Trindade, Margarida Ferreira; (2000) Functional analysis of a homeobox-containing gene expressed during early Xenopus laevis development. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Xom is a homeobox-containing gene expressed during early stages of Xenopus laevis development which is involved in the specification of ventral tissues. Expression of Xom is induced as an immediate-early response to Bone Morphogenetic Protein-4, a member of the Transforming Growth Factor-β family. Moreover, Xom contains a novel homeodomain, which might affect its DNA binding specificity. This thesis describes a functional analysis of Xom. First, a preferred Xom DNA binding site was determined and the ability of Xom to bind potential binding sequences was tested in a series of in vitro assays. Together, these results showed that the sequence CTAATT(A/G) is critical for Xom to bind DNA, but that binding is greatly enhanced by the presence of an ATTA motif 6 or 7 nucleotides downstream of the core TAAT. A cell culture assay further demonstrated that Xom interacts with the selected sequence in vivo. Second, the ability of Xom to regulate transcription was analysed. Xom was shown to behave as a transcriptional repressor in Xenopus embryos and its repressing activity was mapped to N-terminal and C-terminal regions flanking the homeodomain. Xom's transcriptional repressing activity, together with its ventral expression pattern and ventralising activity in the early Xenopus embryo, suggested that Xom could function by down-regulating the expression of genes that are required for dorsal development in Xenopus. Consistent with this suggestion, over-expression of Xom RNA, or of a dominant-negative version, indicated that Xom regulates the expression of goosecoid, a homeobox-containing gene expressed in the organizer capable of partially mimicking the activity of the organizer. Finally, to test whether Xom acts by repressing goosecoid transcription directly, reporter constructs containing a goosecoid promoter fragment containing or lacking point mutations in potential Xom binding sites were co-injected with different effector RNAs into Xenopus embryos. These experiments suggested that at least part of the ability of Xom to repress goosecoid is direct, and identified a possible site to which Xom binds.

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