ABSTRACT
Triple-negative breast cancer (TNBC) is characterized by lack of the estrogen (ER) receptor, progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), and standard receptor-targeted therapies are ineffective. FOXC1, a transcription factor aberrantly overexpressed in many cancers, drives growth, metastasis, and stem-cell-like properties in TNBC. However, the molecular function of FOXC1 is unknown, partly due to heterogeneity of TNBC. Here, we show that although FOXC1 regulates many cancer hallmarks in TNBC, its function is varied in different cell lines, highlighted by the differential response to CDK4/6 inhibitors upon FOXC1 loss. Despite this functional heterogeneity, we show that FOXC1 regulates key oncogenes and tumor suppressors and identify a set of core FOXC1 peaks conserved across TNBC cell lines. We identify the ER-associated and drug-targetable nuclear receptor NR2F2 as a cofactor of FOXC1. Finally, we show that core FOXC1 targets in TNBC are regulated in parallel by the pioneer factor FOXA1 and the nuclear receptor NR2F2 in ER + breast cancer.
ABSTRACT
Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.
Subject(s)
Astrocytes , Basic Helix-Loop-Helix Transcription Factors , Cellular Reprogramming , Nerve Tissue Proteins , Neurons , YY1 Transcription Factor , Animals , YY1 Transcription Factor/metabolism , YY1 Transcription Factor/genetics , Astrocytes/metabolism , Mice , Cellular Reprogramming/physiology , Neurons/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Epigenome , Chromatin Assembly and Disassembly , Epigenesis, Genetic , Cells, CulturedABSTRACT
Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. In vivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.
Subject(s)
Neuroblastoma , Piperazines , Pyridines , Tretinoin , Animals , Mice , Humans , Cell Line, Tumor , Cell Differentiation , Tretinoin/pharmacology , Neuroblastoma/drug therapy , Adrenergic Agents/therapeutic useABSTRACT
Neuroblastoma is believed to arise from sympathetic neuroblast precursors that fail to engage the neuronal differentiation programme, but instead become locked in a pro-proliferative developmental state. Achaete-scute homolog 1 (ASCL1) is a proneural master regulator of transcription which modulates both proliferation and differentiation of sympathetic neuroblast precursor cells during development, while its expression has been implicated in the maintenance of an oncogenic programme in MYCN-amplified neuroblastoma. However, the role of ASCL1 expression in neuroblastoma is not clear, especially as its levels vary considerably in different neuroblastoma cell lines. Here, we have investigated the role of ASCL1 in maintaining proliferation and controlling differentiation in both MYCN amplified and Anaplastic Lymphoma Kinase (ALK)-driven neuroblastoma cells. Using CRISPR deletion, we generated neuroblastoma cell lines lacking ASCL1 expression, and these grew more slowly than parental cells, indicating that ASCL1 contributes to rapid proliferation of MYCN amplified and non-amplified neuroblastoma cells. Genome-wide analysis after ASCL1 deletion revealed reduced expression of genes associated with neuronal differentiation, while chromatin accessibility at regulatory regions associated with differentiation genes was also attenuated by ASCL1 knock-out. In neuroblastoma, ASCL1 has been described as part of a core regulatory circuit of developmental regulators whose high expression is maintained by mutual cross-activation of a network of super enhancers and is further augmented by the activity of MYC/MYCN. Surprisingly, ASCL1 deletion had little effect on the transcription of CRC gene transcripts in these neuroblastoma cell lines, but the ability of MYC/MYCN and CRC component proteins, PHOX2B and GATA3, to bind to chromatin was compromised. Taken together, our results demonstrate several roles for endogenous ASCL1 in neuroblastoma cells: maintaining a highly proliferative phenotype, regulating DNA binding of the core regulatory circuit genes to chromatin, while also controlling accessibility and transcription of differentiation targets. Thus, we propose a model where ASCL1, a key developmental regulator of sympathetic neurogenesis, plays a pivotal role in maintaining proliferation while simultaneously priming cells for differentiation in neuroblastoma.
