Kevin F. Kelly

β-Catenin Enhances Oct-4 Activity and Reinforces Pluripotency through a TCF-Independent Mechanism

Understanding the mechanisms regulating pluripotency in embryonic and induced pluripotent stem cells is required to ensure their safe use in clinical applications. Glycogen synthase kinase-3 (GSK-3) has emerged as an important regulator of pluripotency, based primarily on studies with small-molecule GSK-3 inhibitors. Here, we use mouse embryonic stem cells (ESCs) lacking GSK-3 to demonstrate that a single GSK-3 substrate, β-catenin, controls the ability of ESCs to exit the pluripotent state and to differentiate into neurectoderm. Unexpectedly, the effects of β-catenin on pluripotency do not appear to be dependent on TCF-mediated signaling, based on experiments utilizing a β-catenin C-terminal truncation mutant or highly efficient dominant-negative TCF strategies. Alternatively, we find that stabilized β-catenin forms a complex with and enhances the activity of Oct-4, a core component of the transcriptional network regulating pluripotency. Collectively, our data suggest previously underappreciated, divergent TCF-dependent and TCF-independent roles for β-catenin in ESCs.

NLS-dependent nuclear localization of p120ctn is necessary to relieve Kaiso-mediated transcriptional repression

The Armadillo catenin p120ctn regulates cadherin adhesive strength at the plasma membrane and interacts with the novel BTB/POZ transcriptional repressor Kaiso in the nucleus. The dual localization of p120ctn at cell-cell junctions and in the nucleus suggests that its nucleocytoplasmic trafficking is tightly regulated. Here we report on the identification of a specific and highly basic nuclear localization signal (NLS) in p120ctn. The functionality of the NLS was validated by its ability to direct the nuclear localization of a heterologous β-galactosidase-GFP fusion protein. Mutating two key positively charged lysines to neutral alanines in the NLS of full-length p120ctn inhibited both p120ctn nuclear localization as well as the characteristic p120ctn-induced branching phenotype that correlates with increased cell migration. However, while these findings and others suggested that nuclear localization of p120ctn was crucial for the p120ctn-induced branching phenotype, we found that forced nuclear localization of both wild-type and NLS-mutated p120ctn did not induce branching. Recently, we also found that one role of p120ctn was to regulate Kaiso-mediated transcriptional repression. However, it remained unclear whether p120ctn sequestered Kaiso in the cytosol or directly inhibited Kaiso transcriptional activity in the nucleus. Using minimal promoter assays, we show here that the regulatory effect of p120ctn on Kaiso transcriptional activity requires the nuclear translocation of p120ctn. Therefore, an intact NLS in p120ctn is requisite for its first identified regulatory role of the transcriptional repressor Kaiso.

Regulation of the Rapsyn Promoter by Kaiso and δ-Catenin

ABSTRACT Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that δ-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and δ-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and δ-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and δ-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, δ-catenin, and myogenic transcription factors at the neuromuscular junction.

The Human Enhancer Blocker CTC-binding Factor Interacts with the Transcription Factor Kaiso

CTC-binding factor (CTCF) is a DNA-binding protein of vertebrates that plays essential roles in regulating genome activity through its capacity to act as an enhancer blocker. We performed a yeast two-hybrid screen to identify protein partners of CTCF that could regulate its activity. Using full-length CTCF as bait we recovered Kaiso, a POZ-zinc finger transcription factor, as a specific binding partner. The interaction occurs through a C-terminal region of CTCF and the POZ domain of Kaiso. CTCF and Kaiso are co-expressed in many tissues, and CTCF was specifically co-immunoprecipitated by several Kaiso monoclonal antibodies from nuclear lysates. Kaiso is a bimodal transcription factor that recognizes methylated CpG dinucleotides or a conserved unmethylated sequence (TNGCAGGA, the Kaiso binding site). We identified one consensus unmethylated Kaiso binding site in close proximity to the CTCF binding site in the human 5′ β-globin insulator. We found, in an insulation assay, that the presence of this Kaiso binding site reduced the enhancer-blocking activity of CTCF. These data suggest that the Kaiso-CTCF interaction negatively regulates CTCF insulator activity.

Nuclear import of the BTB/POZ transcriptional regulator Kaiso.

