Karen Rother

Differential regulation of transcription and induction of programmed cell death by human p53‐family members p63 and p73

The p53 tumor suppressor acts as a transcription factor and has a central function in controlling apoptosis. With p63 and p73 two genes coding for proteins homologous to p53 have been identified. We describe the properties of seven human p63 and p73 proteins as transcriptional activators of p21WAF1/CIP1 expression and apoptotic inducers in direct comparison to p53 in the same assay systems employing DLD‐1‐tet‐off colon cells. Programmed cell death is detected in cells expressing high levels of p53 and p73α. Cells overexpressing TAp63α, TAp63γ, TA*p63α, TA*p63γ, ΔNp63α, and ΔNp63γ display low or no detectable apoptosis.

Identification of Tcf-4 as a transcriptional target of p53 signalling.

T-cell factor (Tcf)-4 is a main transcription factor to pass on Wnt/β-catenin signalling. The tumour suppressor protein p53 contributes as a transcription factor to cell-cycle arrest and apoptosis induction. Mutations of components in p53 and Wnt/β-catenin signalling networks play a part in tumour formation. Here, we identify the Tcf-4 gene as a downstream effector of p53. Induction of wild-type p53 in a tet-off regulated human colon cell system leads to the reduction of Tcf-4 mRNA and protein levels. Also, mRNA of the Tcf-4 target gene uPAR is downregulated after p53 induction. Expression of a luciferase reporter controlled by the Tcf-4 promoter is repressed by wild-type p53, but not by a p53 mutant deficient in DNA binding. Such a regulation is seen in cell lines of different origin. These findings directly link Wnt/β-catenin signalling and p53 tumour suppressor function and may provide a mechanism by which loss of p53 function contributes to progression in the adenoma/carcinoma sequence in colon tumours. Furthermore, since Tcf-4 is expressed in many tissues and downregulation of Tcf-4 by p53 is seen in several different cell types, this regulation likely plays a role in proliferation control of all tissues that can express p53 and Tcf-4.

p53 downregulates expression of the G1/S cell cycle phosphatase Cdc25A.

Overexpression of Cdc25A phosphatase is often observed in cancer and results in poor prognosis. Cdc25A mainly dephosphorylates and thereby activates Cyclin-dependent kinase 2 and thus induces progression in the cell cycle from G1 to S phase. Here, we demonstrate that the tumor suppressor p53 downregulates expression from the Cdc25A gene. In a p53-inducible cell system, Cdc25A expression on the mRNA and protein level is downregulated upon p53 expression. Promoter-reporter assays show that this regulation is dependent on the Cdc25A promoter. Mutant p53 fails to reduce Cdc25A transcription. In contrast to p53, neither p63 nor p73 can repress Cdc25A transcription. The Cdc25A promoter displays no p53 binding site, and p53 does not bind directly to the promoter DNA as shown by chromatin immunoprecipitation assays. Previously, the contribution of p53 to G1/S arrest has been mostly linked to activating the expression of the Cdk inhibitor p21WAF1/CIP1. By downregulating Cdc25A expression, p53 may impair transition from G1 to S phase independently of p21WAF1/CIP1. Therefore, the data suggest that, as long as p53 is intact, Cdc25A transcriptional downregulation might play a role in cancer prevention.

One function--multiple mechanisms: the manifold activities of p53 as a transcriptional repressor.

Maintenance of genome integrity is a dynamic process involving complex regulation systems. Defects in one or more of these pathways could result in cancer. The most important tumor-suppressor is the transcription factor p53, and its functional inactivation is frequently observed in many tumor types. The tumor suppressive function of p53 is mainly attributed to its ability to regulate numerous target genes at the transcriptional level. While the mechanism of transcriptional induction by p53 is well characterized, p53-dependent repression is not understood in detail. Here, we review the manifold mechanisms of p53 as a transcriptional repressor. We classify two different categories of repressed genes based on the underlying mechanism, and novel mechanisms which involve regulation through noncoding RNAs are discussed. The complete elucidation of p53 functions is important for our understanding of its tumor-suppressor activity and, therefore, represents the key for the development of novel therapeutic approaches.

Expression of cyclin-dependent kinase subunit 1 (Cks1) is regulated during the cell cycle by a CDE/CHR tandem element and is downregulated by p53 but not by p63 or p73.

Cks1 is a member of the Cyclin-dependent kinase subunit family. These proteins are essential components of cyclin/cyclin-dependent kinase (cdk) complexes contributing to cell cycle control in all eukaryotes. Cks1 protein is found overexpressed in a number of tumors. Expression of Cks1 mRNA starts in late G1 reaching a peak in S/G2-phases of the cell cycle. We find that this expression pattern depends on transcriptional regulation and is controlled by a combination of a cell cycle-dependent element (CDE) together with a cell cycle genes homology region (CHR) in the Cks1 promoter. Furthermore, we observe Cks1 mRNA and protein to be downregulated after induced expression of the tumor suppressor p53. This repression is due to p53 downregulating transcription from the Cks1 promoter. p53-dependent repression is seen in a dose-dependent manner and in several cell types of different origin. In contrast to p53, its homologues p63 and p73 do not significantly repress transcription from the Cks1 promoter. The Cks1 promoter does not contain a p53 binding site. For some promoters the CCAAT box-binding transcription factor NF-Y had been implicated in p53-dependent repression. NF-Y is the main activator for Cks1 transcription but does not influence p53-dependent repression from the Cks1 promoter. Generally, the observation that the potential oncogene Cks1 is downregulated by the tumor suppressor p53 corresponds well with the idea that p53 employs multiple ways in order to halt the cell cycle.

