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At least 19 recordsLinked to original sources
PubMed2026

Manipulation of protein translation and stem cell self-renewal by CRISPR activation of rRNA transcription.

Ribosomal RNA (rRNA) transcription rates vary during development, and their dysregulation is linked to diseases such as cancer and ribosomopathies. Owing to their high abundance and genomic redundancy, the functional significance of rRNA levels remains unclear. We developed TAPIR (Targeted Activation of Protein Translation), a CRISPR-based approach to elevate rRNA levels by inducing 47S ribosomal DNA transcription. TAPIR increased nucleolar size and enhanced protein synthesis, even in rapidly proliferating cells. In neural stem cells, elevated translation promoted self-renewal and proliferation in vitro and in vivo. Furthermore, TAPIR enabled the modeling and partial rescue of associated disease phenotypes. Our findings reveal that rRNA levels directly regulate translational output and that protein synthesis capacity can act as a key determinant of mammalian stem cell behavior.

Animals↗
PubMed2026

E2F7 promotes lung adenocarcinoma progression by affecting phosphorylation and stabilization of β-catenin.

BACKGROUND: E2F transcription factor 7 (E2F7) has been implicated in the tumorigenesis and progression of multiple cancer types; however, the molecular mechanisms through which E2F7 regulates malignant phenotypes in cancer cells remain largely undefined. In this study, we investigated the biological functions and underlying mechanisms of E2F7 in lung adenocarcinoma (LUAD). METHODS: E2F7 expression in LUAD was analyzed using The Cancer Genome Atlas (TCGA) datasets and further validated in clinical specimens via quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. The effects of E2F7 on cancer cell self‑renewal and epithelial-mesenchymal transition (EMT) were assessed using sphere formation and Transwell assays, respectively. In vivo tumorigenicity and metastasis were evaluated using xenograft models combined with extreme limiting dilution analysis to assess tumor-initiating capacity. Wnt/β‑catenin pathway activity was measured using T-cell factor optimal promoter luciferase reporter plasmid/far-from optimal promoter luciferase reporter plasmid (TOP/FOP) flash reporter assays. β‑Catenin expression, stability, and ubiquitination were examined via western blotting, cycloheximide chase assays, and ubiquitination assays. Protein-protein interactions among E2F7, β‑catenin, and glycogen synthase kinase 3 beta (GSK3β) were verified through co‑immunoprecipitation (Co‑IP), glutathione S‑transferase (GST) pull‑down, and immunofluorescence assays. Truncated mutants were generated to map the functional binding domains of E2F7. In vitro immunoprecipitation and kinase assays were further performed to confirm that E2F7 regulates GSK3β autophosphorylation and β‑catenin phosphorylation. RESULTS: Bioinformatic analyses revealed that E2F7 was significantly upregulated in LUAD tissues, and elevated E2F7 expression correlated with poor patient prognosis. Functional assays demonstrated that E2F7 promoted LUAD cell self‑renewal and EMT. Mechanistically, cytoplasmic E2F7 directly associated with β‑catenin through its DNA‑binding domain (DBD) and PHA03247 domain. E2F7 modulated β‑catenin phosphorylation at Ser675 and Ser33/37/T41, thereby inhibiting ubiquitin‑mediated degradation and enhancing β‑catenin protein stability. Furthermore, E2F7 interacted with GSK3β and suppressed its autophosphorylation at Tyr216, concomitant with reduced β-catenin phosphorylation at Ser33/37/T41 and its accumulation. CONCLUSION: Collectively, these findings indicate that E2F7 drives LUAD malignant progression through regulation of the GSK3β/β‑catenin signaling axis and stabilization of β‑catenin. This study unveils a novel oncogenic mechanism of E2F7 in LUAD and identifies E2F7 as a promising therapeutic target for clinical intervention in LUAD.

E2F7↗
PubMed1979

Proliferation kinetics of early hemopoietic precursor cells with self sustaining capacity in the mouse, studied with 125-I-labeled iodo-deoxyuridine.

