Bradley J. Merrill

Repression of Nanog Gene Transcription by Tcf3 Limits Embryonic Stem Cell Self-Renewal

ABSTRACT The dual function of stem cells requires them not only to form new stem cells through self-renewal but also to form lineage-committed cells through differentiation. Embryonic stem cells (ESC), which are derived from the blastocyst inner cell mass, retain properties of self-renewal and the potential for lineage commitment. To balance self-renewal and differentiation, ESC must carefully control the levels of several transcription factors, including Nanog, Sox2, and Oct4. While molecular mechanisms promoting transcription of these genes have been described, mechanisms preventing excessive levels in self-renewing ESC remain unknown. By examining the function of the TCF family of transcription factors in ESC, we have found that Tcf3 is necessary to limit the steady-state levels of Nanog mRNA, protein, and promoter activity in self-renewing ESC. Chromatin immunoprecipitation and promoter reporter assays showed that Tcf3 bound to a promoter regulatory region of the Nanog gene and repressed its transcriptional activity in ESC through a Groucho interaction domain-dependent process. The absence of Tcf3 caused delayed differentiation of ESC in vitro as elevated Nanog levels persisted through 5 days of embryoid body formation. These new data support a model wherein Tcf3-mediated control of Nanog levels allows stem cells to balance the creation of lineage-committed and undifferentiated cells.

At the Roots of a Never-Ending Cycle

Recent studies have yielded a number of important insights into the mechanisms of hair follicle development and cycling and have highlighted the particularly important roles played by stem cells and Wnt signaling pathways.

Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal

The co-occupancy of Tcf3 with Oct4, Sox2 and Nanog on embryonic stem cell (ESC) chromatin indicated that Tcf3 has been suggested to play an integral role in a poorly understood mechanism underlying Wnt-dependent stimulation of mouse ESC self-renewal of mouse ESCs. Although the conventional view of Tcf proteins as the β-catenin-binding effectors of Wnt signalling suggested Tcf3–β-catenin activation of target genes would stimulate self-renewal, here we show that an antagonistic relationship between Wnt3a and Tcf3 on gene expression regulates ESC self-renewal. Genetic ablation of Tcf3 replaced the requirement for exogenous Wnt3a or GSK3 inhibition for ESC self-renewal, demonstrating that inhibition of Tcf3 repressor is the necessary downstream effect of Wnt signalling. Interestingly, both Tcf3–β-catenin and Tcf1–β-catenin interactions contributed to Wnt stimulation of self-renewal and gene expression, and the combination of Tcf3 and Tcf1 recruited Wnt-stabilized β-catenin to Oct4 binding sites on ESC chromatin. This work elucidates the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network.

Tcf3: a transcriptional regulator of axis induction in the early embryo

The roles of Lef/Tcf proteins in determining cell fate characteristics have been described in many contexts during vertebrate embryogenesis, organ and tissue homeostasis, and cancer formation. Although much of the accumulated work on these proteins involves their ability to transactivate target genes when stimulated by β-catenin, Lef/Tcf proteins can repress target genes in the absence of stabilized β-catenin. By ablating Tcf3 function, we have uncovered an important requirement for a repressor function of Lef/Tcf proteins during early mouse development. Tcf3-/- embryos proceed through gastrulation to form mesoderm, but they develop expanded and often duplicated axial mesoderm structures, including nodes and notochords. These duplications are preceded by ectopic expression of Foxa2, an axial mesoderm gene involved in node specification, with a concomitant reduction in Lefty2, a marker for lateral mesoderm. By contrast, expression of a β-catenin-dependent, Lef/Tcf reporter (TOPGal), is not ectopically activated but is faithfully maintained in the primitive streak. Taken together, these data reveal a unique requirement for Tcf3 repressor function in restricting induction of the anterior-posterior axis.

Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia

Single-layered embryonic skin either stratifies to form epidermis or responds to Wnt signaling (stabilized β-catenin) to form hair follicles. Postnatally, stem cells continue to differentially use Wnt signaling in long-term tissue homeostasis. We have discovered that embryonic progenitor cells and postnatal hair follicle stem cells coexpress Tcf3 and Tcf4, which can act as transcriptional activators or repressors. Using loss-of-function studies and transcriptional analyses, we uncovered consequences to the absence of Tcf3 and Tcf4 in skin that only partially overlap with those caused by β-catenin deficiency. We established roles for Tcf3 and Tcf4 in long-term maintenance and wound repair of both epidermis and hair follicles, suggesting that Tcf proteins have both Wnt-dependent and Wnt-independent roles in lineage determination.

Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation

Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon–exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.