Kaiso is a BTB/POZ transcription factor that functions in vitro as a transcriptional repressor of the matrix metalloproteinase gene matrilysin and the non-canonical Wnt signaling gene Wnt-11, and as an activator of the acetylcholine-receptor-clustering gene rapsyn. Similar to other BTB/POZ proteins (e.g. Bcl-6, PLZF, HIC-1), endogenous Kaiso localizes predominantly to the nuclei of mammalian cells. To date, however, the mechanism of nuclear import for most POZ transcription factors, including Kaiso, remain unknown. Here, we report the identification and characterization of a highly basic nuclear localization signal (NLS) in Kaiso. The functionality of this NLS was verified by its ability to target a heterologous β-galactosidase/green-fluorescent-protein fusion protein to nuclei. The mutation of one positively charged lysine to alanine in the NLS of full-length Kaiso significantly inhibited its nuclear localization in various cell types. In addition, wild-type Kaiso, but not NLS-defective Kaiso, interacted directly with the nuclear import receptor Importin-α2 both in vitro and in vivo. Finally, minimal promoter assays using a sequence-specific Kaiso-binding-site fusion with luciferase as reporter demonstrated that the identified NLS was crucial for Kaiso-mediated transcriptional repression. The identification of a Kaiso NLS thus clarifies the mechanism by which Kaiso translocates to the nucleus to regulate transcription of genes with diverse roles in cell growth and development.

Hypoxia inducible factor (HIF)-2α is required for the development of the catecholaminergic phenotype of sympathoadrenal cells

The basic helix‐loop‐helix transcription factor, hypoxia inducible factor (HIF)‐2α has been implicated in the development of the catecholaminergic phenotype in cells of the sympathoadrenal (SA) lineage; however, the underlying mechanisms and HIF‐2α targets remain unclear. Using an immortalized rat adrenomedullary chromaffin cell line (MAH cells) derived from a fetal SA progenitor, we examined the role of HIF‐2α in catecholamine biosynthesis. Chronic hypoxia (2% O2, 24 h) induced HIF‐2α in MAH cells but expression of the rate‐limiting enzyme, tyrosine hydroxylase (TH) and catecholamine levels were unaltered. Interestingly, HIF‐2α depleted MAH cells showed dramatically lower (5–12 times) levels of dopamine and noradrenaline compared with wild‐type and scrambled controls, even in normoxia (21% O2). This was correlated with a marked reduction in the expression of DOPA decarboxylase (DDC) and dopamine β hydroxylase (DβH) but not TH. Chromatin immunoprecipitation assays revealed that HIF‐2α was bound to the DDC gene promoter which contains two putative hypoxia response elements. These data suggest that a basal level of HIF‐2α function is required for the normal developmental expression of DDC and DβH in SA progenitor cells, and that loss of this function leads to impaired catecholamine biosynthesis.

Kaiso regulates Znf131-mediated transcriptional activation

Kaiso is a dual-specificity POZ-ZF transcription factor that regulates gene expression by binding to sequence-specific Kaiso binding sites (KBS) or methyl-CpG dinucleotide pairs. Kaiso was first identified as a binding partner for the epithelial cell adhesion regulator p120(ctn). The p120(ctn)/Kaiso interaction is reminiscent of the beta-catenin/TCF interaction and several studies have suggested that Kaiso is a negative regulator of the Wnt/beta-catenin TCF signaling pathway. To gain further insight into Kaisos function, we performed a yeast two-hybrid screen using the Kaiso POZ domain as bait. This screen identified the POZ-ZF protein, Znf131, as a Kaiso-specific binding partner. GST pull-down assays confirmed that the interaction is mediated via the POZ domain of each protein, and co-immunoprecipitation experiments further supported an in vivo Kaiso-Znf131 interaction. Using a Cyclic Amplification and Selection of Targets (CAST) approach, we identified the 12-base pair DNA palindrome sequence GTCGCR-(X)(n)-YGCGAC as a potential Znf131 binding element (ZBE). In vitro studies using electrophoretic mobility shift assay (EMSA) demonstrated that Znf131 binds the ZBE via its zinc finger domain. Znf131 DNA-binding specificity was confirmed using competition assays and ZBE mutational analyses. An artificial promoter-reporter construct containing four tandem copies of the ZBE was constructed and used to assess Znf131 transcriptional properties. We observed dose-dependent transcriptional activation of this artificial promoter-reporter by Znf131 in both epithelial and fibroblast cells, suggesting that Znf131 is a transcriptional activator. Kaiso overexpression significantly decreased the Znf131-mediated transcriptional activation, and interestingly, co-expression of the Kaiso-specific interaction partner p120(ctn) relieved Kaisos inhibition of Znf131-mediated transcriptional activation. These findings indicate that Znf131 is a transcriptional activator, a less common function of POZ-ZF proteins, that is negatively regulated by its heterodimerization partner Kaiso.