With a new labeling technique in radiation chimeras, an attempt was made to determine the duration of the phases of the stem cell cycle including shortest and mean generation time and to estimate the number of hemopoietic stem cells per unit of bone marrow cellularity. The DNA of bone marrow cells in DNA synthesis was labeled with 5-125I-2'-deoxyuridine. The labeled cells were followed after being transfused into fatally irradiated mice. The stem cells were found to have a half-time of about 4.3 days in the donor mice. The average time in the population, i.e. the turnover time of the stem cells, was 6.2 days. The half-time did not change significantly even after transfusion of bone marrow into lethally irradiated recipient mice. Tritiated thymidine (3H-TdR) suicide technique revealed that bone marrow stem cells seeding to the spleens and to the femurs of lethally irradiated recipients behaved differently--S-phase in cells seeding to femurs being shorter. The radiosensitivity of stem cells in S-phase had a D0 of 80 rad whereas stem cells distributed throughout the whole cell cycle had a D0 of 185 rad. The respective extrapolation numbers were 1.23 and 1.14. It is calculated that 2--7% of all nucleated bone marrow cells belong to self renewing stem cell populations. The method described provides a new approach for the study of hematological stem cells.

Animals↗
PubMed1978

Regulation of the self-renewal probability in Hydra stem cell clones.

Hydra interstitial stem cells continuously give rise to daughter stem cells as well as precursors for nerve and nematocyte differentiation. Growth of the stem cell population is controlled by the self-renewal probability (Ps): Ps is the fraction of stem cell daughters that remain stem cells in each generation. We have determined Ps for Hydra interstitial stem cells by using a novel technique based on the cell conposition of clones. Stem cell clones were grown in aggregates of nitrogen mustard-inactivated Hydra tissue. They contain several hundred cells after 14 days of growth, including stem cells, differentiating nematocytes, and differentiating nerve cells. Clone size, size variability, and the ratio of differentiating cells to stem cells are sensitive measures of Ps. We have prepared standard curves relating these parameters to Ps, using computer simulations of clone growth. Comparisoon of the experimentally observed parameter of clones to these curves indicates that Ps decreases from 0.8 in 5- to 6-day clones to 0.6 in 10- to 12-day clones. The decrease in Ps coincides with the increase in clone size and suggest that Ps may be regulated by the density of stem cells in clones. Such a mechanism could be responsible for the observed homeostasis of stem cell populations in vivo.

Cell Differentiation↗
PubMed1977

Proliferation and differentiation of normal granulopoietic cells in continuous bone marrow cultures.

Modified conditions are reported for successful continuous bone marrow cultures with stem cell self-renewal and granulocyte-macrophage differentiation. Cells cultured over several weeks were found to be identical to freshly isolated bone marrow cells. Polymorphic neutrophils derived from cultures and primary bone marrow neutrophils both showed C3 AND IgG receptors and both actively phagocytosed foreign particles. Cultured and normal CFU-c were identical, both in their dose responsiveness to CFS and in their sedimentation rate characteristics.

Bone Marrow Cells↗
PubMed2024

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells.

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Animals↗
PubMed1979

Stem cell growth and differentiation in Hydra attenuata. I. Regulation of the self-renewal probability in multiclone aggregates.

Interstitial stem cells in Hydra are rapidly proliferating multipotent stem cells which continuously give rise to precursors for nerve and nematocyte differentiation. Growth of the stem cell population is controlled by the cell cycle time of the stem cells and the self-renewal probability, Ps (the fraction of stem cells in each generation which divide to yield more stem cells). In normal Hydra the stem cell generation time is 24 h and Ps = 0.6; under these conditions the stem cell population doubles in 3.5 days. In the present experiments we have systematically investigated the dependence of Ps on stem cell density. We culture stem cells in a feeder layer system consisting of aggregates of nitrogen-mustard (NM)-inactivated Hydra cells. In this system stem cell density can be varied over a wide range by changing the number of clone-forming units (CFU) added to the aggregates. We have measured the growth rate of the stem cell population and the cell cycle of stem cells in NM aggregates after 4--7 days of culture. From these data we calculate the value of Ps. The results indicate that the growth rate decreases 4-fold as the number of CFU seeded per aggregate increases from 10 to 400. Under these same conditions the cell cycle remains constant. The values of Ps calculated from these results indicate the Ps decreases from 0.75 in aggregates seeded with 10--30 CFU to 0.55 in aggregates seeded with 200--400 CFU. These results support a model in which Ps is controlled by negative feedback from neighbouring stem cells. In addition, our experiments indicate that Ps decreases during the growth of stem cell clones. When only a few stem cells are seeded in aggregates, they give rise to isolated clones distributed throughout the aggregate. Ps decreases markedly within such clones as they grow in size presumably due to increasing stem cell content of the clones. Since Ps in such isolated clones declines with growth, we infer that the local stem cell concentration is what controls Ps and that the spatial range of the negative feedback signal is short compared to the dimensions of NM aggregates.

Animals↗
PubMed1978

Proliferative capacity of murine hematopoietic stem cells.