ABSTRACT
Neuroblastoma is a pediatric tumour that accounts for more than 15% of cancer-related deaths in children. High-risk tumours are often difficult to treat, and patients' survival chances are less than 50%. Retinoic acid treatment is part of the maintenance therapy given to neuroblastoma patients; however, not all tumours differentiate in response to retinoic acid. Within neuroblastoma tumors, two phenotypically distinct cell types have been identified based on their super-enhancer landscape and transcriptional core regulatory circuitries: adrenergic (ADRN) and mesenchymal (MES). We hypothesized that the distinct super-enhancers in these different tumour cells mediate differential response to retinoic acid. To this end, three different neuroblastoma cell lines, ADRN (MYCN amplified and non-amplified) and MES cells, were treated with retinoic acid, and changes in the super-enhancer landscape upon treatment and after subsequent removal of retinoic acid was studied. Using ChIP-seq for the active histone mark H3K27ac, paired with RNA-seq, we compared the super-enhancer landscape in cells that undergo neuronal differentiation in response to retinoic acid versus those that fail to differentiate and identified unique super-enhancers associated with neuronal differentiation. Among the ADRN cells that respond to treatment, MYCN-amplified cells remain differentiated upon removal of retinoic acid, whereas MYCN non-amplified cells revert to an undifferentiated state, allowing for the identification of super-enhancers responsible for maintaining differentiation. This study identifies key super-enhancers that are crucial for retinoic acid-mediated differentiation.
ABSTRACT
Neuroblastoma is a solid, neuroendocrine tumor with divergent clinical behavior ranging from asymptomatic to fatal. The diverse clinical presentations of neuroblastoma are directly linked to the high intra- and inter-tumoral heterogeneity it presents. This heterogeneity is strongly associated with therapeutic resistance and continuous relapses, often leading to fatal outcomes. The development of successful risk assessment and tailored treatment strategies lies in evaluating the extent of heterogeneity via the accurate genetic and epigenetic profiling of distinct cell subpopulations present in the tumor. Recent studies have focused on understanding the molecular mechanisms that drive tumoral heterogeneity in pursuing better therapeutic and diagnostic approaches. This review describes the cellular, genetic, and epigenetic aspects of neuroblastoma heterogeneity. In addition, we summarize the recent findings on three crucial factors that can lead to heterogeneity in solid tumors: the inherent diversity of the progenitor cells, the presence of cancer stem cells, and the influence of the tumor microenvironment.
Subject(s)
Neuroblastoma , Humans , Neuroblastoma/genetics , Neuroblastoma/pathology , Neoplastic Stem Cells/pathology , Tumor Microenvironment/geneticsABSTRACT
Adult stem cells constantly react to local changes to ensure tissue homeostasis. In the main body of the stomach, chief cells produce digestive enzymes; however, upon injury, they undergo rapid proliferation for prompt tissue regeneration. Here, we identified p57Kip2 (p57) as a molecular switch for the reserve stem cell state of chief cells in mice. During homeostasis, p57 is constantly expressed in chief cells but rapidly diminishes after injury, followed by robust proliferation. Both single-cell RNA sequencing and dox-induced lineage tracing confirmed the sequential loss of p57 and activation of proliferation within the chief cell lineage. In corpus organoids, p57 overexpression induced a long-term reserve stem cell state, accompanied by altered niche requirements and a mature chief cell/secretory phenotype. Following the constitutive expression of p57 in vivo, chief cells showed an impaired injury response. Thus, p57 is a gatekeeper that imposes the reserve stem cell state of chief cells in homeostasis.
