Figure 1.
Gene structure of CTCF and BORIS.
(A) CTCF and BORIS share a similar ZF domain, but different N- and C-terminal domains. (B) All vertebrate CTCF orthologues posses ten exons. Intron-exon boundaries are identical between all CTCF and BORIS orthologues within the ZF domain (grey). Note, genomic coverage of platypus BORIS is incomplete at the 5′ end (*).
Figure 2.
Neighbour-joining tree showing relationships between members of the CTCF and BORIS gene family.
Sequence we determined experimentally (blue) and discovered by in silico similarity searches (black) form two distinct clusters with previously annotated CTCF and BORIS orthologues (bold). These clusters are separated from each other by a branch with 100% bootstrap value (thick line). Accession numbers and scientific names for these sequences and species are shown in Table S2.
Figure 3.
Human genomic sequence encompassing PCK1-BORIS-RBM38 (top) compared to the orthologous regions in other amniotes.
High similarity over a 100bp window is seen for most exonic sequence (blue) and some untranslated regions (UTR, light blue) or non coding regions (NCR, pink). Despite no similarity to any other region of human BORIS to the chicken PCK1-RBM38 region, the peak labelled with a star is homologous to the first ZF of BORIS.
Table 1.
Average pairwise similarity (%) between regions of human CTCF/BORIS and other vertebrate orthologues.
Figure 4.
Expression analysis of CTCF and BORIS.
(A) Conventional RT-PCR of CTCF and BORIS after 35 cycles. Note, cattle brain, platypus ovary and bearded dragon muscle could not be tested due to tissue unavailability or poor RNA quality. (B) BORIS transcript levels relative to the positive control gene GAPDH as quantified by real-time PCR. To assist comparisons between species, we set BORIS expression in the testis to 1 and adjusted all other values within the same species proportionally. As found in humans, expression of BORIS in somatic tissues of cattle and wallaby did not exceed 0.3% of that found in testis, although ovarian expression is just on or above this threshold. In contrast, levels of BORIS expression in platypus (liver and kidney) and bearded dragon (brain, kidney and ovary) are well above this threshold and in some cases match CTCF expression (Figure S2).
Figure 5.
Proposed model of CTCF and BORIS evolution in amniotes.
The expression of CTCF and BORIS is indicated (black = expressed, white = not expressed) within various taxa. The ancestral expression pattern of BORIS is wide, including multiple somatic tissues (reptiles and monotremes), but becomes progressively restricted in therian mammals with gonad specific expression in marsupials and cattle (Figure 4) and testis-specific expression in humans and mice [16],[38],[47]. Significant events in the evolution of CTCF and BORIS are marked with respect to the phylogenetic tree.