The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferative quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cell progress in the continuum in one direction and such progression is not reversible.

Animals↗
PubMed2025

Differentiation latency and dormancy signatures define fetal liver hematopoietic stem cells at single-cell resolution.

Decoding the mechanisms governing the self-renewal of hematopoietic stem cells (HSCs) during their expansion in the fetal liver (FL) could unlock novel therapeutic strategies to expand transplantable HSCs, a long-standing challenge. To explore intrinsic and extrinsic regulation of FL-HSC self-renewal at single-cell resolution, we engineered a culture platform replicating the FL endothelial niche that supports the amplification of serially engraftable HSCs. Leveraging this platform together with single-cell index flow cytometry, live imaging, transplantation assays, and single-cell RNA sequencing, we demonstrate that differentiation latency, cell-division symmetry, and transcriptional signatures of biosynthetic dormancy are distinguishing properties of rare FL-HSCs capable of serial multilineage hematopoietic reconstitution. Our findings support a paradigm in which intrinsic programs and niche-derived signals together facilitate the symmetric self-renewal of FL-HSCs while delaying their active participation in hematopoiesis. Our study also provides a resource for future investigations into intrinsic and extrinsic signaling pathways governing FL-HSC self-renewal.

Hematopoietic Stem Cells↗
PubMed1979

Antibodies against pluripotent stem cells: their use in studying stem cell function.

The biologic characteristics and specificity of rabbit anti-mouse brain (RAMB) serum for pluripotent hemopoietic stem cells (CFU-s) is reviewed. The application of RAMB serum to the functional analysis of stem cell differentiation and self renewal characteristics is discussed. Preliminary data are presented which suggest the existence of two stem cell subcompartments. The majority of stem cells express membrane determinants that are detected by RAMB serum. A minor (5%-10%) stem cell subpopulation lacks the stem cell antigen and exhibits a greater self-renewal capacity than those cells expressing the antigen.

Animals↗
PubMed2026

Mapping self-associating chromatin hubs identifies Id proteins as key determinants of exhausted CD8+ T cell fate.

Within days of exposure to chronic viral infections, activated CD8+ T cells differentiate into Tcf1-Slamf6loTim3hi exhaustion-prone effector T (TEX_EFF) cells or self-renewing Tcf1+Slamf6hiTim3lo precursor exhausted T (TPEX) cells. Here we showed that early CD8+ TEX cell fates were imprinted by forming subset-specific, self-associating chromatin hubs. Chromatin hub assembly coincided with effector or stemness gene induction and identified the transcription cofactors Id2 and Id3 as key regulators that promoted CD8+ TEX_EFF and CD8+ TPEX cell fates, respectively. Id2 drove CD8+ TEX_EFF cell specification by activating effector genes, while suppressing genes involved in exhaustion and stemness. In contrast, Id3-repressed effector genes but upregulated IL-7Rα and AhR, thereby maintaining the CD8+ TPEX cell pool. Mechanistically, Id2 and Id3 exhibited a distinct impact on the chromatin accessibility landscape in early CD8+ TEX cells by engaging Runx3 and Tcf1 transcription factors along with E proteins. These findings indicated that reshaping chromatin architecture represents a critical means for specifying CD8+ TEX cell fates and ensuring lineage stability.

Animals↗
PubMed2026

Mediator at the Helm: Coordinating transcription and biomolecular condensates in hematopoiesis.

Hematopoiesis relies on precisely coordinated transcriptional programs that balance stem cell self-renewal, lineage commitment, and terminal differentiation. Central to this regulation is the Mediator complex, a large multi-subunit transcriptional co-regulator that integrates signals from transcription factors and chromatin regulators to control RNA polymerase Ⅱ (Pol Ⅱ) activity. The dynamic and modular composition of Mediator enables context-dependent transcriptional outputs, while individual subunits can exert specialized regulatory functions during hematopoietic lineage specification, thereby contributing to cell-fate-specific transcriptional outputs. Recent advances further reveal that transcriptional regulation is shaped by the spatial organization of regulatory machinery with biomolecular condensates formed through liquid-liquid phase separation (LLPS), particularly at super-enhancers. In this emerging framework, Mediator functions not only as a transcriptional integrator but also as a key coordinator of transcriptional machinery within condensates at cell-fate-related gene loci. In this chapter, we summarize how distinct Mediator subunits confer specific modes of transcriptional regulation and discuss how the interplay between Mediator and phase-separated condensates shapes transcriptional control during hematopoiesis. We highlight how specific subunits, including MED1 and MED26, participate in distinct regulatory modes in erythropoiesis, spanning super-enhancer-driven transcriptional activation, progenitor expansion, and condensate-associated mechanisms that influence Pol Ⅱ pausing and global transcription repression during terminal differentiation. Together, these findings support a model in which Mediator integrates transcriptional regulation with nuclear organization through condensate-mediated mechanisms, providing a conceptual framework for understanding hematopoietic cell fate decisions and transcriptional dysregulation in hematological diseases.

