Control Cell Fate Research Articles

Synthesis and bioactivity of pyrrole-conjugated phosphopeptides.

Here we report the synthesis and effect on the cell viability of pyrrole-conjugated phosphopeptides. Encouraged by the selective inhibition of cancer cells by a naphthyl-capped phosphopeptide (Nap-ffpy, 1), we conjugated the heteroaromatic dipyrrole or tripyrrole motif at the N-terminal of short peptides containing phosphotyrosine or phosphoserine and examined the bioactivity of the resulting phosphopeptides (2–10). Although most of the phosphopeptides exhibit comparable activities with that of 1 against HeLa cells at 200 μM, they, differing from 1, are largely compatible with HeLa cells at 400 μM. Enzymatic dephosphorylation of 2–10, at 400 μM is unable to induce a dramatic morphological transition of the peptide assemblies observed in the case of 1. These results suggest that a heteroaromatic motif at the N-terminal of peptides likely disfavors the formation of extensive nanofibers or morphological changes during enzymatic self-assembly, thus provide useful insights for the development of phosphopeptides as substrates of phosphatases for controlling cell fate.

Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification.

SummaryElucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Unfortunately, existing computational gene regulatory network (GRN) reconstruction methods are imprecise, computationally burdensome, and fail to reveal dynamic regulatory topologies. Here, we present Epoch, a reconstruction tool that uses single-cell transcriptomics to accurately infer dynamic networks. We apply Epoch to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESCs) guided by multiple signaling pathways, and we demonstrate that modulating these pathways drives topological changes that bias cell fate potential. We also find that Peg3 rewires the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRNs, we trace how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we identify regulatory circuits of patterning and axis formation that distinguish in vitro and in vivo mesoderm specification.

The Two Non-Visual Arrestins Engage ERK2 Differently

Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. β-arrestin1 and β-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.

Synthetic multistability in mammalian cells.

In multicellular organisms, gene regulatory circuits generate thousands of molecularly distinct, mitotically heritable states through the property of multistability. Designing synthetic multistable circuits would provide insight into natural cell fate control circuit architectures and would allow engineering of multicellular programs that require interactions among distinct cell types. We created MultiFate, a naturally inspired, synthetic circuit that supports long-term, controllable, and expandable multistability in mammalian cells. MultiFate uses engineered zinc finger transcription factors that transcriptionally self-activate as homodimers and mutually inhibit one another through heterodimerization. Using a model-based design, we engineered MultiFate circuits that generate as many as seven states, each stable for at least 18 days. MultiFate permits controlled state switching and modulation of state stability through external inputs and can be expanded with additional transcription factors. These results provide a foundation for engineering multicellular behaviors in mammalian cells.

Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division.

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2-p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.

Genome-wide chromatin contacts of super-enhancer-associated lncRNA identify LINC01013 as a regulator of fibrosis in the aortic valve.

Calcific aortic valve disease (CAVD) is characterized by a fibrocalcific process. The regulatory mechanisms that drive the fibrotic response in the aortic valve (AV) are poorly understood. Long noncoding RNAs derived from super-enhancers (lncRNA-SE) control gene expression and cell fate. Herein, multidimensional profiling including chromatin immunoprecipitation and sequencing, transposase-accessible chromatin sequencing, genome-wide 3D chromatin contacts of enhancer-promoter identified LINC01013 as an overexpressed lncRNA-SE during CAVD. LINC01013 is within a loop anchor, which has contact with the promoter of CCN2 (CTGF) located at ~180 kb upstream. Investigation showed that LINC01013 acts as a decoy factor for the negative transcription elongation factor E (NELF-E), whereby it controls the expression of CCN2. LINC01013-CCN2 is part of a transforming growth factor beta 1 (TGFB1) network and exerts a control over fibrogenesis. These findings illustrate a novel mechanism whereby a dysregulated lncRNA-SE controls, through a looping process, the expression of CCN2 and fibrogenesis of the AV.

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

YAP/TAZ in Bone and Cartilage Biology.

