Alternative Cell Fates Research Articles

Conserved regulatory state expression controlled by divergent developmental gene regulatory networks in echinoids.

Evolution of the animal body plan is driven by changes in developmental gene regulatory networks (GRNs), but how networks change to control novel developmental phenotypes remains, in most cases, unresolved. Here, we address GRN evolution by comparing the endomesoderm GRN in two echinoid sea urchins, Strongylocentrotus purpuratus and Eucidaris tribuloides, with at least 268 million years of independent evolution. We first analyzed the expression of twelve transcription factors and signaling molecules of the S. purpuratus GRN in E. tribuloides embryos, showing that orthologous regulatory genes are expressed in corresponding endomesodermal cell fates in the two species. However, perturbation of regulatory genes revealed that important regulatory circuits of the S. purpuratus GRN are significantly different in E. tribuloides For example, mesodermal Delta/Notch signaling controls exclusion of alternative cell fates in E. tribuloides but controls mesoderm induction and activation of a positive feedback circuit in S. purpuratus These results indicate that the architecture of the sea urchin endomesoderm GRN evolved by extensive gain and loss of regulatory interactions between a conserved set of regulatory factors that control endomesodermal cell fate specification.

NOTCH activity differentially affects alternative cell fate acquisition and maintenance

Here, Cheung et al. use sophisticated tissue specific genetic ablation in pituitary cell lineages to uncover an unexpected differential sensitivity of NOTCH activation signalling pathway in different cell lineages during the embryonic murine pituitary development. These findings are important as they further demonstrate the importance of NOTCH pathway in progenitor differentiation during pituitary development and consequently reveals new aspects of endocrine lineages and plasticity. Further work on the molecular differences on cell specific lineages and cell fate acquisition will be important to fine tune the mechanisms that generate pituitary plasticity. Importantly it will be useful to understand if these mechanisms are maintained in adult pituitary stem cells upon endocrine challenges to maintain adult organ homeostasis.

BACE1 Regulates Proliferation and Neuronal Differentiation of Newborn Cells in the Adult Hippocampus in Mice.

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of β-amyloid (Aβ), one of the major pathogenic molecules of Alzheimer’s disease (AD), and is therefore being actively pursued as a drug target for AD. Adult hippocampal neurogenesis (AHN) is a lifelong process that is known to be important for learning and memory and may have the potential to regenerate damaged neural tissue. In this study, we examined whether BACE1 regulates AHN, which holds important implications for its suitability as a drug target in AD. Cohorts of 2-month-old wild-type (BACE1+/+), heterozygous, and homozygous BACE1 knockout mice (BACE1+/– and BACE1–/–, respectively) were injected with 5-bromo-2′-deoxyuridine (BrdU) and sacrificed 1 day later to examine the impact of loss of BACE1 on neural precursor cell (NPC) proliferation in the adult brain. Parallel cohorts of mice were sacrificed 4 weeks after BrdU injection to determine the effects of BACE1 on survival and differentiation of newborn NPCs. We found that NPC proliferation was increased in BACE1–/– mice compared to BACE1+/+ mice, while no difference was observed in NPC survival across genotypes. Differentiation of NPCs to neuronal lineage was impaired in BACE1–/– mice. However, no differences were observed in astrogenesis, the proportion of immature neurons, or the production of oligodendrocytes across genotypes. Importantly, corresponding with a decrease in neuronal differentiation in the absence of a complementary increase in an alternate cell fate, BACE1–/– mice were found to have a pool of undifferentiated NPCs in the hippocampus compared to BACE1+/+ and BACE1+/– mice.

The Histone Variant MacroH2A Blocks Cellular Reprogramming by Inhibiting Mesenchymal-to-Epithelial Transition.

