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. 2017 May 24;6:746. [Version 1] doi: 10.12688/f1000research.11034.1

Table 1. Approaches to identify Wnt target genes directly activated by the pathway.

We define “direct Wnt targets” as genes whose regulatory DNA can be physically associated with T-cell factors (TCFs) or other transcription factors (TFs) and whose expression is modulated by the recruitment of β-catenin to regulatory chromatin by these TFs. The approaches outlined below each have their advantages and disadvantages, and a combination of them is required to establish with confidence that a gene is a Wnt target gene in a particular context.

Approach Advantages Disadvantages
Computational searches for TCF binding sites
Position-weight matrices constructed based on
validated lists of TCF binding sites can be used
to screen cis-regulatory DNA for additional sites
(e.g. 109). The efficiency of this approach can be
improved by adding multiple sequences bound by
TFs (e.g. helper sites in invertebrates; see 69).
The functional relevance of binding sites can be
verified with reporter assays.
• Quickly identifies potentially
regulated genes
• The identification of binding
sites also establishes
candidates for mutagenesis
to rigorously test their
functionality
• Most effective when the search
space is restricted to short
stretches of DNA (<20 kb) rather
than the whole genome
• Not all consensus TCF sites will
be functional
• TCFs and other TFs have
degenerate binding sites that could
be functional, which could be
missed if the calling criteria are too
stringent
Transcriptome analyses of Wnt-regulated genes
Microarrays or RNA sequencing can be used to
identify genes whose expression changes in Wnt-on
and Wnt-off conditions in cell culture (e.g. 74) or
embryos (e.g. 30)
• Identifies the full array of
genes regulated by Wnt
pathway activation
• Many genetic and
biochemical reagents are
available to manipulate the
Wnt pathway
• Does not distinguish between
direct and indirect targets of Wnt
signaling
•  In vivo analyses in animal tissues
are limited by the specificity of
the genetic drivers used for the
manipulations
Chromatin immunoprecipitation sequencing
(ChIP-seq) analyses of TCF or β-catenin genomic
occupancy
ChIP-seq with TCFs and β-catenin with or
without Wnt activation can identify candidate
Wnt-regulated enhancers. This approach can be
combined with ChIP-seq for other TFs (e.g. 76) or
with transcriptome analyses to assign genes to
regulatory DNA sequences (e.g. 30).
• Biochemically establishes
the presence of Wnt effectors
at cis-regulatory elements
• Provides evidence of
direct regulation by the Wnt
pathway
• Many TCF/β-catenin binding sites
have no detectable function
• Quality of the antibody used plays
a major role
• While this approach can identify
putative Wnt-dependent cis-regulatory
elements, identifying which gene
the element regulates can be
difficult, especially for long-range
enhancers