Tracing oncogene-driven remodelling of the intestinal stem cell niche.

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.

Elevated ASCL1 activity creates de novo regulatory elements associated with neuronal differentiation.

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.

The developmental origin of brain tumours: a cellular and molecular framework.

The development of the nervous system relies on the coordinated regulation of stem cell self-renewal and differentiation. The discovery that brain tumours contain a subpopulation of cells with stem/progenitor characteristics that are capable of sustaining tumour growth has emphasized the importance of understanding the cellular dynamics and the molecular pathways regulating neural stem cell behaviour. By focusing on recent work on glioma and medulloblastoma, we review how lineage tracing contributed to dissecting the embryonic origin of brain tumours and how lineage-specific mechanisms that regulate stem cell behaviour in the embryo may be subverted in cancer to achieve uncontrolled proliferation and suppression of differentiation.

N-terminal phosphorylation of xHes1 controls inhibition of primary neurogenesis in Xenopus.

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.

Multi-site phosphorylation controls the neurogenic and myogenic activity of E47.

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.

Cell cycle-dependent phosphorylation and regulation of cellular differentiation.

Embryogenesis requires an exquisite regulation of cell proliferation, cell cycle withdrawal and differentiation into a massively diverse range of cells at the correct time and place. Stem cells also remain to varying extents in different adult tissues, acting in tissue homeostasis and repair. Therefore, regulated proliferation and subsequent differentiation of stem and progenitor cells remains pivotal throughout life. Recent advances have characterised the cell cycle dynamics, epigenetics, transcriptome and proteome accompanying the transition from proliferation to differentiation, revealing multiple bidirectional interactions between the cell cycle machinery and factors driving differentiation. Here, we focus on a direct mechanistic link involving phosphorylation of differentiation-associated transcription factors by cell cycle-associated Cyclin-dependent kinases. We discuss examples from the three embryonic germ layers to illustrate this regulatory mechanism that co-ordinates the balance between cell proliferation and differentiation.

Nervous decision-making: to divide or differentiate.

The intricate balance between proliferation and differentiation is of fundamental importance in the development of the central nervous system (CNS). The division versus differentiation decision influences both the number and identity of daughter cells produced, thus critically shaping the overall microstructure and function of the CNS. During the past decade, significant advances have been made to characterise the changes in the cell cycle during differentiation, and to uncover the multiple bidirectional links that coordinate these two processes. Here, we explore the nature and mechanistic basis of these links in the context of the developing CNS, highlighting new insights into transcriptional, post-translational, and epigenetic levels of interaction.

The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro.

Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. Here we demonstrate the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, we see that this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.

An oncologist׳s friend: How Xenopus contributes to cancer research.

One of the most striking features of the Xenopus system is the versatility in providing a unique range of both in vitro and in vivo models that are rapid, accessible and easily manipulated. Here we present an overview of the diverse contribution that Xenopus has made to advance our understanding of tumour biology and behaviour; a contribution that goes beyond the traditional view of Xenopus as a developmental model organism. From the utility of the egg and oocyte extract system to the use of whole embryos as developmental or induced tumour models, the Xenopus system has been fundamental to investigation of cell cycle mechanisms, cell metabolism, cell signalling and cell behaviour, and has allowed an increasing appreciation of the parallels between early development and the pathogenesis of tumour progression and metastasis. Although not the prototypical oncological model system, we propose that Xenopus is an adaptable and multifunctional tool in the oncologist׳s arsenal.

MyoD phosphorylation on multiple C terminal sites regulates myogenic conversion activity.

MyoD is a master regulator of myogenesis with a potent ability to redirect the cell fate of even terminally differentiated cells. Hence, enhancing the activity of MyoD is an important step to maximising its potential utility for in vitro disease modelling and cell replacement therapies. We have previously shown that the reprogramming activity of several neurogenic bHLH proteins can be substantially enhanced by inhibiting their multi-site phosphorylation by proline-directed kinases. Here we have used Xenopus embryos as an in vivo developmental and reprogramming system to investigate the multi-site phospho-regulation of MyoD during muscle differentiation. We show that, in addition to modification of a previously well-characterised site, Serine 200, MyoD is phosphorylated on multiple additional serine/threonine sites during primary myogenesis. Through mutational analysis, we derive an optimally active phospho-mutant form of MyoD that has a dramatically enhanced ability to drive myogenic reprogramming in vivo. Mechanistically, this is achieved through increased protein stability and enhanced chromatin association. Therefore, multi-site phospho-regulation of class II bHLH proteins is conserved across cell lineages and germ layers, and manipulation of phosphorylation of these key regulators may have further potential for enhancing mammalian cell reprogramming.

