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HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

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Cdx4 is a Cdx2 target gene.

Joanne G A Savory¹, Melissa Mansfield, Catherine St Louis

¹Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5.

Mechanisms of Development

|October 12, 2010

Summary

Cdx2 downregulates Cdx4 expression by directly binding to the Cdx4 promoter, independent of the Wnt signaling pathway. This reveals a novel cross-regulatory interaction in vertebrate development.

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Area of Science:

Developmental Biology
Gene Regulation
Vertebrate Embryogenesis

Background:

Cdx genes (Cdx1, Cdx2, Cdx4) are crucial for vertebrate development, involved in anterior-posterior patterning and axial elongation.
These genes relay signals from Wnt, retinoic acid (RA), and FGF pathways.
Previous studies indicated autoregulation and cross-regulation among Cdx genes, with Cdx4 being a Wnt pathway target and Cdx1 interacting with LEF/TCF.

Purpose of the Study:

To investigate how Cdx2 influences Cdx4 expression.
To determine if Cdx2 interacts with the canonical Wnt signaling pathway in regulating the Cdx4 promoter.

Main Methods:

Transient transfection assays to assess Cdx4 promoter activity.
Electrophoretic mobility shift assays (EMSAs) to detect Cdx2 binding to the Cdx4 promoter.
Chromatin immunoprecipitation (ChIP) assays in embryos to confirm in vivo Cdx2 occupancy of the Cdx4 promoter.

Main Results:

Cdx4 expression was significantly downregulated in Cdx2(-/-) mutants, suggesting cross-regulation.
Cdx2 directly regulated the Cdx4 promoter in transfection assays.
EMSAs and ChIP assays confirmed that Cdx2 binds to specific elements on the Cdx4 promoter in vitro and in vivo.
No interaction was observed between Cdx2 and components of the canonical Wnt signaling pathway.

Conclusions:

Cdx2 directly regulates Cdx4 gene expression by binding to its promoter.
Both canonical Wnt signaling and Cdx2 can modulate Cdx4 promoter activity but operate through distinct mechanisms.
This study uncovers a previously unrecognized cross-regulatory interaction between Cdx2 and Cdx4 in vertebrate development.