Subject(s)
Chief Cells, Gastric , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Animals , Cell Lineage , Chief Cells, Gastric/metabolism , Mice , Organoids , Stem Cells , StomachABSTRACT
BACKGROUND: The pro-neural transcription factor ASCL1 is a master regulator of neurogenesis and a key factor necessary for the reprogramming of permissive cell types to neurons. Endogenously, ASCL1 expression is often associated with neuroblast stem-ness. Moreover, ASCL1-mediated reprogramming of fibroblasts to differentiated neurons is commonly achieved using artificially high levels of ASCL1 protein, where ASCL1 acts as an "on-target" pioneer factor. However, the genome-wide effects of enhancing ASCL1 activity in a permissive neurogenic environment has not been thoroughly investigated. Here, we overexpressed ASCL1 in the neuronally-permissive context of neuroblastoma (NB) cells where modest endogenous ASCL1 supports the neuroblast programme. RESULTS: Increasing ASCL1 in neuroblastoma cells both enhances binding at existing ASCL1 sites and also leads to creation of numerous additional, lower affinity binding sites. These extensive genome-wide changes in ASCL1 binding result in significant reprogramming of the NB transcriptome, redirecting it from a proliferative neuroblastic state towards one favouring neuronal differentiation. Mechanistically, ASCL1-mediated cell cycle exit and differentiation can be increased further by preventing its multi-site phosphorylation, which is associated with additional changes in genome-wide binding and gene activation profiles. CONCLUSIONS: Our findings show that enhancing ASCL1 activity in a neurogenic environment both increases binding at endogenous ASCL1 sites and also results in additional binding to new low affinity sites that favours neuronal differentiation over the proliferating neuroblast programme supported by the endogenous protein. These findings have important implications for controlling processes of neurogenesis in cancer and cellular reprogramming.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Neural Stem Cells , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cellular Reprogramming/genetics , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neurons/metabolismABSTRACT
The growth of glioblastoma (GBM), one of the deadliest adult cancers, is fuelled by a subpopulation of stem/progenitor cells, which are thought to be the source of resistance and relapse after treatment. Re-engagement of a latent capacity of these cells to re-enter a trajectory resulting in cell differentiation is a potential new therapeutic approach for this devastating disease. ASCL1, a proneural transcription factor, plays a key role in normal brain development and is also expressed in a subset of GBM cells, but fails to engage a full differentiation programme in this context. Here, we investigated the barriers to ASCL1-driven differentiation in GBM stem cells. We see that ASCL1 is highly phosphorylated in GBM stem cells where its expression is compatible with cell proliferation. However, overexpression of a form of ASCL1 that cannot be phosphorylated on Serine-Proline sites drives GBM cells down a neuronal lineage and out of cell cycle more efficiently than its wild-type counterpart, an effect further enhanced by deletion of the inhibitor of differentiation ID2, indicating mechanisms to reverse the block to GBM cell differentiation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Brain Neoplasms/metabolism , Brain Neoplasms/physiopathology , Glioblastoma/metabolism , Glioblastoma/physiopathology , Inhibitor of Differentiation Protein 2/genetics , Neoplastic Stem Cells/metabolism , Amino Acid Motifs , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Brain Neoplasms/genetics , Cell Cycle , Cell Differentiation , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Humans , Inhibitor of Differentiation Protein 2/metabolism , Neoplastic Stem Cells/cytology , PhosphorylationABSTRACT
Interactions between tumour cells and the surrounding microenvironment contribute to tumour progression, metastasis and recurrence1-3. Although mosaic analyses in Drosophila have advanced our understanding of such interactions4,5, it has been difficult to engineer parallel approaches in vertebrates. Here we present an oncogene-associated, multicolour reporter mouse model-the Red2Onco system-that allows differential tracing of mutant and wild-type cells in the same tissue. By applying this system to the small intestine, we show that oncogene-expressing mutant crypts alter the cellular organization of neighbouring wild-type crypts, thereby driving accelerated clonal drift. Crypts that express oncogenic KRAS or PI3K secrete BMP ligands that suppress local stem cell activity, while changes in PDGFRloCD81+ stromal cells induced by crypts with oncogenic PI3K alter the WNT signalling environment. Together, these results show how oncogene-driven paracrine remodelling creates a niche environment that is detrimental to the maintenance of wild-type tissue, promoting field transformation dominated by oncogenic clones.
Subject(s)
Colorectal Neoplasms/pathology , Intestine, Small/pathology , Neoplastic Stem Cells/pathology , Oncogenes , Stem Cell Niche , Animals , Clone Cells/pathology , Colorectal Neoplasms/genetics , Female , Intestine, Small/metabolism , Male , Mice , Mutation , Neoplastic Stem Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Reproducibility of Results , Single-Cell Analysis , Stem Cell Niche/genetics , Tumor Microenvironment , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt Signaling PathwayABSTRACT
Problems of cell biology and the molecular controls underpinning them have been studied in the remarkably versatile Xenopus systems for many years. This versatility is showcased in several accompanying protocols, which are introduced here. One protocol demonstrates how the Xenopus embryonic ectoderm can be used to study the effects of mechanical cell deformation; another illustrates how the developing eye can be used as a platform for determining cell-cycle length. Two protocols show how extracts from Xenopus embryos can be exploited to characterize the behavior of specific intracellular proteins-specifically, to determine protein phosphorylation status and the ability to bind to chromatin. Finally, because specific antibodies to Xenopus proteins are pivotal reagents for cell biology and biochemistry applications, four protocols describing how to generate, purify, and assay the specificity of antibodies raised against Xenopus proteins are included in hopes of stimulating the expansion of these critical resources across the Xenopus community.
