The evolutionarily conserved leprecan gene: Its regulation by Brachyury and its role in the developing Ciona notochord

Abstract

In Ciona intestinalis, leprecan was identified as a target of the notochord-specific transcription factor Ciona Brachyury (Ci-Bra) (Takahashi et al., 1999). By screening ~14 kb of the Ci-leprecan locus for cis-regulatory activity, we have identified a 581-bp minimal notochord-specific cis-regulatory module (CRM) whose activity depends upon T-box binding sites located at the 3’-end of its sequence. These sites are specifically bound in vitro by a GST-Ci-Bra fusion protein, and mutations that abolish binding in vitro result in loss or decrease of regulatory activity in vivo. Serial deletions of the 581-bp notochord CRM revealed that this sequence is also able to direct expression in muscle cells through the same T-box sites that are utilized by Ci-Bra in the notochord, which are also bound in vitro by the muscle-specific T-box activators Ci-Tbx6b and Ci-Tbx6c. Additionally, we created plasmids aimed to interfere with the function of Ci-leprecan and categorized the resulting phenotypes, which consist of variable dislocations of notochord cells along the anterior-posterior axis. Together, these observations provide mechanistic insights generally applicable to T-box transcription factors and their target sequences, as well as a first set of clues on the function of Leprecan in early chordate development.

Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidianCiona intestinalis

“…The presence of putative T-box binding sites in the minimal Ci-Bra CRM (data not shown) indicates that autoregulation of Brachyury transcription might occur also in Ciona. Ci-Bra in turn activates transcription of at least 50 notochord genes (Takahashi et al, 1999a;Di Gregorio and Levine, 1999;Hotta et al, 2000;Hotta et al, 2008;Oda-Ishii and Di Gregorio, 2007;Kugler et al, 2008), among which are Cileprecan (Dunn and Di Gregorio, 2009) and Ci-tune (presented here). Both the Ci-leprecan and the Ci-tune notochord CRMs contain sites that are efficiently bound in vitro by Ci-FoxA-a and by Ci-Bra (Fig.…”

“…We employed the previously described GST-Ci-Bra (Di Gregorio and Levine, 1999) and GST-Ci-FoxA-a (Dunn and Di Gregorio, 2009) fusion proteins and the following radiolabeled double-stranded oligonucleotides (only the 5Ј-3Ј strand is reported, mutations are underlined):…”

“…Protein-DNA complexes were formed at room temperature for 30 minutes, then resolved on polyacrylamide gels as previously described (Dunn and Di Gregorio, 2009). For the competition experiments, the concentrations of unlabeled double-stranded oligonucleotides were 1 M and 5 M for the low and high concentrations, respectively.…”

“…However, in the case of Ci-leprecan, the Ci-FoxA-a site contributes only minimally to notochord activity (Fig. 8, faded blue oval) (Dunn and Di Gregorio, 2009); in the case of Ci-tune, the Ci-FoxA-a site is indispensable (Fig. 8, dark blue oval).…”

Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly

“…Because ancestors of basement membrane type IV collagen and fibril-forming collagens are recognizable in sponges (Porifera) (33), we speculate that the A1 sequence is in evolutionary terms a relatively new substrate for P3H that became recognizable perhaps when the P3H1/ CRTAP/cyclophilin B complex (or its ancestral form) first appeared. Presumably the event that created hydroxylation activity at this site occurred before the series of whole or partial genomic duplications that led to the divergence of A-clade collagen genes (␣1(I), ␣2(I), ␣1(II), ␣1(III), and ␣2(V)) in vertebrates (35) and perhaps also before or soon after the ancestral leprecan (P3H) gene was duplicated twice and eventually diverged into three copies (4,34,36,37). Because the sequence motif at site A1 differs from that at sites A2-A4 and B1-B3 (Fig.…”