Gene expression of cyclin-dependent kinase subunit Cks2 is repressed by the tumor suppressor p53 but not by the related proteins p63 or p73

Cks2 proteins are essential components of cyclin/cyclin‐dependent kinase complexes and contribute to cell cycle control. We identify Cks2 as a transcriptional target downregulated by the tumor suppressor p53. Cks2 expression was found to be repressed by p53 both at the mRNA and the protein levels. p53 downregulates transcription from the Cks2 promoter in a dose‐dependent manner and in all cell types tested. This repression appears to be independent of p53 binding to the Cks2 promoter. In contrast to p53, neither p63 nor p73 proteins can repress Cks2 transcription. Thus p53, rather than its homologues p63 and p73, may contribute to control of the first metaphase/anaphase transition of mammalian meiosis by downregulation of Cks2 expression.

The retinal dehydrogenase/reductase retSDR1/DHRS3 gene is activated by p53 and p63 but not by mutants derived from tumors or EEC/ADULT malformation syndromes.

Retinol and its metabolites have important roles in many processes including embryonic development, cellular differentiation, apoptosis and maintenance of epithelia. Retinal short-chain dehydrogenase/reductase retSDR1, also known as dehydrogenase/reductase member 3 (DHRS3), is involved in maintaining the cellular supply of retinol metabolites. We observe that retSDR1 expression is activated by members of the p53 family. Particularly p53 and TAp63γ regulate transcription through two separate response elements in the retSDR1 promoter. Both proteins bind the promoter in vitro and in vivo. Induction of DNA damage leads to recruitment of p53 and p63 to the retSDR1 promoter. A tumor-derived p53 mutant is unable to activate retSDR1 transcription. As mutants of p63 in humans exhibit phenotypes that cause several autosomal dominantly inherited syndromes leading to developmental malformations, we tested the transcriptional response of TAp63γ mutants derived from the EEC, SHFM and ADULT syndromes. EEC syndrome-specific mutations of TAp63γ fail to transactivate retSDR1 and an ADULT syndrome-derived mutant stimulates retSDR1 transcription significantly less than the wild-type variant of p63. Taken together, the results suggest a potential role of the p53/p63-mediated retSDR1 activation in tumor suppression as well as in developmental processes.

The CCN3 gene coding for an extracellular adhesion-related protein is transcriptionally activated by the p53 tumor suppressor

The CCN3 protein (Nov, Nephroblastoma overexpressed) is a member of the CCN family (Cyr61, CTGF, Nov) of growth regulators and exerts antiproliferative properties. We show here that the tumor suppressor p53 transcriptionally upregulates the CCN3 gene. p53 is an important transcription factor contributing to cell cycle arrest and apoptosis after cell damage through the regulation of numerous target genes. We show that CCN3 mRNA and protein are upregulated following p53 expression. A DNA binding-deficient p53 mutant fails to regulate CCN3. CCN3 protein is located in the perinuclear space after induction and is also exported to the extracellular matrix. Furthermore, the CCN3 promoter is inducible by p53 and the response element is located in the first exon of the CCN3 gene. Chromatin immunoprecipitations show that p53 binds to the CCN3 promoter in vivo. As CCN3 was shown to inhibit cell growth, transcriptional induction by p53 may serve as an antiproliferative signal in the extracellular matrix. Furthermore, CCN3 depletion was also reported to reduce collagen type IV-dependent adhesion of melanocytes. Thus, elevated levels of CCN3 protein regulated by p53 might influence cell adhesion.

Bistable Epigenetic States Explain Age‐Dependent Decline in Mesenchymal Stem Cell Heterogeneity

The molecular mechanisms by which heterogeneity, a major characteristic of stem cells, is achieved are yet unclear. We here study the expression of the membrane stem cell antigen‐1 (Sca‐1) in mouse bone marrow mesenchymal stem cell (MSC) clones. We show that subpopulations with varying Sca‐1 expression profiles regenerate the Sca‐1 profile of the mother population within a few days. However, after extensive replication in vitro, the expression profiles shift to lower values and the regeneration time increases. Study of the promoter of Ly6a unravels that the expression level of Sca‐1 is related to the promoter occupancy by the activating histone mark H3K4me3. We demonstrate that these findings can be consistently explained by a computational model that considers positive feedback between promoter H3K4me3 modification and gene transcription. This feedback implicates bistable epigenetic states which the cells occupy with an age‐dependent frequency due to persistent histone (de‐)modification. Our results provide evidence that MSC heterogeneity, and presumably that of other stem cells, is associated with bistable epigenetic states and suggest that MSCs are subject to permanent state fluctuations. Stem Cells 2017;35:694–704

Stem cell competition in the gut: insights from multi-scale computational modelling

Three-dimensional (3D) computational tissue models can provide a comprehensive description of tissue dynamics at the molecular, cellular and tissue level. Moreover, they can support the development of hypotheses about cellular interactions and about synergies between major signalling pathways. We exemplify these capabilities by simulation of a 3D single-cell-based model of mouse small intestinal crypts. We analyse the impact of lineage specification, distribution and cellular lifespan on clonal competition and study effects of Notch- and Wnt activation on fixation of mutations within the tissue. Based on these results, we predict that experimentally observed synergistic effects between autonomous Notch- and Wnt signalling in triggering intestinal tumourigenesis originate in the suppression of Wnt-dependent secretory lineage specification by Notch, giving rise to an increased fixation probability of Wnt-activating mutations. Our study demonstrates that 3D computational tissue models can support a mechanistic understanding of long-term tissue dynamics under homeostasis and during transformation.