Subject(s)
Cell Biology , Cytological Techniques/methods , Ectoderm/metabolism , Embryo, Nonmammalian/metabolism , Xenopus Proteins/metabolism , Animals , Antibodies/immunology , Antibodies/metabolism , Biochemistry/methods , Chromatin/metabolism , Ectoderm/embryology , Embryo, Nonmammalian/embryology , Humans , Immunity/immunology , Models, Animal , Phosphorylation , Protein Binding , Xenopus Proteins/immunology , Xenopus laevisABSTRACT
Emerging three-dimensional (3D) cultures of glioblastoma are becoming powerful models to study glioblastoma stem cell behavior and the impact of cell-cell and cell-microenvironment interactions on tumor growth and invasion. Here we describe a method for culturing human glioblastoma stem cells (GSCs) in 3D by co-culturing them with pluripotent stem cell-derived brain organoids. This requires multiple coordinated steps, including the generation of cerebral organoids, and the growth and fluorescence tagging of GSCs. We highlight how to recognize optimal organoid generation and how to efficiently mark GSCs, before describing optimized co-culture conditions. We show that GSCs can efficiently integrate into brain organoids and maintain a significant degree of cell fate heterogeneity, paving the way for the analysis of GSC fate behavior and lineage progression. These results establish the 3D culture system as a viable and versatile GBM model for investigating tumor cell biology and GSC heterogeneity.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Brain Neoplasms/pathology , Coculture Techniques , Glioblastoma/pathology , Neoplastic Stem Cells/pathology , Organoids , Biomarkers , Cell Differentiation , Cell Line, Tumor , Cell Lineage/genetics , HumansABSTRACT
Pediatric cancers often resemble trapped developmental intermediate states that fail to engage the normal differentiation program, typified by high-risk neuroblastoma arising from the developing sympathetic nervous system. Neuroblastoma cells resemble arrested neuroblasts trapped by a stable but aberrant epigenetic program controlled by sustained expression of a core transcriptional circuit of developmental regulators in conjunction with elevated MYCN or MYC (MYC). The transcription factor ASCL1 is a key master regulator in neuroblastoma and has oncogenic and tumor-suppressive activities in several other tumor types. Using functional mutational approaches, we find that preventing CDK-dependent phosphorylation of ASCL1 in neuroblastoma cells drives coordinated suppression of the MYC-driven core circuit supporting neuroblast identity and proliferation, while simultaneously activating an enduring gene program driving mitotic exit and neuronal differentiation. IMPLICATIONS: These findings indicate that targeting phosphorylation of ASCL1 may offer a new approach to development of differentiation therapies in neuroblastoma. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/12/1759/F1.large.jpg.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Neuroblastoma/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Phosphorylation , Protein Processing, Post-Translational , Up-RegulationABSTRACT
Pancreatic islets play an essential role in regulating blood glucose level. Although the molecular pathways underlying islet cell differentiation are beginning to be resolved, the cellular basis of islet morphogenesis and fate allocation remain unclear. By combining unbiased and targeted lineage tracing, we address the events leading to islet formation in the mouse. From the statistical analysis of clones induced at multiple embryonic timepoints, here we show that, during the secondary transition, islet formation involves the aggregation of multiple equipotent endocrine progenitors that transition from a phase of stochastic amplification by cell division into a phase of sublineage restriction and limited islet fission. Together, these results explain quantitatively the heterogeneous size distribution and degree of polyclonality of maturing islets, as well as dispersion of progenitors within and between islets. Further, our results show that, during the secondary transition, α- and ß-cells are generated in a contemporary manner. Together, these findings provide insight into the cellular basis of islet development.
