Treating Male Infertility: A Novel Regulatory Circuit for Early Post-Implantation Pluripotent State Acquisition and Male Germ Cell Specification.

N6-Methyladenosine Safeguards Formative Competence for Lineage Entry.We have identified a critical role for the RNA modification N6-Methyladenosine (m6A) in enabling robust somatic and germline entry. By using embryonic stem cells, our study characterizes how m6A is vital to preserve a unique pluripotent state called formative pluripotency. Here, precursors to eggs and sperm arise, enabling reproductive capacity.In the formative phase of pluripotency, mammalian embryonic stem cells acquire the competence for somatic cell differentiation as well as germline entry. This involves transcription factor (TF) network ‘re-wiring’ and epigenetic enhancer remodeling. How these processes are negotiated to establish lineage competence remains poorly defined. In view of exploring formative pluripotency control, we identify key roles for the RNA modification N6-Methyladenosine (m6A) in moderating mouse and human signaling kinase activation. This ensures efficient epigenetic silencing, required for formative state acquisition. Furthermore, m6A prevents the precocious up-regulation of developmental genes, safeguarding the integrity of both mouse and human lineage competence and germline entry. Interestingly, depletion of m6A also promotes the up-regulation of genes involved in germ cell maturation, like DDX469. Hence, timed m6A level manipulation during later differentiation stages may benefit the isolation of post-mitotic, patient derived germ cells for therapy. Understanding m6A dependent regulation of germline competence and maturation will help decode the pathogenesis of infertility and germ cell cancer. These mechanistic insights have propelled our study into revisions this year for publication in the prestigious journal Developmental Cell. We look forrwad therefore for final reporting on this project next year.

N6-Methyladenosine Promotes Murine and Human Germline Entry in Formative Pluripotency.We have found that the RNA modification N6-Methyladenosine is critical for germ cell lineage entry in the mouse. To elevate the impact of the study further, we have invested time to characterize this effect also in human germ cell specification. Our research study will be submitted soon to a high impact journal.Our knock-down screening in the mouse germ cell system showed that Mettl3 and Mettl14 are essential for proper germ cell specification. These enzymes form a complex that globally deposits the modification N-6-Methyladenosine (m6A) on RNA. From this work, we now show that depleting m6A abundances also significantly interferes with human germ cell specification via diverse mechanisms which interestingly converge upon the same key signaling pathway in both species. o elevate the impact of the study further, we have invested time to characterize this effect also in human germ cell specification. Our research study will be submitted soon to a high impact journal.

N6-Methyladenosine Modulates Eras Safeguarding Entry to the Mouse GermlineWe have had tremendous success in this project. We have generated a high impact publication, PNAS, IF:12.4. A PhD student under my supervision is finished and due to defend his thesis in April. We have a second publication in submission to a prestigious, high impact factor journal. We define N6-Methyladenosine (m6A) biology in stem and germ cells.N6-Methyladenosine (m6A) abundances were recently shown to regulate pluripotency departure by controlling the levels of Nanog, a key transcription factor. After pluripotency exit, the role of m6A deposition in cell lineage determination remains poorly understood. Competence for lineage entry is acquired when embryonic stem cells differentiate into a transient epiblast-like cell population. During this phase of transcription factor re-wiring and key epigenetic changes, cells gain the competence for both somatic and germ cell lineage specification. We found that m6A depletion in epiblast-like cells by Mettl3/Mettl14 knock-down impedes germ cell lineage induction. m6A depletion enables the downregulation of pluripotency transcription factors and irreversible pluripotency departure. However, a significant proportion of cells fails undergo key epigenetic changes, necessary for the acquisition of germ cell lineage competence. Whole-transcriptome profiling identified elevated expression of embryonic ras (Eras) which drives signaling activation. Eras knock-down restores enhancer resetting and germ cell lineage induction efficiency in m6A depleted epiblast-like cells. Ythdf1 and 2 m6A readers are both specifically required to regulate the efficient turnover of Eras mRNA transcripts. By modulating Eras activity, m6A deposition ensures epigenetic integrity, safeguarding competence for germ cell lineage specification. Our findings reveal a novel, indispensable role for m6A abundances enabling the acquisition of competence for mouse lineage commitment.

N-6-Methyladenosine Regulates Oct3/4 Distal Enhancer Activity and Germ Cell Lineage Commitment.N6-methyladenosine (m6A) is the most abundant mRNA modification, regulating pluripotency exit in embryonic stem cells. The effects of decreasing m6A abundances during early primordial germ cell (PGC) lineage specification is unknown as m6A deficient embryos die early. We examine m6A depletion effects on PGC lineage specification in vitro.N6-methyladenosine (m6A) abundances regulate pluripotency exit and lineage commitment, although m6A dependent mechanisms involved in cell state control are poorly understood. Here, we show that m6A depletion by Mettl3 or Mettl14 knock-down (KD) decreases mouse embryonic stem cell (mESC) differentiation into the primordial germ-cell-like-cell (PGCLC) lineage. This is due to a significant proportion of m6A depleted cells failing to decommission Oct3/4 distal enhancer activity, required for PGCLC differentiation. Transcriptional profiling shows that m6A depleted mouse embryonic stem cells (mESCs) retain increased expression of the embryonic ras (Eras) oncogene during pluripotency exit. Increased mRNA stability promotes elevated Eras protein levels and phosphatidylinositol-3-OH kinase (PI3K)-Akt activation. PI3K-Akt blocks the cessation of Oct3/4 distal enhancer activity during mESC differentiation. Eras or Akt KD reinstates Oct3/4 distal enhancer decommissioning and restores germ cell lineage commitment propensity in m6A depleted cells. Ythdf1 and 2 m6A reader proteins are specifically required for efficient Eras mRNA turnover and downregulation during differentiation. We define a novel m6A dependent mechanism required for the cessation of naïve epigenetic regulation and robust germ cell lineage commitment.

