Abstract
The role of serum response factor (Srf), a central mediator of actin dynamics and mechanical signaling, in cell identity regulation is debated to be either a stabilizer or a destabilizer. We ...investigated the role of Srf in cell fate stability using mouse pluripotent stem cells. Despite the fact that serum-containing cultures yield heterogeneous gene expression, deletion of Srf in mouse pluripotent stem cells leads to further exacerbated cell state heterogeneity. The exaggerated heterogeneity is detectible not only as increased lineage priming but also as the developmentally earlier 2C-like cell state. Thus, pluripotent cells explore more variety of cellular states in both directions of development surrounding naïve pluripotency, a behavior that is constrained by Srf. These results support that Srf functions as a cell state stabilizer, providing rationale for its functional modulation in cell fate intervention and engineering.
Graphical Abstract
Graphical Abstract
Erythropoietic proliferation and differentiation are coordinated and regulated by a complex compendium of molecular components and networks. Understanding the underlying mechanisms and the dependence ...of erythroid maturation on cell-cycle behavior can provide a detailed insight into normal and ineffective erythropoiesis. The dynamic cell cycle speed of erythroid progenitors reflects the erythron's response to external stimuli, such as severe anemia or bleeding. Aberrant cell cycle speed also defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. Current methods to resolve cell cycle length heterogeneity at a single-cell level in real-time present with limitations, including cellular toxicity, insufficient intensity, and dilution over subsequent cell divisions. We utilized a unique live-cell reporter of cell cycle speed using a histone H2B-FT fusion protein containing the color-changing Fluorescent Timer (FT) protein. The FT protein emits blue fluorescence when newly synthesized and matures into a stable red fluorescent protein over 1.2 hours. The fusion protein thus distinguishes faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence.
Knock-in mice expressing H2B-FT from a universally active locus under the control of a dox inducible promoter were previously generated and characterized. We successfully characterized the stress erythropoietic response of the spleen and bone marrow (BM) after inducing hemolytic anemia by phenylhydrazine (PHZ) administration in these transgenic mice. Flow cytometric investigation of successive stages of erythroblasts revealed that all stages of erythroblasts maintain rapid cell division after the hemolytic insult (***p<0.0001, Mann-Whitney test) and not only early progenitors, as previously thought. We also observed that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. Most importantly, we observed that the last nucleated cell stage, orthochromatic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate the anemia.
Blue-red (BR) profiles of the different erythroblasts from the PHZ-treated animals showed a marked distribution into fast-cycling (high blue fluorescence) and slow-cycling (high red fluorescence) subpopulations. Histograms of normalized BR ratios revealed significantly differentially cycling subpopulations in the polychromatic erythroblasts from spleen and orthochromatic erythroblasts from BM under stress. Mass spectrometric analysis of the differentially cycling subpopulations sorted from the respective erythroblasts shows upregulation of genes encoding cell cycle related and phospho-proteins. We are currently performing comparative analyses with openly available proteomic data.
With the Erythropoietin (Epo) model for inducing stress erythropoiesis, we do find a modest increase in blue-red ratios for each of the erythroblast populations in Epo-treated timer mice as compared to the PHZ model.
A recent study on steroid resistance in DBA reported that dexamethasone (dex) treatment of peripheral blood progenitors caused the specific upregulation of p57Kip2 leading to higher expansion and accelerated erythroid differentiation. We will utilize in vitro human CD34+ primary cell culture to assess the erythropoietic response to known treatments of anemia of chronic kidney disease and Diamond-Blackfan Anemia, like Epo and dex, respectively.
These findings shed new light on the normal response to external stress, underscoring the possibility of precise quantification of cell cycle speed in animal models of anemia. We highlight the use of a sophisticated fluorescent system that can help elucidate the role of cell cycle speed in stress hematopoiesis, and determine the mechanistic pathways acting at single-cell or population level. Further phosphoproteomic investigation can lead to identification of discrete molecular targets regulating erythroid cell proliferation and differentiation with potential therapeutic implications. The tool can aid in answering important questions delineating cell cycle dynamics as the cause or consequence of erythroid differentiation in normal and pathophysiological conditions.
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No relevant conflicts of interest to declare.
As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA ...methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.