Transcription factor-induced reprogramming of somatic cells to pluripotency is mediated via profound alterations in the epigenetic landscape. The histone variant macroH2A1 (mH2A1) is a barrier to the cellular reprogramming process. We demonstrate here that mH2A1 blocks reprogramming and contributes to the preservation of cell identity by trapping cells at the very early stages of the process, namely, at the mesenchymal-to-epithelial transition (MET). We provide a comprehensive analysis of the genomic sites occupied by the mH2A1 nucleosomes in human fibroblasts and embryonic stem (ES) cells and how they affect the reprogramming of fibroblasts to pluripotency. We have integrated chromatin immunoprecipitation sequencing (ChIP-seq) data with transcriptome sequencing (RNA-seq) data using cells containing reduced levels of mH2A1 and have inferred mH2A1-centered gene-regulatory networks that support the fibroblast and ES cell fates. We found that the exact positions of mH2A1 nucleosomes in regulatory regions of specific network genes with key regulatory roles guarantee the functional robustness of the regulatory networks. Using the reconstructed networks, we can predict and validate several components and their interactions in the establishment of stable cell types by limiting progression to alternative cell fates.

NOTCH activity differentially affects alternative cell fate acquisition and maintenance.

The pituitary is an essential endocrine gland regulating multiple processes. Regeneration of endocrine cells is of therapeutic interest and recent studies are promising, but mechanisms of endocrine cell fate acquisition need to be better characterised. The NOTCH pathway is important during pituitary development. Here, we further characterise its role in the murine pituitary, revealing differential sensitivity within and between lineages. In progenitors, NOTCH activation blocks cell fate acquisition, with time-dependant modulation. In differentiating cells, response to activation is blunted in the POU1F1 lineage, with apparently normal cell fate specification, while POMC cells remain sensitive. Absence of apparent defects in Pou1f1-Cre; Rbpjfl/fl mice further suggests no direct role for NOTCH signalling in POU1F1 cell fate acquisition. In contrast, in the POMC lineage, NICD expression induces a regression towards a progenitor-like state, suggesting that the NOTCH pathway specifically blocks POMC cell differentiation. These results have implications for pituitary development, plasticity and regeneration. Activation of NOTCH signalling in different cell lineages of the embryonic murine pituitary uncovers an unexpected differential sensitivity, and this consequently reveals new aspects of endocrine lineages development and plasticity.

A Poroelastic Model of a Fibrous-Porous Tissue Engineering Scaffold

Tissue engineering scaffolds are used in conjunction with stem cells for the treatment of various diseases. A number of factors provided by the scaffolds affect the differentiation of stem cells. Mechanical cues that are part of the natural cellular microenvironment can both accelerate the differentiation toward particular cell lineages or induce differentiation to an alternative cell fate. Among such factors, there are externally applied strains and mechanical (stiffness and relaxation time) properties of the extracellular matrix. Here, the mechanics of a fibrous-porous scaffold is studied by applying a coordinated modeling and experimental approach. A force relaxation experiment is used, and a poroelastic model associates the relaxation process with the fluid diffusion through the fibrous matrix. The model parameters, including the stiffness moduli in the directions along and across the fibers as well as fluid diffusion time, are estimated by fitting the experimental data. The time course of the applied force is then predicted for different rates of loading and scaffold porosities. The proposed approach can help in a reduction of the technological and experimental efforts to produce 3-D scaffolds for regenerative medicine as well as in a higher accuracy of the estimation of the local factors sensed by stem cells.

Developmental History Provides a Roadmap for the Emergence of Tumor Plasticity

We show that the loss or gain of transcription factor programs that govern embryonic cell-fate specification is associated with a form of tumor plasticity characterized by the acquisition of alternative cell fates normally characteristic of adjacent organs. In human non-small cell lung cancers, downregulation of the lung lineage-specifying TF NKX2-1 is associated with tumors bearing features of various gut tissues. Loss of Nkx2-1 from murine alveolar, but not airway, epithelium results in conversion of lung cells to gastric-like cells. Superimposing oncogenic Kras activation enables further plasticity in both alveolar and airway epithelium, producing tumors that adopt midgut and hindgut fates. Conversely, coupling Nkx2-1 loss with foregut lineage-specifying SOX2 overexpression drives the formation of squamous cancers with features of esophageal differentiation. These findings demonstrate that elements of pathologic tumor plasticity mirror the normal developmental history of organs in that cancer cells acquire cell fates associated with developmentally related neighboring organs.

A Phyllopod-Mediated Feedback Loop Promotes Intestinal Stem Cell Enteroendocrine Commitment in Drosophila.