N-6-Methyladenosine (m6A) regulates the establishment of epigenetic frameworks critical for lineage specification.This cutting-edge, dynamic, single cell resolution study has unravelled precise effects on lineage specification. We abandoned 'old-school' cell population average approaches, which do not reflect true results on individual and heterogeneous cells. Currently we are elucidating a mechanism linking m6A abundances to the regulation of the epigenome.We have cooperated closely with project partners at the Helmholtz Research Center in Munich. Short research stays of 1-2 days were occasionally required to strategize on technology development and progress. This has enormously strengthened ties for future projects and application of our cutting edge technology. Thus far, work conducted between our groups has generated a high impact publication under international peer-review in Cell Reports. Importantly, we have delineated how complex stem cell state dynamics are affected by manipulating the levels of the RNA modification, m6A. We achieved this by applying a cutting-edge, single cell resolution platform established by my lab in Norway. This was possible due to the funding of this project. Using biochemical and next generation sequencing approaches, our study also revealed mechanistic insight for the stem cell and RNA fields. It showed that opposing pathways responsible for steering stem cell states are activated due to m6A manipulation. One pathway wins the tug of war more often than not, leading stem cells into a primed state for differentiation. Conceptually, our study is timely for the scientific conceptual advancement and warrants publication in a high impact international peer review journal. Rather than attributing cell state change to a transcription factor transcript stability mechanism, we show for the first time that changes occur due to the elevation of key signaling pathways. This drives change in cell lineage regulation by affecting the production of hundreds of transcription factors in one swoop. As an offshoot project, this study is well on target to be the second major publication from our lab. Importantly, we are applying our single cell resolution platform to define how germ cell and neural lineage specification is blocked when m6A abundances are decreased. We are using a defined cell lineage specification system for this purpose. In a recent breakthrough, preliminary data shows a connection between m6A abundance regulation and shaping of the epigenome to affect cell lineage choice. We are investigating the mechanisms involved in these key changes on the molecular level.

N6-Methyladenosine (m6A) Mediated Regulation in Germ Cell SpecificationWe are characterising the effects of depleting m6A in embryonic stem cells and examining effects on germ cell specification at single cell resolution using unique live cell imaging technology and reporter cell systems. We have found that decreasing m6A significantly reduces germ cell program induction from embryonic stem cells.N6-methyladenosine (m6A) methylation affects transcription factor (TF) mRNA turnover, although how m6A abundances regulate pluripotent state change and cell lineage commitment is incompletely understood. Current models of m6A dependent pluripotency control and developmental progression converge on regulating the stability of m6A modified TF transcripts. However, orchestrating selective m6A deposition on particular TF populations over others to sustain correct feedforward developmental progression is still poorly understood. Norway has some of the highest rates of male infertility globally. To understand underlying causes and develop treatments, we must assess the impact of changing m6A abundances on germ cell differentiation. Recent studies suggest that changing m6A levels can modulate key regulatory control layers upstream of TFs, including signalling molecule phosphorylation in cancer and histone modifier levels in neural stem cells. Here, we use germ cell reporter mouse ESC lines and have performed germ cell induction in m6A depleted cells to assess effects on germ cell differentiation. The same repertoire is being conducted also on germ cell reporters generated in human embryonic stem cells to compare the human scenario. In the mouse, analyses revealed a central role for m6A levels during a specific and critical stage of germ cell induction. We are currently investigating which regulators of the germ cell induction process are affected by depleted m6A levels. We aim to characterize, how these m6A level dependent changes mechanistically affect germ cell differentiation.

A Novel RNA Regulatory Circuit for Pluripotent Cell State Traverse and Male Germ Cell Specification.We have discovered a novel molecular switch function for an early cell lineage specification from pluripotent embryonic stem cells. By modulating the levels of RNA methyltransferase machinery components and employing quantitative live cell imaging and molecular approaches, we define mechanistic underpinnings for a unique lineage choice.RNA layer control mechanisms involved in pluripotent cell state traverse are poorly understood. The RNA methyltransferase-like 3 and 14 (Mettl3 and Mettl14 ) are critical components of a methyltransferase complex which deposits the dynamic 6-Methyladenosine (m6A) modification unto RNA. In the naïve pluripotent state, Mettl3 loss and complex disruption was shown to promote a hyper-pluripotent cancer-like phenotype. In primed pluripotency and epiblast stem cells (EpiSCs), Mettl3 depletion resulted in cell differentiation or death. We examined the effects of Mettl3 and/or Mettl14 depletion on pluripotency heterogeneity in Serum+LIF conditions using protein fusion, double knock-in mESC reporters (NanogKATUSHKA/Oct4VENUS). By transcription factor, quantitative live-cell imaging over many cell generations, we show for the first time, the emergence of a NanogKATUSHKA-/Oct4VENUSHIGH epiblast like cell state (EpiLC), at single cell resolution. Mettl3 and/or Mettl14 depletion culminates in the upregulation of epiblast transcription factors (TFs) Otx2/Oct6 and changes in pAKT, pErk and pStat signaling pathways. We functionally define how these downstream changes co-operatively promote the EpiLC cell state traverse and maintenance. A current key question in the final stages of our study is whether the EpiLC state is prone to differentiate into a particular cell lineage over another and why. Specifically, we ask if RNA methyltransferase modulation as a molecular switch function promotes germ cell differentiation competence under conditions which do not normally foster such lineage specification. We have now established assays enabling us to assess if RNA methyltransferase modulation further enhances germ cell competence or promotes lineage choice into other cell states, such as neural stem cells.