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•Depletion of Dppa2 and Dppa4 reduced reprogramming efficiency•Overexpression of Dppa2 and Dppa4 improved reprogramming efficiency and kinetics•Dppa2/4 overexpression induced ɣH2AX and chromatin decompaction via Parp1•Depletion of Parp1 or H2AX abolished the effect of Dppa2/4 on chromatin remodeling
During cellular reprogramming, the epigenome of a somatic cell is reset to a state compatible with pluripotency maintenance. The molecular machinery underlying this process remains poorly defined. Hernandez et al. identify chromatin-associated factors Dppa2 and Dppa4 as the key components mediating the reset of somatic chromatin to a pluripotent configuration.
The hematopoietic stem cell–enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia ...(AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9. By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9–driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b. Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non–cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2. Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b. In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non–cell-intrinsic leukemia-promoting effects by an oncogenic miRNA.
•miR-125b overexpression accelerates MLL-AF9–driven AML and endows partial addiction to its overexpression.•A miR-125b-TET2-VEGFA pathway promotes leukemogenesis involving a non–cell-intrinsic mechanism.
Aberrant cell cycle speed during hematopoiesis defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. ...We utilized a unique live-cell reporter of cell cycle speed using a Histone H2B-FT fusion protein containing the color-changing Fluorescent Timer (FT) protein, distinguishing faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence.
In transgenic mice expressing this reporter, we characterized the stress erythropoietic response of spleen and bone marrow following phenylhydrazine-induced hemolytic anemia. Using flow cytometric sorting of erythroblast stages, we found that all stages divide more rapidly after the hemolytic insult, not only early progenitors as previously thought. We also observe that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. And the last nucleated cell stage, orthochromatophilic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate anemia. Mass spectrometry of sorted populations shows upregulation of cell cycle and metabolic pathway related genes. Using this system in in vitro human CD34+ primary cell culture, we assessed the erythropoietic response to known treatments of β-thalassemia and Diamond Blackfan Anemia, like Epo and dexamethasone. We found an increase in cell cycling rates with an increase in dexamethasone concentration but not that of Epo.
These findings shed new light on the normal response to external stress, underscoring the possibility of precise quantification of cell cycle speed in animal models of anemia. This system can help in understanding stress hematopoiesis, allowing identification of cellular factors that facilitate response to external stress.
Erythroid terminal differentiation (ETD) entails cell divisions coupled to decreasing cell size. The tight link between the number of cell divisions and red cell size is apparent in nutritional ...deficiencies or genetic variants in which fewer cycles result in larger red cells. Here we investigated novel EpoR functions, finding that EpoR signaling disrupts the relationship between cell cycle number and cell size, simultaneously promoting rapid cycling and the formation of larger red cells.
EpoR is essential for erythroblast survival, but it is unclear whether it has other non-redundant functions. To address this, we developed a genetic system in which we rescue mouse Epor -/- fetal liver progenitors from apoptosis by transduction with the anti-apoptotic protein Bcl-x L, and compare their ensuing differentiation with that of Epor -/- progenitors rescued with EpoR (Fig 1a). We found that the Bcl-x L survival signal, in the absence EpoR, supported formation of enucleated red cells. However, key ETD features were abnormal.
First, Bcl-x L-transduced Epor -/- erythroblasts underwent slower and fewer cell cycles (Figure 1b), differentiating prematurely into enucleated red cells. Premature induction of the cyclin-dependent-kinase inhibitor p27 KIP1 was in part responsible for the fewer cycles in the absence of EpoR signaling. We confirmed that EpoR also stimulates rapid cycling in wild-type erythroblasts in vivo, using a mouse transgenic for a live-cell reporter of cell cycle speed.
Second, using imaging flow cytometry, we found that Bcl-x L-transduced Epor -/- erythroblasts were smaller than EpoR-transduced Epor -/- cells (Fig 1c,d). By doubly transducing Epor -/- erythroblasts with both Bcl-x L and EpoR, we verified that EpoR absence, and not Bcl-x L overexpression, is responsible for the smaller size of Bcl-x L-transduced Epor -/- erythroblasts and reticulocytes.
Bcl-x L-transduced Epor -/- erythroblasts failed to upregulate the transferrin receptor, suggesting that iron deficiency may be responsible for their smaller size. However, neither iron supplementation, nor transduction with the transferrin receptor, rescued their smaller size. Iron regulates cell size through Heme-regulated eIF2α kinase (HRI). To definitively test the role of iron and HRI, we generated mice doubly deleted for both EpoR and HRI. We then rescued both Epor -/- and Epor -/-Hri -/- -fetal liver cells in parallel, by transduction with either Bcl-x L or EpoR. In agreement with the known role of HRI as a negative regulator of erythroblast size, both Bcl-x L- transduced and EpoR-transduced erythroblasts were larger on the Epor -/-Hri -/- genetic background. However, the difference in size between Bcl-x L and EpoR-rescued erythroblasts persisted in Epor -/-Hri -/- erythroblasts and reticulocytes (Fig 1c,d), conclusively showing that EpoR signaling regulates cell size independently of the HRI pathway.