Tcf3 Functions as a Steady State Limiter of Transcriptional Programs of Mouse Embryonic Stem Cell Self Renewal

Elucidating the underlying transcriptional control of pluripotent cells is necessary for the development of new methods of inducing and maintaining pluripotent cells in vitro. Three transcription factors, Nanog, Oct4, and Sox2, have been reported to form a feedforward circuit promoting pluripotent cell self‐renewal in embryonic stem cells (ESC). Previously, we found that a transcriptional repressor activity of Tcf3, a DNA‐binding effector of Wnt signaling, reduced Nanog promoter activity and Nanog levels in mouse embryonic stem cells (mESC). The objective of this study was to determine the scope of Tcf3 effects on gene expression and self‐renewal beyond the regulation of Nanog levels. We show that Tcf3 acts broadly on a genome‐wide scale to reduce the levels of several promoters of self‐renewal (Nanog, Tcl1, Tbx3, Esrrb) while not affecting other ESC genes (Oct4, Sox2, Fgf4). Comparing effects of Tcf3 ablation with Oct4 or Nanog knockdown revealed that Tcf3 counteracted effects of both Nanog and Oct4. Interestingly, the effects of Tcf3 were more strongly correlated with Oct4 than with Nanog, despite the normal levels of Oct4 in TCF3−/− mESC. The deranged gene expression allowed TCF3−/− mESC self‐renewal even in the absence of leukemia inhibitory factor and delayed differentiation in embryoid bodies. These findings identify Tcf3 as a cell‐intrinsic inhibitor of pluripotent cell self‐renewal that functions by limiting steady‐state levels of self‐renewal factors.

Canonical Wnt/β-Catenin Regulation of Liver Receptor Homolog-1 Mediates Pluripotency Gene Expression

Delineating the signaling pathways that underlie ESC pluripotency is paramount for development of ESC applications in both the research and clinical settings. In culture pluripotency is maintained by leukemia inhibitory factor (LIF) stimulation of two separate signaling axes: Stat3/Klf4/Sox2 and PI3K/Tbx3/Nanog, which converge in the regulation of Oct4 expression. However, LIF signaling is not required in vivo for self‐renewal, thus alternate signaling axes likely mediate these pathways. Additional factors that promote pluripotency gene expression have been identified, including the direct regulation of Oct4 by liver receptor homolog‐1 (Lrh‐1) and β‐catenin regulation of Nanog. Here, we present genetic, molecular, and pharmacological studies identifying a signaling axis in which β‐catenin promotes pluripotency gene expression in an Lrh‐1‐dependent manner. Furthermore, Lrh‐1 was identified as a novel β‐catenin target gene, and Lrh‐1 regulation is required for maintaining proper levels of Oct4, Nanog, and Tbx3. Elucidation of this pathway provides an alternate mechanism by which the primary pluripotency axis may be regulated in vivo and may pave the way for small molecule applications to manipulate pluripotency or improve the efficiency of somatic cell reprogramming. STEM CELLS 2010;28:1794–1804

Function of Wnt/β-catenin in counteracting Tcf3 repression through the Tcf3–β-catenin interaction

The canonical Wnt/β-catenin signaling pathway classically functions through the activation of target genes by Tcf/Lef–β-catenin complexes. In contrast to β-catenin-dependent functions described for Tcf1, Tcf4 and Lef1, the known embryonic functions for Tcf3 in mice, frogs and fish are consistent with β-catenin-independent repressor activity. In this study, we genetically define Tcf3–β-catenin functions in mice by generating a Tcf3ΔN knock-in mutation that specifically ablates Tcf3–β-catenin. Mouse embryos homozygous for the knock-in mutation (Tcf3ΔN/ΔN) progress through gastrulation without apparent defects, thus genetically proving that Tcf3 function during gastrulation is independent of β-catenin interaction. Tcf3ΔN/ΔN mice were not viable, and several post-gastrulation defects revealed the first in vivo functions of Tcf3–β-catenin interaction affecting limb development, vascular integrity, neural tube closure and eyelid closure. Interestingly, the etiology of defects indicated an indirect role for Tcf3–β-catenin in the activation of target genes. Tcf3 directly represses transcription of Lef1, which is stimulated by Wnt/β-catenin activity. These genetic data indicate that Tcf3–β-catenin is not necessary to activate target genes directly. Instead, our findings support the existence of a regulatory circuit whereby Wnt/β-catenin counteracts Tcf3 repression of Lef1, which subsequently activates target gene expression via Lef1–β-catenin complexes. We propose that the Tcf/Lef circuit model provides a mechanism downstream of β-catenin stability for controlling the strength of Wnt signaling activity during embryonic development.

Regulation of Tcf7l1 DNA Binding and Protein Stability as Principal Mechanisms of Wnt/β-Catenin Signaling

Wnt/β-catenin signal transduction requires direct binding of β-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. β-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-β-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/β-catenin in breast cancer.