SummaryThe intestinal epithelium in the Drosophila midgut is maintained by intestinal stem cells (ISCs), which are capable of generating both enterocytes and enteroendocrine cells (EEs) via alternative cell fate specification. Activation of Delta-Notch signaling directs ISCs for enterocyte generation, but how EEs are generated from ISCs remains poorly understood. Here, we identified Phyllopod (Phyl) as a key regulator that drives EE generation from ISCs. Phyl, which is normally suppressed by Notch, functions as an adaptor protein that bridges Tramtrack 69 (Ttk69) and E3 ubiquitin ligase Sina for degradation. Degradation of Ttk69 allows the activation of the Achaete-Scute Complex (AS-C)-Pros regulatory axis, which promotes EE specification. Interestingly, expression of AS-C genes in turn further induces Phyl expression, thereby establishing a positive feedback loop for continuous EE fate specification and commitment. This positive feedback circuit-driven regulatory mechanism could represent a common strategy for reliable and irreversible cell fate determination from progenitor cells.

Stem Cells from Dental Pulp: What Epigenetics Can Do with Your Tooth.

Adult stem cells have attracted scientific attention because they are able to self-renew and differentiate into several specialized cell types. In this context, human dental tissue-derived mesenchymal stem cells (hDT-MSCs) have emerged as a possible solution for repairing or regenerating damaged tissues. These cells can be isolated from primary teeth that are naturally replaced, third molars, or other dental tissues and exhibit self-renewal, a high proliferative rate and a great multilineage potential. However, the cellular and molecular mechanisms that determine lineage specification are still largely unknown. It is known that a change in cell fate requires the deletion of existing transcriptional programs, followed by the establishment of a new developmental program to give rise to a new cell lineage. Increasing evidence indicates that chromatin structure conformation can influence cell fate. In this way, reversible chemical modifications at the DNA or histone level, and combinations thereof can activate or inactivate cell-type-specific gene sequences, giving rise to an alternative cell fates. On the other hand, miRNAs are starting to emerge as a possible player in establishing particular somatic lineages. In this review, we discuss two new and promising research fields in medicine and biology, epigenetics and stem cells, by summarizing the properties of hDT-MSCs and highlighting the recent findings on epigenetic contributions to the regulation of cellular differentiation.

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes

Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate fate genes to activation during direct cell conversion. Ourproteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMXand RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties.

Alteration in MicroRNA Expression Governs the Nature and Timing of Cellular Fate Commitment.

In the central nervous system, the expression level of transcriptional repressor Hes1 (hairy and enhancer of split-1) tightly controls the alternative cell fate commitment during differentiation as well as the time required for such cellular transitions. A microRNA, miR-9, that interacts with Hes1 in a mutually antagonistic manner, influences both the process of lineage specification and timing of differentiation significantly, but the impact of the miR-9 in guiding these events still remains poorly understood. Here, we proposed a stochastic mathematical model of the miR-9/Hes1 double-negative feedback interaction network that at the outset shows how alternative cell fate such as quiescence, progenitor, and neuronal states can be accomplished through fine-tuning the Hes1 dynamics by altering the expression level of miR-9. The model simulations further foretell a correlated variation of the period of oscillation of Hes1, and the time delay observed between Hes1 mRNA and protein as the transcription rate of miR-9 increases during the neural progenitor state attainment. Importantly, the model simulations aided by the systematic sensitivity analysis predict that the timing of differentiation to the neuronal state crucially depends on the negative regulators (miR-9 and Hes6) of the Hes1. Our results indicate that miR-9/Hes1 interaction network can be effectively exploited for an efficient and well-timed neuronal transformation.

Loss of Kdm5c Causes Spurious Transcription and Prevents the Fine-Tuning of Activity-Regulated Enhancers in Neurons

During development, chromatin-modifying enzymes regulate both the timely establishment of cell-type-specific gene programs and the coordinated repression of alternative cell fates. To dissect the role of one such enzyme, the intellectual-disability-linked lysine demethylase 5C (Kdm5c), in the developing and adult brain, we conducted parallel behavioral, transcriptomic, and epigenomic studies in Kdm5c-null and forebrain-restricted inducible knockout mice. Together, genomic analyses and functional assays demonstrate that Kdm5c plays a critical role as arepressor responsible for the developmental silencing of germline genes during cellular differentiation and in fine-tuning activity-regulated enhancers during neuronal maturation. Although the importance of these functions declines after birth, Kdm5c retains an important genome surveillance role preventing the incorrect activation of non-neuronal and cryptic promoters in adult neurons.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

The Akt/mTOR pathway is a key driver of murine CD4+ T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions.