EpoR promoted increased erythroblast and reticulocyte cell size in wild-type mice in vitro and in vivo, in response to Epo concentrations ranging from 10 to 10,000 mU/ml. We also evaluated the effect of Epo on red cell size in humans, in two independent studies, where healthy volunteers were administered Epo for either 3 weeks (20 IU /kg every 48 hours, 25 subjects, Study #1) or for 7 weeks (weekly Epo dosing that increased hemoglobin by 10 -15%; 24 subjects, Study #2). In a third intervention, 21 subjects participated in a randomized double-blind placebo-controlled crossover study in which 900 ml of whole blood was withdrawn from the treatment group by venipuncture. In all three studies, the increase in MCV in the treatment groups persisted long after Epo and reticulocyte levels returned to baseline (Figure 2). There was no correlation between MCV and the reticulocyte count, whose time courses were clearly divergent (r < 0.1, Pearson's product-moment correlation). Further, computational simulation suggests that the extent and duration of the increase in MCV is unlikely to be the result of skewing of the circulating red cell population in favor of younger, larger red cells.
Our work reveals a paradoxical EpoR-driven increase in erythroblast cycling simultaneously with increased erythroblast and red cell size. It suggests that EpoR alters the relationship between cell cycle and biomass in erythroblasts. It further suggests that hypoxia, anemia and other high-Epo syndromes are new diagnostic interpretations of increased MCV in the clinic.
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No relevant conflicts of interest to declare.
The conventional view of oncogenesis posits that driver mutations, when present in appropriate cell types, initiate malignant transformation. At the apex of the differentiation hierarchy, stem cells, ...such as hematopoietic stem cells, have been suggested to be the cell-of-origin for a variety of hematopoietic malignancies. However, since the mutation bearing hematopoietic stem cells can differentiate to give rise to downstream progeny, it suggests the possibility that some of the downstream cells be the relevant cell-of-origin permitting malignant transformation. Given that cancer cell-of-origin has been a product of logic deduction evading rigorous experimentation, whether and how their unique cellular properties, if any, contribute to transformation remain undetermined.
To experimentally define the key properties of the cell-of-origin permissive to oncogenic transformation, we constructed a doxycycline (Dox) inducible MLL-AF9 knock-in mouse model, which allows precise temporal control of oncogene expression in desired hematopoietic cells, in vitro and in vivo . Whole body induction by Dox led to 100% lethality due to acute myeloid leukemia (AML) within 6-8 weeks, while the un-induced littermates remained healthy, confirming the inducibility and potency of the MLL-AF9 oncogene. To minimize the differences contributed by cellular differentiation stages and to focus solely on the contributions by specific cellular properties, we narrowed our analysis on a phenotypically homogeneous population, the granulocyte-macrophage progenitors (GMPs), which have been shown to be critically important in supporting leukemic development (Ugale et al ., 2014, Cell Reports; Ye et al ., 2015, Cell Stem Cell). We found that although MLL-AF9 expression can be induced in all GMPs, only a subset (~25%) of them is permissive to transformation. These results prompted us to investigate the underlying cellular and molecular properties that enable oncogenic transformation.
Inspired by our earlier work demonstrating that the cell fate plasticity of GMPs is related to their rapid cell cycle behavior (Guo et al ., 2014, Cell), we tested whether the rapid proliferation of GMPs underlies their permissiveness to oncogenic transformation. If so, transformation potential should be enriched in the faster-proliferating GMP subsets. To test this possibility, we designed single cell assays to investigate the transformation efficiencies of GMPs with regard to their cell cycle speeds. Specifically, the intrinsic cell cycle speed was measured within 24 hours of Dox addition, and transformation status of hundreds of individual GMPs was determined by methylcellulose colony formation assay. Within the first 24 hours of Dox addition, the cell cycle speed remained unchanged, reflecting the intrinsic cell cycle kinetics, rather than a proliferative response to oncogene expression. Our results show that the faster a GMP divides, the more likely it transforms in response to MLL-AF9 expression. Strikingly, most GMPs that can divide more than 3 times within 24 hours are transformed. These data indicate the fastest cycling GMPs provide the oncogene with a permissive cellular context to exert its function. Furthermore, prospectively isolated faster cycling GMPs enriched for transformed colony formation in vitro, and induced earlier lethal AML in vivo . Importantly, transient deceleration of GMPs for 48 hours by low-dose CDK4/6 inhibitor, Palbociclib, significantly reduced transformation efficiency by ~ 60%. Taken together, these results demonstrate that MLL-AF9 initiated transformation is dependent on specific cellular properties which are associated with a rapid cell cycle. Genomic analyses of the transformation-permissive GMPs, using non-permissive GMPs as controls, are being performed to define the permissiveness in molecular details, on both transcriptional and epigenetic levels.