YAP and TAZ were initially described as the main regulators of organ growth during development and more recently implicated in bone biology. YAP and TAZ are regulated by mechanical and cytoskeletal cues that lead to the control of cell fate in response to the cellular microenvironment. The mechanical component represents a major signal for bone tissue adaptation and remodelling, so YAP/TAZ contributes significantly in bone and cartilage homeostasis. Recently, mice and cellular models have been developed to investigate the precise roles of YAP/TAZ in bone and cartilage cells, and which appear to be crucial. This review provides an overview of YAP/TAZ regulation and function, notably providing new insights into the role of YAP/TAZ in bone biology.

GFI1 Cooperates with IKZF1/IKAROS to Activate Gene Expression in T-cell Acute Lymphoblastic Leukemia.

Combinatorial diversity and cooperation between DNA binding proteins and complexes assembled by them can direct context-dependent transcriptional outputs to control cell fate and may offer new insights for therapeutic targeting in cancer.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

1021 – SWI/SNF CHROMATIN REMODELING COMPLEXES GOVERN LYMPHOID LINEAGE COMMITMENT

Lymphocyte development from hemopoietic stem cells (HSCs) is accompanied by a loss of self-renewal capacity and a progressive restriction of developmental potential. Although key transcriptional pathways controlling lymphoid development have been identified, detailed molecular mechanisms explaining how multipotent HSCs initially commit to the lymphoid lineage are still lacking. Work from our laboratory indicates that combinatorial assembly of SWI/SNF chromatin remodeling complexes is a key epigenetic mechanism controlling cell fate decisions in the hemopoietic tissue. Single-cell RNA-seq analyses in bone marrow cells revealed that expression of the Smarcd1 subunit is enriched in lymphoid-primed multipotent progenitors. Using a conditional knock-out mouse model, we showed that Smarcd1 is specifically required for the development of the lymphoid lineage. Acute deletion of Smarcd1 in adult hematopoiesis causes lymphopenia with near complete absence of lymphoid-primed multipotent progenitors, indicating a role in lymphoid lineage commitment. Genome-wide studies revealed a functional collaboration between Smarcd1 and the bHLH transcription factor E2A in activating the lymphoid program of gene expression. Mechanistically, we show that Smarcd1 binds to and regulates chromatin accessibility of E2A at H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hemopoietic stem cells. Altogether, these studies identify Smarcd1 as a chromatin remodeler that collaborates with E2A to determine the lymphoid cell fate during hemopoiesis. Lymphocyte development from hemopoietic stem cells (HSCs) is accompanied by a loss of self-renewal capacity and a progressive restriction of developmental potential. Although key transcriptional pathways controlling lymphoid development have been identified, detailed molecular mechanisms explaining how multipotent HSCs initially commit to the lymphoid lineage are still lacking. Work from our laboratory indicates that combinatorial assembly of SWI/SNF chromatin remodeling complexes is a key epigenetic mechanism controlling cell fate decisions in the hemopoietic tissue. Single-cell RNA-seq analyses in bone marrow cells revealed that expression of the Smarcd1 subunit is enriched in lymphoid-primed multipotent progenitors. Using a conditional knock-out mouse model, we showed that Smarcd1 is specifically required for the development of the lymphoid lineage. Acute deletion of Smarcd1 in adult hematopoiesis causes lymphopenia with near complete absence of lymphoid-primed multipotent progenitors, indicating a role in lymphoid lineage commitment. Genome-wide studies revealed a functional collaboration between Smarcd1 and the bHLH transcription factor E2A in activating the lymphoid program of gene expression. Mechanistically, we show that Smarcd1 binds to and regulates chromatin accessibility of E2A at H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hemopoietic stem cells. Altogether, these studies identify Smarcd1 as a chromatin remodeler that collaborates with E2A to determine the lymphoid cell fate during hemopoiesis.

Patterning of Phase-Separated Condensates by Dnd1 Controls Cell Fate

Patterning of Phase-Separated Condensates by Dnd1 Controls Cell Fate

Positional signals establishment in the regulation of female germline specification

The female germline specification process of a single megaspore mother cell (MMC) of ovule primordium (nucellus) is intriguingly complex because it involves the interaction of different pathways tightly linked with positional information. Various Arabidopsis genes, including the stem cell promoting factor WUSCHEL, have already been shown to be involved in this precise regulation process. Recently, there have been some reviews on MMC specialization, mainly from the aspects of epigenetics, microRNAs and gene regulatory networks. However, those reviews have not taken into consideration the function of positional signals in female germline specification. Here, we review major progress in the cell fate control of female germline specification, highlighting the functions of positional cues.