Capacity of Pneumococci to Activate Macrophage Nuclear Factor κB: Influence on Necroptosis and Pneumonia Severity.

During pneumococcal pneumonia, antibacterial defense requires the orchestrated expression of innate immunity mediators, initiated by alveolar macrophages and dependent on transcription driven by nuclear factor κB (NF-κB). Such immune pressure may select for pneumococci, which avoid or subvert macrophage NF-κB activation. Analyzing pneumococci collected from children in Massachusetts, we found that the activation of macrophage NF-κB by Streptococcus pneumoniae is highly diverse, with a preponderance of low NF-κB activators that associate particularly with complicated pneumonia. Low NF-κB activators cause more severe lung infections in mice, and they drive macrophages toward an alternate and detrimental cell fate of necroptosis. Both outcomes can be reversed by activation of macrophages with pneumococci that are high NF-κB activators. These results suggest that low NF-κB activation is a virulence property of pneumococci and that the appropriate activation of macrophages, including NF-κB, may hold promise as an adjunct therapeutic avenue for pneumococcal pneumonia.

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or "tipping point" at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations.

Measurement of bistability in a multidimensional parameter space.

Bistability plays an important role to generate two stable states for alternative cell fates, or to promote cellular diversity and cell cycle oscillations. Positive feedback loops are necessary for the existence of bistability and ultrasensitive reactions in the loops broaden the parameter range of bistability. The broader parameter range a system's bistability covers, the more robust the two states are. It is challenging to determine the bistable range of a parameter because noise and transient processes induce transitions between the two states. We found that a threshold of transition rates coincides with the bistability boundaries determined by the open-loop approach. With this threshold, we estimated the boundaries for various synthetic single-gene positive feedback loops in yeast in a two dimensional parameter space: the inducer concentration and promoter dynamic range. While the bistable range of inducer concentration was influenced by many factors, the promoter dynamic range was more informative. The narrowest promoter dynamic range at which bistability can emerge revealed whether the full potential of an ultrasensitive reaction, such as dimerization, is exploited in the feedback loop. The convenient control of basal expression to adjust the promoter dynamic range permits a practical and reliable comparison of robustness of related positive feedback loops.

LIM domain-binding 1 maintains the terminally differentiated state of pancreatic β cells.

The recognition of β cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain β cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic β cells. Inducible ablation of LDB1 in mature β cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted β cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of β cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative β cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of β cells and is a component of active enhancers in both murine and human islets.

The Direct Notch1 Cofactor Zmiz1 Differentially Regulates Notch1 Signals in a Stage-Specific Manner to Preserve Early T-Cell Precursors and Expand Committed T Cells

