ADNP modulates SINE B2-derived CTCF-binding sites during blastocyst formation in mice.

CTCF is crucial for chromatin structure and transcription regulation in early embryonic development. However, the kinetics of CTCF chromatin occupation in preimplantation embryos have remained unclear. In this study, we used CUT&RUN technology to investigate CTCF occupancy in mouse preimplantation development. Our findings revealed that CTCF begins binding to the genome prior to zygotic genome activation (ZGA), with a preference for CTCF-anchored chromatin loops. Although the majority of CTCF occupancy is consistently maintained, we identified a specific set of binding sites enriched in the mouse-specific short interspersed element (SINE) family B2 that are restricted to the cleavage stages. Notably, we discovered that the neuroprotective protein ADNP counteracts the stable association of CTCF at SINE B2-derived CTCF-binding sites. Knockout of Adnp in the zygote led to impaired CTCF binding signal recovery, failed deposition of H3K9me3, and transcriptional derepression of SINE B2 during the morula-to-blastocyst transition, which further led to unfaithful cell differentiation in embryos around implantation. Our analysis highlights an ADNP-dependent restriction of CTCF binding during cell differentiation in preimplantation embryos. Furthermore, our findings shed light on the functional importance of transposable elements (TEs) in promoting genetic innovation and actively shaping the early embryo developmental process specific to mammals.

The roles of TET family proteins in development and stem cells.

Ten-eleven translocation (TET) methylcytosine dioxygenases are enzymes that catalyze the demethylation of 5-methylcytosine on DNA. Through global and site-specific demethylation, they regulate cell fate decisions during development and in embryonic stem cells by maintaining pluripotency or by regulating differentiation. In this Primer, we provide an updated overview of TET functions in development and stem cells. We discuss the catalytic and non-catalytic activities of TETs, and their roles as epigenetic regulators of both DNA and RNA hydroxymethylation, highlighting how TET proteins function in regulating gene expression at both the transcriptional and post-transcriptional levels.

Decoding human in vitro terminal erythropoiesis originating from umbilical cord blood mononuclear cells and pluripotent stem cells.

Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single-cell resolution. Interestingly, we detected a few progenitor cells and non-erythroid cells from both origins. In PSC-derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN-derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic-related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony-forming unit-erythroid cells. Inhibition of CD99 reduced the proliferation of PSC-derived cells and facilitated erythroid maturation. Furthermore, CD99-CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression.

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 µg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

PROTAC-mediated vimentin degradation promotes terminal erythroid differentiation of pluripotent stem cells.

BACKGROUND: Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. METHODS: In this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells. RESULTS: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. CONCLUSIONS: VIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.

The pluripotency factor Tex10 finetunes Wnt signaling for PGC and male germline development.

Testis-specific transcript 10 (Tex10) is a critical factor for pluripotent stem cell maintenance and preimplantation development. Here, we dissect its late developmental roles in primordial germ cell (PGC) specification and spermatogenesis using cellular and animal models. We discover that Tex10 binds the Wnt negative regulator genes, marked by H3K4me3, at the PGC-like cell (PGCLC) stage in restraining Wnt signaling. Depletion and overexpression of Tex10 hyperactivate and attenuate the Wnt signaling, resulting in compromised and enhanced PGCLC specification efficiency, respectively. Using the Tex10 conditional knockout mouse models combined with single-cell RNA sequencing, we further uncover critical roles of Tex10 in spermatogenesis with Tex10 loss causing reduced sperm number and motility associated with compromised round spermatid formation. Notably, defective spermatogenesis in Tex10 knockout mice correlates with aberrant Wnt signaling upregulation. Therefore, our study establishes Tex10 as a previously unappreciated player in PGC specification and male germline development by fine-tuning Wnt signaling.

Comparative functional genomics identifies unique molecular features of EPSCs.

Extended pluripotent or expanded potential stem cells (EPSCs) possess superior developmental potential to embryonic stem cells (ESCs). However, the molecular underpinning of EPSC maintenance in vitro is not well defined. We comparatively studied transcriptome, chromatin accessibility, active histone modification marks, and relative proteomes of ESCs and the two well-established EPSC lines to probe the molecular foundation underlying EPSC developmental potential. Despite some overlapping transcriptomic and chromatin accessibility features, we defined sets of molecular signatures that distinguish EPSCs from ESCs in transcriptional and translational regulation as well as metabolic control. Interestingly, EPSCs show similar reliance on pluripotency factors Oct4, Sox2, and Nanog for self-renewal as ESCs. Our study provides a rich resource for dissecting the regulatory network that governs the developmental potency of EPSCs and exploring alternative strategies to capture totipotent stem cells in culture.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

DUX-miR-344-ZMYM2-Mediated Activation of MERVL LTRs Induces a Totipotent 2C-like State.

Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos, although the molecular mechanism underlying such an expanded 2CLC potency remains elusive. We examine global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We find that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with activation of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by DUX and post-transcriptionally represses ZMYM2 and its partner LSD1, and ZMYM2 recruits LSD1/HDAC corepressor complex to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the previously unappreciated DUX-miR-344-Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.

Esrrb plays important roles in maintaining self-renewal of trophoblast stem cells (TSCs) and reprogramming somatic cells to induced TSCs.

Trophoblast stem cells (TSCs), which can be derived from the trophoectoderm of a blastocyst, have the ability to sustain self-renewal and differentiate into various placental trophoblast cell types. Meanwhile, essential insights into the molecular mechanisms controlling the placental development can be gained by using TSCs as the cell model. Esrrb is a transcription factor that has been shown to play pivotal roles in both embryonic stem cell (ESC) and TSC, but the precise mechanism whereby Esrrb regulates TSC-specific transcriptome during differentiation and reprogramming is still largely unknown. In the present study, we elucidate the function of Esrrb in self-renewal and differentiation of TSCs, as well as during the induced TSC (iTSC) reprogramming. We demonstrate that the precise level of Esrrb is critical for stem state maintenance and further trophoblast differentiation of TSCs, as ectopically expressed Esrrb can partially block the rapid differentiation of TSCs in the absence of fibroblast growth factor 4. However, Esrrb depletion results in downregulation of certain key TSC-specific transcription factors, consequently causing a rapid differentiation of TSCs and these Esrrb-deficient TSCs lose the ability of hemorrhagic lesion formation in vivo. This function of Esrrb is exerted by directly binding and activating a core set of TSC-specific target genes including Cdx2, Eomes, Sox2, Fgfr4, and Bmp4. Furthermore, we show that Esrrb overexpression can facilitate the MEF-to-iTSC conversion. Moreover, Esrrb can substitute for Eomes to generate GEsTM-iTSCs. Thus, our findings provide a better understanding of the molecular mechanism of Esrrb in maintaining TSC self-renewal and during iTSC reprogramming.

Inhibition of Aberrant DNA Re-methylation Improves Post-implantation Development of Somatic Cell Nuclear Transfer Embryos.

Somatic cell nuclear transfer (SCNT) enables cloning of differentiated cells by reprogramming their nuclei to a totipotent state. However, successful full-term development of SCNT embryos is a low-efficiency process and arrested embryos frequently exhibit epigenetic abnormalities. Here, we generated genome-wide DNA methylation maps from mouse pre-implantation SCNT embryos. We identified widespread regions that were aberrantly re-methylated, leading to mis-expression of genes and retrotransposons important for zygotic genome activation. Inhibition of DNA methyltransferases (Dnmts) specifically rescued these re-methylation defects and improved the developmental capacity of cloned embryos. Moreover, combining inhibition of Dnmts with overexpression of histone demethylases led to stronger reductions in inappropriate DNA methylation and synergistic enhancement of full-term SCNT embryo development. These findings show that excessive DNA re-methylation is a potent barrier that limits full-term development of SCNT embryos and that removing multiple epigenetic barriers is a promising approach to achieve higher cloning efficiency.

Direct induction of neural progenitor cells transiently passes through a partially reprogrammed state.

The generation of functional neural progenitor cells (NPCs) holds great promise for both research and clinical applications in neurodegenerative diseases. Traditionally, NPCs are derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), or NPCs can be directly converted from somatic cells by sets of transcription factors or by a combination of chemical cocktails and/or hypoxia. However, the ethical issues of ESCs, the risk of tumorigenesis from iPSCs and transgenic integration from exogenous genes as well as complicated manipulation and time-consuming of chemical induced NPCs (ciNPCs) limit the applications of these strategies. Here, we describe a novel method for generating growth factor-induced neural progenitor cells (giNPCs) from mouse embryonic and adult fibroblasts by using inductive and/or permissive signaling culture conditions. These giNPCs closely resemble brain-derived NPCs in terms of transcription networks and neural lineage differentiation potentials. Moreover, this somatic cell to NPC induction is a gradual process that includes initiation, intermediate, maturation and stabilization stages. Importantly, gene expression and histone modification analyses further indicate a partially reprogrammed state during the generation process of induced NPCs, in which lineage specific genes and pluripotency associated genes are transiently activated. Our study therefore describes the potential safety problems that also exist in the transgene-free direct induction strategy and highlights the importance of excluding the possibility of residual partially reprogrammed and/or teratoma-like cells from the generated NPCs for future clinical trials.