in human naïve PSCs. Most of the CDS, and several of the functional properties, are conserved, implying that NANOGP1 could be supporting or cooperating with its ancestral gene copy in stabilising pluripotency. At the same time, differences in the N-terminal of the CDS, binding occupancy, and distinct expression patterns, could potentially contribute to functional diversification. These differences could have significant evolutionary consequences for creating species-specific developmental strategies, such as novel cell type-specific activity, expanded protein interaction networks and interplay with signalling pathways. Collectively, these potential new properties might extend functional potential and, hence, could encourage diversification of developmental mechanisms. Taken together, my work has demonstrated that NANOG/NANOGP1 duplication serves as a paradigm for exploring how pseudogenes could support their ancestral copies, as well as expand the evolutionary potential of conserved developmental programmes
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NANOGP1, a tandem duplicate of NANOG, exhibits partial functional conservation in human naïve pluripotent stem cells.
Peer reviewed: TrueFunder: Darwin TrustFunder: Cambridge Commonwealth, European and International Trust; Id: http://dx.doi.org/10.13039/501100003343Funder: Erasmus+Funder: Babraham Institute; Id: http://dx.doi.org/10.13039/100012067Gene duplication events can drive evolution by providing genetic material for new gene functions, and they create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naïve-state pluripotent stem cells. Sequence analysis and epitope-tagging revealed that NANOGP1 is protein coding with an intact homeobox domain. The duplication that created NANOGP1 occurred earlier in primate evolution than previously thought and has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways, including homeodomain point mutations. NANOGP1 is a strong inducer of naïve pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naïve pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development
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Sox2 modulation increases naïve pluripotency plasticity.
Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity.We thank Yael Costa for critical reading of the manuscript. Peter Humphreys for assistance with imaging. William Mansfield for blastocyst injections. This study was supported by a Wellcome Trust Fellowship (WT101861) to J.C.R.S. B.K.K is supported by a European Research Council grant (639050). K.M. is a recipient of a Darwin Trust of Edinburgh Ph.D. studentship. K.T. is a recipient of a MRC Ph.D. studentship. H.T.S. and L.E.B. were supported
by BBSRC and MRC research grants, BB/R018588/1 and MR/R017735/1 respectively. G.G.S. is funded by BBSRC research grant RG53615
Distinct Molecular Trajectories Converge to Induce Naive Pluripotency.
Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest. Here we show that acquisition of naive pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells (EpiSCs), one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency. These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature. We propose that fine-tuned regulation of this "transition factor" underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions.HTS is
funded by MRC PhD Studentship 1233706, JCRS by Wellcome Fellowship WT101861, and BG by Bloodwise, CRUK, Wellcome and NIH-NIDDK. The authors gratefully acknowledge core support from the Wellcome-MRC Cambridge Stem Cell Institute