When stem cells first enter the thymus and become early T-cell precursor (ETP) cells, they are exposed to high levels of Notch1 ligand. Notch1 signal strength must be tightly regulated because on one hand, excessive Notch1 signals drive premature T-cell commitment, resulting in loss of ETP cells and alternative cell fates. On the other hand, complete loss of Notch1 signals impairs ETP proliferation, also resulting in loss of ETP cells. Thus, keeping Notch signals finely balanced in ETP cells preserves "stemness". However, after ETP cells commit to the T-cell lineage by the DN3 cell stage, Notch1 signals ramp up dramatically to drive proliferation. It is unclear how Notch1 signals are initially restrained and then amplified. We previously showed that the PIAS-like coregulator Zmiz1 is a direct, context-dependent cofactor of Notch1 in T-cell leukemia. In contrast to drosophila Zmiz1, mammalian Zmiz1 evolved a tetratricopeptide repeat (TPR) domain that binds directly to Notch1 and selectively induces oncogenic target genes such as Myc through its transactivation domain (TAD).To understand the role of Zmiz1 in T-cell development, we bred conditional Zmiz1 and Notch1 knockout mice to Cre strains that delete floxed genes in hematopoietic cells (VavCre/MxCre), in early T cells (LckCre), and in late T-cells (CD4Cre). Like deletion of Notch1, deletion of Zmiz1 caused an early defect at the ETP stage and a late defect at the DN3 stage. The defect with Zmiz1 deletion was less severe than with Notch1 deletion (~4-fold reduction for Zmiz1 deletion versus ~8-fold reduction for Notch1 deletion). Unexpectedly, the ETP defect in Zmiz1-deficient mice partially phenocopied excessive Notch1 activation with increased differentiation to DN2 cells and loss of ETP cells and alternative cell fates (myeloid and NK). To confirm this effect, we plated Zmiz1-deficient hematopoietic stem and progenitor cells (HSPCs) directly on OP9-DL stromal cells. Accordingly, these cultures recapitulated the in vivo phenotype, including suppression of myeloid cells. Reducing Notch signals slightly with modest doses of Notch inhibitors restored myeloid differentiation. In contrast to the ETP defect, the DN3 defect resembled Notch1 loss-of function. Accordingly, overexpression of activated Notch1 or a fusion protein containing only the Notch-interacting domain (TPR) and the TAD was sufficient to rescue the DN3 block.To determine mechanism, we performed RNA-Seq in sorted ETP and DN3 cells from Zmiz1-deficient mice and mice treated with the anti-NRR Notch1 antibody. Zmiz1 coregulated ~16% of Notch1 target genes in ETP cells and ~24% in DN3 cells. In ETP cells, Zmiz1 primarily acted as a repressor of Notch1 target genes. Enrichment analyses showed that Notch1 promoted changes associated with T-lineage commitment, such as induction of Dtx1, Notch3, and Ptcra. In contrast, Zmiz1 reversed these changes. Although generally antagonistic with each other, Zmiz1 and Notch1 concordantly activated a minority of target genes important for proliferation, such as Myc. Upon differentiation to the DN3 cell stage, Zmiz1 switched from primarily a repressor of Notch1 target genes to primarily an activator, inducing Myc and Wnt pathway genes. Accordingly, overexpression of Myc in Zmiz1-deficient DN3 cells was sufficient to rescue the DN3 block in OP9-DL culture. To determine whether Zmiz1 needed to bind Notch1 in order activate or repress Notch1 target genes, we used HSQC NMR to identify amino acids in the TPR that were required for Notch1 binding. Two amino acids, R14 and E34, were confirmed by reporter and co-IP assays to be critical for the Zmiz1-Notch1 interaction. The Zmiz1(R14A+E34A) mutant, which was incapable of binding Notch1, failed to induce Myc. In contrast, this mutant retained full ability to repress Notch target genes associated with T-cell commitment.These data suggest that in ETP cells, Zmiz1 preferentially restrains Notch1 T-cell commitment genes. However, after T-lineage commitment, Zmiz1 switches primarily into a Notch1-interaction mode that preferentially promotes Notch1 signals. It has been puzzling how Notch1 can drive seemingly conflicting biological processes of self-renewal and commitment. Zmiz1 appears to be one solution that evolved to differentially regulate Notch1 signals at a given Notch dosage strength in order to preserve ETP "stemness" while at the same time expanding committed T cells. DisclosuresNo relevant conflicts of interest to declare.

Interaction of CCR4-NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis.

Transcription factor EBF1 (early B-cell factor 1) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. To identify proteins that regulate the diverse functions of EBF1, we used SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4-NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and we identified histidine 240 in EBF1 as a critical residue for this interaction. Complementation of Ebf1-/- progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but we also detected genes with altered occupancy, suggesting that the CCR4-NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which we found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4-NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation.

Contribution of Bistability and Noise to Cell Fate Transitions Determined by Feedback Opening

Alternative cell fates represent a form of non-genetic diversity, which can promote adaptation and functional specialization. It is difficult to predict the rate of the transition between two cell fates due to the strong effect of noise on feedback loops and missing parameters. We opened synthetic positive feedback loops experimentally to obtain open-loop functions. These functions allowed us to identify a deterministic model of bistability by bypassing noise and the requirement to resolve individual processes in the loop. Combining the open-loop function with kinetic measurements and reintroducing the measured noise, we were able to predict the transition rates for the feedback systems without parameter tuning. Noise in gene expression was the key determinant of the transition rates inside the bistable range. Transitions between two cell fates were also observed outside of the bistable range, evidenced by bimodality and hysteresis. In this case, a slow transient process was the rate-limiting step in the transitions. Thus, feedback opening is an effective approach to identify the determinants of cell fate transitions and to predict their rates.