Genome-wide analysis of canonical Wnt target gene regulation in Xenopus tropicalis challenges β-catenin paradigm

Abstract

Wnt/β-catenin signaling is an important cell-to-cell signaling mechanism that controls gene expression during embryonic development and is critically implicated in human diseases. Developmental, cellular, and transcriptional responses to Wnt signaling are remarkably context-specific in different biological processes. While nuclear localization of β-catenin is the key to activation of the Wnt/β-catenin pathway and target gene expression, the molecular mechanisms of how the same Wnt/β-catenin signaling pathway induces specific responses remain undetermined. Recent advances in high-throughput sequencing technologies and the availability of genome information for Xenopus tropicalis have enabled us to uncover a genome-wide view of Wnt/β-catenin signaling in early vertebrate embryos, which challenges previous concepts about molecular mechanisms of Wnt target gene regulation. In this review, we summarize our experimental approaches, introduce the technologies we employed and focus on recent findings about Wnt target gene regulation from Xenopus research. We will also discuss potential functions of widespread β-catenin binding in the genome that we discovered in this species.

Keywords: ChIP-seq; RNA-seq; Wnt signaling; Xenopus; gastrula; β-catenin.

Figure 1

Nuclear β‐catenin is the hallmark of canonical Wnt signaling. Diagram depicting two cells and an outline of the canonical Wnt signal transduction pathway. (a) In a cell that is not responding to extracellular Wnt signaling any cytoplasmic β‐catenin protein is proteolytically degraded. (b) In response to extracellular Wnt signaling, any cytoplasmic β‐catenin protein is stabilized and enters the nucleus to regulate Wnt target gene expression in a complex with DNA‐binding transcription factors. For a more detailed recent review of Wnt signaling mechanisms, consult Hoppler and Nakamura (2014)

Figure 2

Proposed mechanisms for regulating context‐specific Wnt target gene expression. (a) The previously established paradigm asserted that transcription of context‐specific Wnt target genes is regulated by restricted access of nuclear β‐catenin protein to potential Wnt target gene sequences, with β‐catenin association to Wnt target gene sequences therefore considered both required and sufficient for context‐specific Wnt target gene regulation. (b) Genome‐wide analysis of context‐specific Wnt target gene regulation reveals more wide‐spread genome association of nuclear β‐catenin; including to Wnt target gene sequences that are not transcriptionally regulated in the particular cellular context studied. While β‐catenin association to Wnt target gene sequences is required, context‐specific mechanisms are additionally required for Wnt target gene transcription

Figure 3

Dramatic shift in response to Wnt signaling in early Xenopus embryogenesis. Early Xenopus development is regulated by maternal gene products. Maternal Wnt signaling regulates dorsal axis establishment. After the MBT and the onset of zygotic transcription, Wnt signaling regulates essentially the opposite, ventral development or more precisely ventrolateral mesoderm development. Responses to maternal and to early zygotic Wnt signaling are mediated by essentially the same β‐catenin‐dependent signal transduction mechanism depicted in Figure 1

Figure 4

Identification of direct context‐specific Wnt/β‐catenin target genes in a genome‐wide approach. (a) Transcriptome analysis (with RNA‐seq) of early gastrula‐stage embryos. Comparing the transcriptome of control embryos with embryos with wnt8a knockdown and those with experimentally reinstated Wnt8a identifies genes regulated by Wnt8a signaling in post‐MBT embryos. (b) ChIP‐seq analysis with a β‐catenin‐specific antibody identifies DNA‐sequences associated with β‐catenin and nearby potentially regulated genes in early gastrula‐stage embryos. (c) Venn diagram comparing Wnt8a‐regulated genes (in beige) from the RNA‐seq analysis (see panel A) with the β‐catenin associated genes (in green) from the ChIP‐seq analysis (see panel B) with the overlap or intersection identifying direct Wnt8a target genes. Note that there are many more identified β‐catenin associated genes than identified Wnt8a signaling‐regulated genes in early gastrula embryos. (d) Example of a direct Wnt8a‐target gene (hoxd1) in genome view, with from top to bottom, transcripts from control embryos, transcripts from the wnt8a knock down (note reduced expression), transcripts from embryos with experimentally reinstated Wnt8a expression, β‐catenin‐associated DNA sequences (“β‐catenin” peaks), sequences of the control sample of the ChIP‐seq experiment (Input control) and the hoxd1 gene model. Note β‐catenin‐associated DNA sequences downstream (left) of the rest of the hoxd1 gene and transcript sequences of particularly the exon sequences in the control and wnt8a rescue samples