DEV025569 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Sequence comparison of Kaiso proteins from different vertebrate species. (A) Phylograms of vertebrate Kaiso proteins based on the analysis of complete Kaiso protein sequences using the Clustal program http://www.ebi.ac.uk/Tools/sequence.html and (B) Kaiso-ZF domains. (C) The alignment of mouse (mKaiso) and chicken (gKaiso) ZF domains showing that their amino acid sequences are virtually identical.

• Supplemental Figure S2 -

  Fig. S2. EMSA experiments using GST-fused Kaiso ZF domain. (A) EMSA experiment with E. coli extracts containing Gst (Gst only) and Gst-ZF1-3 domains of xKaiso (xKaisoZFs), dKaiso (dKaisoZFs) and gKaiso (gKaisoZFs) with methylated Sm, non-methylated S or human matrilysin (Hmat) probes. Arrow indicates the Kaiso ZF-specific band shift. (B) Phylogenetic tree based on the homology between Kaiso ZF domains and correlated with efficiency of binding to Sm and Hmat for Kaiso proteins from different vertebrates; '++++' and '+++' indicate the strength of efficient binding and '−' indicates no detectable binding. (C) EMSA experiments using Gst-ZF1-3 domain of xKaiso (xKaisoZFs) with methylated Sm probe and CTGCNA-containing probes derived from the promoter regions of Siamois (Siam) and xWnt11 (Wnt) in the presence of E. coli DNA as a non-specific competitor. (D) EMSA using full-length Gst-xKaiso protein with probes as in C. Arrows indicate the Kaiso-specific band shift. Asterisks indicate non-specific binding in the E. coli extracts.

• Supplemental Figure S3 -

  Fig. S3. Kaiso is a ubiquitously expressed methyl-dependent transcriptional repressor. Kaiso whole-mount RNA in situ hybridization on zebrafish (A), chicken (B) and Xenopus (C) embryos. The stages of development for Xenopus and hours after fertilisation for zebrafish embryos are indicated. An, animal pole; Veg, vegetal pole; Ant, anterial part of the embryo; Ecd, ectoderm; np, neural plate; nc, notochord; nt, neural tube; sc, spinal cord; tb, tailbud; mn, motoneurons; psm, perisomitic mesoderm; ba, branchial arches; sm, somites; YE, yolk extension layer; EEM, extraembryonic membranes; Vnt, ventral neural tube.

• Supplemental Figure S4 -

  Fig. S4. Co-injection of xKaiso mRNA can ameliorate the dKMO phenotype in zebrafish. (A) FITC images of rescue experiment shown in Fig. 2D; dKMO is on the left and dKMO plus xKaiso mRNA on the right. Note the microcephaly phenotype of the dKMO embryo due to a lack of brain structure and tissues, which are present in the rescued embryo, which has a phenotypically normal head. (B) Graph representing mean dKMO fluorescence for different phenotypic classes based on embryo size, as measured with ImageJ. Embryos were scored for axis length ranging from no axis extension (class 1), short folded axis (class 3) to slightly curved normal length (class 5). Two series are shown; dKMO only (blue) and dKMO plus xKaiso mRNA (pink). Note that in the presence of xKaiso mRNA the phenotypic effect is reduced for the same dKMO dose. Within each phenotypic class the means are not significantly different owing to the variance. However, the trend is significant over the four phenotypic classes combined.

• Supplemental Figure S5 -

  Fig. S5. CpG sequences bound by murine Kaiso are enriched in methylated CpGs. (A) Bar chart showing the overlap between CpG islands methylated in HEK293 cells (from our array analysis) and the mKaiso-bound sequences (mKaiso ZFH6 Ab). The number of ChIP sequences overlapping with CpG islands methylated in HEK293 cells is shown. This number was normalised to the total number of ChIP sequences for different values of the distance between ChIP sequence and methylated CpG island on the array (distance threshold). Note that mKaiso ChIP sequences are significantly enriched in methylated CpGs as compared with control (mKaiso preimmune serum) experiment at all values of the distance threshold analysed. Similar data were obtained for the dKaiso ChIP experiment (data not shown). (B) The Tex19 promoter (−304 to +198) contains 30 CpGs (indicated by vertical lines) that when methylated includes four core (CGCG) Kaiso binding sites (red boxes). The transcription start site is indicated.

• Supplemental Figure S6 -

  Fig. S6. Survival rates of control or xKMO morphants cultured in the presence of a general caspase inhibitor or xKMO morphants co-injected with an xp53 morpholino (p53MO). (A) Pie chart of survival and phenotypes of control embryos incubated with the caspase inhibitor (Z-DEVD-FMK). (B) Pie charts of survival and phenotypes of embryos injected with the xKaiso morpholino (xKMO) only, and with the caspase inhibitor (Z-DEVD-FMK). (C) Pie chart of survival and phenotypes of embryos injected with the xKaiso morpholino (xKMO) and an xp53 morpholino (p53MO).

• Supplemental Figure S7 -

  Fig. S7. No evidence for mis-expression of xWnt11 in xKaiso-depleted Xenopus laevis embryos. Whole-mount in situ hybridisation on control and xKMO Xenopus pre-MBT embryos using xWnt11 (A) or xID2 (B) probes. The stages of development are indicated. Animal and dorsal views are shown for every group of embryos. Note that only xID2 is ectopically expressed in pre-MBT xKMO morphants.