Subject(s)
Cell Differentiation , Glucagon-Secreting Cells/physiology , Insulin-Secreting Cells/physiology , Pancreas/embryology , Animals , Cell Lineage/physiology , Computer Simulation , Embryo, Mammalian , Embryonic Development , Female , Genes, Reporter/genetics , Imaging, Three-Dimensional , Luminescent Proteins/genetics , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Models, Animal , Models, Biological , Organogenesis , Pancreas/diagnostic imaging , Stem Cells/physiologyABSTRACT
Only one class of targeted agents (anti-GD2 antibodies) has been incorporated into front-line therapy for neuroblastoma since the 1980s. The Neuroblastoma New Drug Development Strategy (NDDS) initiative commenced in 2012 to accelerate the development of new drugs for neuroblastoma. Advances have occurred, with eight of nine high-priority targets being evaluated in paediatric trials including anaplastic lymphoma kinase inhibitors being investigated in front-line, but significant challenges remain. This article reports the conclusions of the second NDDS forum, which expanded across the Atlantic to further develop the initiative. Pre-clinical and clinical data for 40 genetic targets and mechanisms of action were prioritised and drugs were identified for early-phase trials. Strategies to develop drugs targeting TERT, telomere maintenance, ATRX, alternative lengthening of telomeres (ALT), BRIP1 and RRM2 as well as direct targeting of MYCN are high priority and should be championed for drug discovery. Promising pre-clinical data suggest that targeting of ALT by ATM or PARP inhibition may be potential strategies. Drugs targeting CDK2/9, CDK7, ATR and telomere maintenance should enter paediatric clinical development rapidly. Optimising the response to anti-GD2 by combinations with chemotherapy, targeted agents and other immunological targets are crucial. Delivering this strategy in the face of small patient cohorts, genomically defined subpopulations and a large number of permutations of combination trials, demands even greater international collaboration. In conclusion, the NDDS provides an internationally agreed, biologically driven selection of prioritised genetic targets and drugs. Improvements in the strategy for conducting trials in neuroblastoma will accelerate bringing these new drugs more rapidly to front-line therapy.
Subject(s)
Brain Neoplasms/drug therapy , Drug Development , Neuroblastoma/drug therapy , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/therapeutic use , Brain Neoplasms/pathology , Child , Congresses as Topic , Drug Development/methods , Drug Development/organization & administration , Drug Development/trends , Drug Discovery/methods , Drug Discovery/organization & administration , Drug Discovery/trends , Europe , Humans , Medical Oncology/methods , Medical Oncology/organization & administration , Medical Oncology/trends , Molecular Targeted Therapy/methods , Molecular Targeted Therapy/trends , Neuroblastoma/pathology , Pediatrics/methods , Pediatrics/organization & administration , Pediatrics/trends , Protein Kinase Inhibitors/isolation & purification , Protein Kinase Inhibitors/therapeutic use , Therapies, Investigational/methods , Therapies, Investigational/trendsABSTRACT
The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of "punctuated" neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs.
Subject(s)
Adult Stem Cells/cytology , Gastric Mucosa/cytology , Stomach/cytology , Adult Stem Cells/metabolism , Biomarkers/metabolism , Cell Differentiation , Cell Lineage , Cell Self Renewal , Cells, Cultured , Gastric Mucosa/metabolism , Humans , Ki-67 Antigen/metabolism , Sequence Analysis, RNA , Single-Cell Analysis , Stathmin/metabolism , Stem Cell NicheABSTRACT
Xenopus embryos have long been used to show phenotypic effects following overexpression of proteins of interest such as transcription factors. Posttranslational modification of these proteins can dramatically alter the extent of the observed phenotype by inhibiting or enhancing protein activity. To determine the mechanisms controlling transcription factor activity, it is useful to compare relative levels of chromatin-bound protein, as this can reveal altered chromatin association in addition to changes in overall protein accumulation seen in the cytoplasm. Assaying protein binding to the bulk DNA described here compliments alternative assays such as electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) that measure site-specific DNA binding. This protocol describes a method to prepare and analyze chromatin and cytoplasmic extracts from embryos overexpressing proteins of interest, and it uses a robust fractionation procedure that results in clear separation of cytoplasmic tubulin from histone-H3 enriched chromatin. This assay for relative chromatin-bound protein is most suitable for comparing modified forms of a single protein (e.g., to investigate the effects of point mutations on chromatin association). Optimization is required for the specific protein of interest but guide ranges are provided.