Post-translational modification of Ngn2 differentially affects transcription of distinct targets to regulate the balance between progenitor maintenance and differentiation.

Neurogenin 2 (Ngn2) controls neuronal differentiation cell-autonomously by transcriptional activation of targets such as NeuroD, while simultaneously controlling progenitor maintenance non-cell-autonomously by upregulating Delta expression and Notch signalling. Reduction in Cdk-dependent multisite phosphorylation of Ngn2 enhances its promoter binding affinity. This leads specifically to an increase in neuronal differentiation without an apparent increase in progenitor maintenance via Delta-Notch signalling, although the mechanism underlying this imbalance remains unclear. Here we show in Xenopus embryos and mouse P19 cells that the NeuroD promoter is substantially more sensitive to the phosphorylation status of Ngn2 than the Delta promoter, and that this can be attributed to differences in the ease of promoter activation. In addition, we also show that the phosphorylation status of Ngn2 regulates sensitivity to Notch signalling. These observations explain how Ngn2 post-translational modification in response to changes in the cell cycle kinase environment results in enhanced neuronal differentiation upon cell cycle lengthening.

Cell cycle regulation of proliferation versus differentiation in the central nervous system.

Formation of the central nervous system requires a period of extensive progenitor cell proliferation, accompanied or closely followed by differentiation; the balance between these two processes in various regions of the central nervous system gives rise to differential growth and cellular diversity. The correlation between cell cycle lengthening and differentiation has been reported across several types of cell lineage and from diverse model organisms, both in vivo and in vitro. Furthermore, different cell fates might be determined during different phases of the preceding cell cycle, indicating direct cell cycle influences on both early lineage commitment and terminal cell fate decisions. Significant advances have been made in the last decade and have revealed multi-directional interactions between the molecular machinery regulating the processes of cell proliferation and neuronal differentiation. Here, we first introduce the modes of proliferation in neural progenitor cells and summarise evidence linking cell cycle length and neuronal differentiation. Second, we describe the manner in which components of the cell cycle machinery can have additional and, sometimes, cell-cycle-independent roles in directly regulating neurogenesis. Finally, we discuss the way that differentiation factors, such as proneural bHLH proteins, can promote either progenitor maintenance or differentiation according to the cellular environment. These intricate connections contribute to precise coordination and the ultimate division versus differentiation decision.

Complex domain interactions regulate stability and activity of closely related proneural transcription factors.

Characterising post-translational regulation of key transcriptional activators is crucial for understanding how cell division and differentiation are coordinated in developing organisms and cycling cells. One important mode of protein post-translational control is by regulation of half-life via ubiquitin-mediated proteolysis. Two key basic Helix-Loop-Helix transcription factors, Neurogenin 2 (Ngn2) and NeuroD, play central roles in development of the central nervous system but despite their homology, Ngn2 is a highly unstable protein whilst NeuroD is, by comparison, very stable. The basis for and the consequences of the difference in stability of these two structurally and functionally related proteins has not been explored. Here we see that ubiquitylation alone does not determine Ngn2 or NeuroD stability. By making chimeric proteins, we see that the N-terminus of NeuroD in particular has a stabilising effect, whilst despite their high levels of homology, the most conserved bHLH domains of these proneural proteins alone can confer significant changes in protein stability. Despite widely differing stabilities of Ngn2, NeuroD and the chimeric proteins composed of domains of both, there is little correlation between protein half-life and ability to drive neuronal differentiation. Therefore, we conclude that despite significant homology between Ngn2 and NeuroD, the regulation of their stability differs markedly and moreover, stability/instability of the proteins is not a direct correlate of their activity.

Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis.

During development of the central nervous system, the transition from progenitor maintenance to differentiation is directly triggered by a lengthening of the cell cycle that occurs as development progresses. However, the mechanistic basis of this regulation is unknown. The proneural transcription factor Neurogenin 2 (Ngn2) acts as a master regulator of neuronal differentiation. Here, we demonstrate that Ngn2 is phosphorylated on multiple serine-proline sites in response to rising cyclin-dependent kinase (cdk) levels. This multi-site phosphorylation results in quantitative inhibition of the ability of Ngn2 to induce neurogenesis in vivo and in vitro. Mechanistically, multi-site phosphorylation inhibits binding of Ngn2 to E box DNA, and inhibition of DNA binding depends on the number of phosphorylation sites available, quantitatively controlling promoter occupancy in a rheostat-like manner. Neuronal differentiation driven by a mutant of Ngn2 that cannot be phosphorylated by cdks is no longer inhibited by elevated cdk kinase levels. Additionally, phosphomutant Ngn2-driven neuronal differentiation shows a reduced requirement for the presence of cdk inhibitors. From these results, we propose a model whereby multi-site cdk-dependent phosphorylation of Ngn2 interprets cdk levels to control neuronal differentiation in response to cell cycle lengthening during development.

Phosphorylation in intrinsically disordered regions regulates the activity of Neurogenin2.

BACKGROUND: Neuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear. RESULTS: Here we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks). CONCLUSIONS: Phosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation.

The cell cycle and pluripotency.

PSCs (pluripotent stem cells) possess two key properties that have made them the focus of global research efforts in regenerative medicine: they have unlimited expansion potential under conditions which favour their preservation as PSCs and they have the ability to generate all somatic cell types upon differentiation (pluripotency). Conditions have been defined in vitro in which pluripotency is maintained, or else differentiation is favoured and is directed towards specific somatic cell types. However, an unanswered question is whether or not the core cell cycle machinery directly regulates the pluripotency and differentiation properties of PSCs. If so, then manipulation of the cell cycle may represent an additional tool by which in vitro maintenance or differentiation of PSCs may be controlled in regenerative medicine. The present review aims to summarize our current understanding of links between the core cell cycle machinery and the maintenance of pluripotency in ESCs (embryonic stem cells) and iPSCs (induced PSCs).

Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1.

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.

Co-ordination of cell cycle and differentiation in the developing nervous system.

During embryonic development, cells must divide to produce appropriate numbers, but later must exit the cell cycle to allow differentiation. How these processes of proliferation and differentiation are co-ordinated during embryonic development has been poorly understood until recently. However, a number of studies have now given an insight into how the cell cycle machinery, including cyclins, CDKs (cyclin-dependent kinases), CDK inhibitors and other cell cycle regulators directly influence mechanisms that control cell fate and differentiation. Conversely, examples are emerging of transcriptional regulators that are better known for their role in driving the differentiated phenotype, which also play complementary roles in controlling cell cycle progression. The present review will summarise our current understanding of the mechanisms co-ordinating the cell cycle and differentiation in the developing nervous system, where these links have been, perhaps, most extensively studied.

Palbociclib releases the latent differentiation capacity of neuroblastoma cells.

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.

Neuroblastoma progress on many fronts: the Neuroblastoma Research Symposium.

Neuroblastoma (NBL) is a pediatric tumor of infancy derived from precursor cells of the sympathetic nervous system. Clinicians and researchers in developmental biology and genetics recently met to facilitate meaningful crosstalk and to discuss considerable progress made in the clinical treatment and basic biology of NBL. For instance, discoveries in familial NBL have identified genetic aberrations in Phox2b and Alk that predispose to NBL, while advances in epigenetics and MYCN regulation have also offered insight into NBL pathogenesis and future treatment. Moreover, novel therapeutic avenues are also being explored, including targeted immunotherapies, and innovative radiotherapeutic and chemotherapeutic approaches. This multi-disciplinary meeting was convened to aid the transfer of new biological findings into the clinic and to use clinical advances to inform the basic biological understanding of this devastating disease.