No relevant conflicts of interest to declare.
Cell proliferation changes concomitantly with fate transitions during reprogramming, differentiation, regeneration, and oncogenesis. Methods to resolve cell cycle length heterogeneity in real time ...are currently lacking. Here, we describe a genetically encoded fluorescent reporter that captures live-cell cycle speed using a single measurement. This reporter is based on the color-changing fluorescent timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. We generated a mouse strain expressing an H2B-FT fusion reporter from a universally active locus and demonstrate that faster cycling cells can be distinguished from slower cycling ones on the basis of the intracellular fluorescence ratio between the FT’s blue and red states. Using this reporter, we reveal the native cell cycle speed distributions of fresh hematopoietic cells and demonstrate its utility in analyzing cell proliferation in solid tissues. This system is broadly applicable for dissecting functional heterogeneity associated with cell cycle dynamics in complex tissues.
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•Stable molecule levels are more sensitive to cell cycle length than labile ones•Stable/labile species can be modeled by the two states of a fluorescent timer (FT)•H2B-FT resolves live-cell cycle speed heterogeneity in cultured cells and in mice•Fast proliferating cells appear bluer, while slow dividing cells are redder
Cell cycle speed greatly influences cell state but remains challenging to measure, particularly in dynamic or complex tissues. Here, Eastman et al. describe H2B-FT, a two-color reporter that resolves cell cycle speed ratiometrically in a single-snapshot measurement, enabling the identification and prospective isolation of live cells with distinct cycling rates.
ABSTRACTFibrosis is a consequence of aberrant wound healing processes that can be debilitating for patients and often are associated with highly morbid disease processes. Myofibroblasts play an ...important role in determining an appropriate physiologic response to tissue injury or an excessive response leading to fibrosis. Specifically, “supermature” focal adhesions, α-smooth muscle actin, and the myocardin-related transcription factor/serum response factor pathway likely play a significant role in the differentiation and survival of myofibroblasts in fibrotic lesions. Thus, targeting each of these and disrupting their functioning could lead to the development of therapeutic options for patients suffering from fibrosis and other sequelae of dysregulated wound healing. In this paper, we review the current literature concerning the roles of these three constituents of fibrotic signaling pathways, work already done in attempting to regulate these processes, and discuss the potential of these biomolecular constituents as therapeutic targets in future translational research.
Yes-associated protein (YAP) is known to promote the stemness of multiple stem cell types, including pluripotent stem cells, while also antagonizing pluripotency during early embryogenesis. How YAP ...accomplishes these distinct functions remains unclear. Here, we report that, depending on the specific cells in which it is expressed, YAP could exhibit opposing effects on pluripotency induction from mouse somatic cells. Specifically, YAP inhibits pluripotency induction cell-autonomously but promotes it non-cell-autonomously. For its non-cell-autonomous role, YAP alters the expression of many secreted and matricellular proteins, including CYR61. YAP's non-cell-autonomous promoting effect could be recapitulated by recombinant CYR61 and abrogated by CYR61 depletion. Thus, we define a YAP-driven effect on enhancing pluripotency induction largely mediated by CYR61. Our work highlights the importance of considering the distinct contributions from heterologous cell types in deciphering cell fate control mechanisms and calls for careful re-examination of the co-existing bystander cells in complex cultures and tissues.
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•YAP inhibits pluripotency induction when expressed cell-autonomously•YAP promotes pluripotency induction when expressed non-cell-autonomously•YAP expression alters the expression of genes that encode extracellular components•CYR61 is secreted by YAP-expressing cells to promote nearby reprogramming
In this article, Hartman and colleagues show that, depending on the specific cells in which it is expressed, Yes-associated protein (YAP) exhibits opposing effects on pluripotency induction from mouse somatic cells. Specifically, YAP inhibits pluripotency induction cell-autonomously but promotes it non-cell-autonomously. Its non-autonomous function is accomplished by altering the expression of many secreted and matricellular proteins, including CYR61, a known YAP target.
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