Fibronectin-Enriched Biomaterials, Biofunctionalization, and Proactivity: A Review

Modern innovation in reconstructive medicine implies the proposition of material-based strategies suitable for tissue repair and regeneration. The development of such systems necessitates the design of advanced materials and the control of their interactions with their surrounding cellular and molecular microenvironments. Biomaterials must actively engage cellular matter to direct and modulate biological responses at implant sites and beyond. Indeed, it is essential that a true dialogue exists between the implanted device and the cells. Biomaterial engineering implies the knowledge and control of cell fate considering the globality of the adhesion process, from initial cell attachment to differentiation. The extracellular matrix (ECM) represents a complex microenvironment able to meet these essential needs to establish a relationship between the material and the contacting cells. The ECM exhibits specific physical, chemical, and biochemical characteristics. Considering the complexity, heterogeneity, and versatility of ECM actors, fibronectin (Fn) has emerged among the ECM protagonists as the most pertinent representative key actor. The following review focuses on and synthesizes the research supporting the potential to use Fn in biomaterial functionalization to mimic the ECM and enhance cell–material interactions.

Temporally Controlled Photouncaged Epidermal Growth Factor Influences Cell Fate in Hydrogels.

Hydrogels are powerful materials that more accurately mimic the cellular microenvironment over static two-dimensional culture. Photochemical strategies enable dynamic complexity to be achieved within hydrogels to better mimic the extracellular matrix; however, many photochemical systems to pattern proteins within hydrogels are complicated by long reaction times to immobilize these proteins wherein the protein can lose activity. As proof-of-concept, we demonstrate an elegant method where photocaged proteins are immobilized in hydrogels and then directly photoactivated. Specifically, we immobilized streptavidin-ortho-nitrobenzyl-modified epidermal growth factor (EGF) to cross-linked hyaluronan hydrogels and cultured two EGF-responsive cancer cells of breast and lung therein. We used light to temporally uncage and control EGF activation, thereby inducing cell death in breast cancer cells and proliferation in lung cancer cells. These results show how temporal, photochemical, protein activation influences cellular response and lays the foundation for further advances in manipulating the in vitro environment to control cell fate.

Blood stem cell PU.1 upregulation is a consequence of differentiation without fast autoregulation.

Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.

In Sickness and in Health: The Oxygen Reactive Species and the Bone

Oxidative stress plays a central role in physiological and pathological bone conditions. Its role in signalment and control of bone cell population differentiation, activity, and fate is increasingly recognized. The possibilities of its use and manipulation with therapeutic goals are virtually unending. However, how redox balance interplays with the response to mechanical stimuli is yet to be fully understood. The present work summarizes current knowledge on these aspects, in an integrative and broad introductory perspective.

Polycomb Group Proteins Establish a Morphogen-Mediated Spatial Map During Early-Stage Brain Development

The first step for development of a functionally and structurally intricate organ, the brain, involves precise control of the neural stem cell (NSC) fate. NSCs produce a variety of neurons and glial cells according to the developmental time and 3-dimensional (3D) position within the brain. Morphogens play a key role in determining the 3D positional information of NSCs and thus regulating brain arealization along the rostrocaudal and dorsoventral axes. In this review article, we summarize recent studies that described essential roles of Polycomb group proteins in the establishment of the 3D atlas of the brain, in part through suppressing brain area-specific genes such as morphogens.

The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function.

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc-Flt4 signalling. Ddx21 function is essential for Vegfc-Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc-Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

In Vivo Monitoring of Cytosolic pH Using the Ratiometric pH Sensor pHluorin.

Cytosolic pH (pHcyt) is a key factor controlling cell fate. The genetically encoded pH-sensor pHluorin has proven highly valuable for studies on pHcyt in many living organisms. pHluorin displays a bimodal excitation spectrum with peaks at 395nm and 475nm, which is dependent on pH. Here we describe two different protocols for determining pHcyt in the soil-borne fungal pathogen Fusarium oxysporum, based either on population or single-cell analysis.