Ectopic γ-catenin Expression Partially Mimics the Effects of Stabilized β-catenin on Embryonic Stem Cell Differentiation

β-catenin, an adherens junction component and key Wnt pathway effector, regulates numerous developmental processes and supports embryonic stem cell (ESC) pluripotency in specific contexts. The β-catenin homologue γ-catenin (also known as Plakoglobin) is a constituent of desmosomes and adherens junctions and may participate in Wnt signaling in certain situations. Here, we use β-catenin(+/+) and β-catenin(−/−) mouse embryonic stem cells (mESCs) to investigate the role of γ-catenin in Wnt signaling and mESC differentiation. Although γ-catenin protein is markedly stabilized upon inhibition or ablation of GSK-3 in wild-type (WT) mESCs, efficient silencing of its expression in these cells does not affect β-catenin/TCF target gene activation after Wnt pathway stimulation. Nonetheless, knocking down γ-catenin expression in WT mESCs appears to promote their exit from pluripotency in short-term differentiation assays. In β-catenin(−/−) mESCs, GSK-3 inhibition does not detectably alter cytosolic γ-catenin levels and does not activate TCF target genes. Intriguingly, β-catenin/TCF target genes are induced in β-catenin(−/−) mESCs overexpressing stabilized γ-catenin and the ability of these genes to be activated upon GSK-3 inhibition is partially restored when wild-type γ-catenin is overexpressed in these cells. This suggests that a critical threshold level of total catenin expression must be attained before there is sufficient signaling-competent γ-catenin available to respond to GSK-3 inhibition and to regulate target genes as a consequence. WT mESCs stably overexpressing γ-catenin exhibit robust Wnt pathway activation and display a block in tri-lineage differentiation that largely mimics that observed upon overexpression of β-catenin. However, β-catenin overexpression appears to be more effective than γ-catenin overexpression in sustaining the retention of markers of naïve pluripotency in cells that have been subjected to differentiation-inducing conditions. Collectively, our study reveals a function for γ-catenin in the regulation of mESC differentiation and has implications for human cancers in which γ-catenin is mutated and/or aberrantly expressed.

The responses of neural stem cells to the level of GSK-3 depend on the tissue of origin

Summary Neural stem cells (NSCs) can be obtained from a variety of sources, but not all NSCs exhibit the same characteristics. We have examined how the level of glycogen synthase kinase-3 activity regulates NSCs obtained from different sources: the mouse embryonic striatum, embryonic hippocampus, and mouse ES cells. Growth of striatal NSCs is enhanced by mild inhibition of GSK-3 but not by strong inhibition that is accompanied by Wnt/TCF transcriptional activation. In contrast, the growth of hippocampal NSCs is enhanced by both mild inhibition of GSK-3 as well as stronger inhibition. Active Wnt/TCF signaling, which occurs normally in the embryonic hippocampus, is required for growth of neural stem and progenitor cells. In the embryonic striatal germinal zone, however, TCF signaling is normally absent and its activation inhibits growth of NSCs from this region. Using a genetic model for progressive loss of GSK-3, we find that primitive ES cell-derived NSCs resemble striatal NSCs. That is, partial loss of GSK-3 alleles leads to an increase in NSCs while complete ablation of GSK-3, and activation of TCF-signaling, leads to their decline. Furthermore, expression of dominant negative TCF-4 in the GSK-3-null background was effective in blocking expression of Wnt-response genes and was also able to rescue neuronal gene expression. These results reveal that GSK-3 regulates NSCs by divergent pathways depending on the tissue of origin. The responses of these neural precursor cells may be contingent on baseline Wnt/TCF signaling occurring in a particular tissue.

Molecular Mechanisms Underlying Pluripotency and Lineage Commitment – The Role of GSK-3

The highly related serine/threonine kinases GSK3┙ and GSK3┚ are transducers of Wnt/┚catenin, PI-3K, Notch and Hedgehog signalling pathways, placing them at the hub of key developmental and metabolic processes. There is accumulating evidence suggesting that GSK-3 inhibitors aid in the acquisition or sustenance of pluripotency in embryonic stem cells of mouse, rat and human origin. However, the mechanism through which GSK-3 inhibitors impart their effects is unclear due to the myriad cellular processes in which GSK-3 plays a role. Here, we review the studies that have examined the consequences of GSK-3 inhibition in pluripotent stem cells with a focus on key signalling pathways, which have been implicated in GSK-3 inhibitor-mediated effects.