Hematopoiesis↗
PubMed2026

Identifying critical lysines in mammalian histone H3 with high-throughput CRISPR prime editing.

Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.

Histones↗
PubMed1979

Self-renewal in culture of proliferative blast progenitor cells in acute myeloblastic leukemia.

We have proposed that colonies of cells with blastlike morphology growing in culture are derived from a blast subpopulation with high proliferative potential. To test whether or not these blast progenitors have the capacity for self-renewal, blast colonies grown from the peripheral blood of the 21 patients with acute myeloblastic leukemia were replated; secondary colonies were observed in 17 instances, and these were similar to primary colonies in size, morphology, and culture requirements. Great patient-to-patient variation was observed in the frequency of secondary colonies, but low secondary plating efficiency was significantly correlated with successful remission induction. We conclude that the blast progenitors detected in the assay have at least limited self-renewal capacity and that this capacity may, along with other risk factors, contribute to clinical outcome.

Adult↗
PubMed2005

Gfi1 coordinates epigenetic repression of p21Cip/WAF1 by recruitment of histone lysine methyltransferase G9a and histone deacetylase 1.

The growth factor independent 1 (Gfi1) transcriptional regulator oncoprotein plays a crucial role in hematopoietic, inner ear, and pulmonary neuroendocrine cell development and governs cell processes as diverse as self-renewal of hematopoietic stem cells, proliferation, apoptosis, differentiation, cell fate specification, and oncogenesis. However, the molecular basis of its transcriptional functions has remained elusive. Here we show that Gfi1 recruits the histone lysine methyltransferase G9a and the histone deacetylase 1 (HDAC1) in order to modify the chromatin of genes targeted for repression by Gfi1. G9a and HDAC1 are both in a repressive complex assembled by Gfi1. Endogenous Gfi1 colocalizes with G9a, HDAC1, and K9-dimethylated histone H3. Gfi1 associates with G9a and HDAC1 on the promoter of the cell cycle regulator p21Cip/WAF1, resulting in an increase in K9 dimethylation at histone H3. Silencing of Gfi1 expression in myeloid cells reverses G9a and HDAC1 recruitment to p21Cip/WAF1 and elevates its expression. These findings highlight the role of epigenetics in the regulation of development and oncogenesis by Gfi1.

Cell Line, Tumor↗
PubMed2025

A Protocol for Detecting DNA Methylation Changes at CpG Sites of Stemness-Related Genes in Aging Stem Cells.

Aging adversely affects the self-renewal and differentiation capabilities of stem cells, which impairs tissue regeneration as well as the homeostasis. Epigenetic mechanisms, specifically DNA methylation, play a key role in the maintenance of pluripotency in stem cells and regulation of pluripotency-related gene expression. Age-related modifications in methylation patterns could influence the expression of genes critical for stem cell potency maintenance, including transcription factors Nanog and Sox2. The following chapter describes a step-by-step bisulfite sequencing protocol for detection of methylation changes in the aging stem cells and provides valuable insights into the stem cells epigenetic profile. Further, the methodology describes the steps of genomic DNA extraction, bisulfite conversion, real-time PCR amplification, and sequencing for an in-depth view of the epigenetic profile derived from aging stem cells.

DNA Methylation↗
PubMed2026

Generation of TWO iPSC lines (CRICKi025-A and CRICKi026-A) from healthy donor bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are key components of the bone marrow (BM), providing structural support and paracrine signals that regulate haematopoietic stem cell maintenance, self-renewal and differentiation. However, primary BM MSCs are rare, heterogeneous, and subject to donor variability and have limited ex-vivo expansion capacity, restricting their utility. Here, we describe two human induced pluripotent stem cells lines, CRICKi0025-A and CRICKi0026-A, reprogrammed from adult BM-derived MSCs using non-integrating Sendai virus vectors. Both lines showcase grade-A morphology, are genomically stable, upregulate essential pluripotent markers and can differentiate into the three germ layers. These lines are a well-characterised resource for generating MSCs.

Journal Article↗
PubMed2024

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

Humans↗