Subject(s)
Chromatin/chemistry , DNA-Binding Proteins/analysis , Xenopus Proteins/analysis , Animals , Protein Binding , Xenopus/embryologyABSTRACT
Xenopus embryos provide a rapid and accessible in vivo model, expressing a plethora of endogenous kinase and phosphatase enzymes that control protein phosphorylation and, in turn, affect physiological function. Traditionally, the detection of protein phosphorylation has been achieved by radioisotope phosphate labeling of proteins, sometimes with in vitro assays using recombinant proteins or with site-specific phospho-antibodies. However, the target phospho-sites and kinases responsible are often unknown, and the use of radioactive isotopes is not always desirable. Therefore, as a first step in determining the functional significance of potential phosphorylation, it is useful to show that a protein can be phosphorylated in vivo in Xenopus eggs and/or embryos. This protocol describes a nonradioactive method to visualize protein phosphorylation by exposing the protein to the egg/embryo kinase environment and then observing a difference in protein migration (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] and western blot analysis) with and without treatment with the lambda phosphatase enzyme. Subsequent investigation of the ability of site-specific phospho-mutant proteins to recapitulate the effect of phosphatase treatment can be used to explore the identity of the phosphorylated sites. Moreover, the detection of multiple bands of the protein of interest even after phosphatase treatment points to the presence of other types of posttranslational modifications.
Subject(s)
Blotting, Western/methods , Electrophoretic Mobility Shift Assay/methods , Protein Processing, Post-Translational , Xenopus Proteins/metabolism , Animals , Electrophoresis, Polyacrylamide Gel , Phosphorylation , Xenopus/embryologyABSTRACT
The superfamily of basic-Helix-Loop-Helix (bHLH) transcription factors influence cell fate in all three embryonic germ layers, and the tissue-specific class II factors have received prominent attention for their potent ability to direct differentiation during development and in cellular reprogramming. The activity of many class II bHLH proteins driving differentiation, and the inhibitory class VI bHLH factor Hes1, is controlled by phosphorylation on multiple sites by Cyclin-dependent kinases (Cdks). As class II proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins, regulation of class I transcription factors such as E47 may influence the activity of multiple tissue-specific bHLH proteins. Using differentiation of nerve and muscle in Xenopus frog embryos as a model system, we set out to explore whether with the ubiquitously expressed class I E protein E47 that hetero-dimerises with Class II bHLHs to control their activity, is also regulated by multi-site phosphorylation. We demonstrate that E47 can be readily phosphorylated by Cdks on multiple sites in vitro, while ectopically-expressed E47 exists in multiple phosphorylated forms in Xenopus embryos. Preventing multi-site phosphorylation using a phospho-mutant version of E47 enhances the neurogenic and myogenic activity of three different class II bHLH reprogramming factors, and also when E47 acts in hetero-dimerisation with endogenous proteins. Mechanistically, unlike phospho-regulation of class II bHLH factors, we find that preventing phosphorylation of E47 increases the amount of chromatin-bound E47 protein but without affecting its overall protein stability. Thus, multi-site phosphorylation is a conserved regulatory mechanism across the bHLH superfamily that can be manipulated to enhance cellular differentiation.
Subject(s)
Muscle Development , Neurogenesis , Transcription Factor 3/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Animals , Mice , Mutation , Phosphorylation , Transcription Factor 3/genetics , Xenopus Proteins/genetics , Xenopus laevis/metabolismABSTRACT
The processes of cell proliferation and differentiation are intimately linked during embryogenesis, and the superfamily of (basic) Helix-Loop-Helix (bHLH) transcription factors play critical roles in these events. For example, neuronal differentiation is promoted by class II bHLH proneural proteins such as Ngn2 and Ascl1, while class VI Hes proteins act to restrain differentiation and promote progenitor maintenance. We have previously described multi-site phosphorylation as a key regulator of tissue specific class II bHLH proteins in all three embryonic germ layers, and this enables coordination of differentiation with the cell cycle. Hes1 homologues also show analogous conserved proline directed kinase sites. Here we have used formation of Xenopus primary neurons to investigate the effects of xHes1 multi-site phosphorylation on both endogenous and ectopic proneural protein-induced neurogenesis. We find that xHes1 is phosphorylated in vivo, and preventing phosphorylation on three conserved SP/TP sites in the N terminus of the protein enhances xHes1 protein stability and repressor activity. Mechanistically, compared to wild-type xHes1, phospho-mutant xHes1 exhibits greater repression of Ngn2 transcription as well as producing a greater reduction in Ngn2 protein stability and chromatin binding. We propose that cell cycle dependent phosphorylation of class VI Hes proteins may act alongside similar regulation of class II bHLH proneural proteins to co-ordinate their activity.