Cnidarian Zic Genes
3.5 Opa as a Transcription Factor
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Cyc. This regular oscillation in gene expression allows the expression of both behavioral and metabolic genes to be linked to the circadian clock (Tataroglu and Emery 2014). opa may be involved in providing tissue specificity to the clock. In a genome-wide ChIP-seq analysis for targets of Clk and Cyc, Stark and colleagues found that opa is additionally required for expression of circadian transcriptional targets in the head (Meireles-Filho et al. 2014). Another GATA family transcription factor, serpent, serves a similar purpose in the body of the fly. Further analysis of targets that require Opa/Clk/Cyc for transcription in the fly head will likely reveal additional complexity to the role of opa in the circadian clock of Drosophila.
site. However, purified Ci zinc finger protein constructs bind to both the optimal Gli/
Ci site and the optimal Opa site with substantially higher affinity than Opa (Sen et al. 2010). Zic proteins thus appear to be weak interactors with DNA and may not always depend on direct DNA binding for activity in the way observed for most DNA sequence-specific transcription factors.
The behavior of our SELEX-determined Opa consensus site in vivo illustrates this point. We multimerized this binding site, placed it upstream of lac-Z in a reporter construct, and introduced it into flies transgenically (Sen et al. 2010).
Multiple chromosomal insertions of this construct had minimal endogenous expres- sion and responded robustly in vivo to ubiquitous full-length Opa expression. They only weakly responded to ubiquitous expression of Ci. Ubiquitous expression of Opa also resulted in spatially specific expression, indicating the requirement for additional factors. An equivalent transgenic construct in which nucleotides critical for in vitro Opa binding had been mutated was also examined: it was not activated by either Opa or Ci in vivo. Therefore, while Opa zinc finger domains do not com- pete with Ci zinc finger domains for binding to optimal Opa sites in vitro, in vivo, full-length Opa more robustly activates gene expression from its optimal binding
Fig. 3.6 Experimentally determined binding motifs for Opa, Gli, and Zic family members are similar. The Opa DNA binding motif as determined by high- throughput SELEX (a) (Nitta et al. 2015), a yeast 1-hybrid screen (b) (Noyes et al. 2008), and SELEX (c) (Sen et al. 2010) all contain a core
CGGGGGGTC sequence.
Closely related
transcription factor binding sites, including Gli, as determined by DNA footprinting (d) (Kinzler and Vogelstein 1990), Zic2 by SELEX (e) (Mizugishi et al. 2001), Zic3 by ChIP-seq (f) (Winata et al.
2013), and Zic3 by ChIP-chip (g) (Lim et al.
2010) are all similar
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sites than full-length Ci, and for this in vivo interaction, DNA binding is essential (Sen et al. 2010).
When the Drosophila genome was interrogated for sites with homology to our SELEX determined Opa site (Fig. 3.6c) (Sen et al. 2010), a site matching the con- sensus was found in slp1, a segmentation gene that genetically requires opa for expression (Swantek and Gergen 2004). This site bound an Opa zinc finger protein in EMSA. Two short (518 bp, 202 bp) lac-Z reporter constructs bearing this site were introduced transgenically into flies and examined in embryos. Similarly to the multimerized site above, these constructs responded to ectopic Opa expression but not to Ci. However, when the same nucleotide substitutions were introduced into the slp1 site as were used to eliminate Opa binding in the multimerized SELEX site, they did not abrogate the ability of Opa to activate the reporter construct. Thus a single Opa binding site in its native context in vivo apparently has different require- ments for DNA binding than an artificial multimerized version (Sen et al. 2010).
Zic proteins can activate gene expression from constructs bearing no obvious Zic sites (Mizugishi et al. 2001), and some Zic3 binding sites are unable to induce reporter expression (Winata et al. 2015). Even in the absence of DNA binding, Opa is able to produce tissue-specific expression cell autonomously in vivo from reporter constructs containing the dpp head enhancer element described above (Sen et al. 2010). This observation, in combination with the slp1 data, suggests that Opa, like Zic proteins, may not absolutely require DNA binding for transcriptional activity. In both cases, additional known DNA binding factors are required to activate transcription; for the head capsule enhancer, the Hox gene labial is required (Stultz et al. 2012), while slp1 requires run transcriptional activity (Swantek and Gergen 2004). Opa may use protein- protein interactions in some cases to activate gene expression, or other transcription factors could introduce a chromatin configuration more conducive to Opa binding. The range of mechanisms by which Opa influences transcription remains to be elucidated.
Opa may also function by collaborating with developmental signaling pathways.
Zic proteins bind Tcf, the transcriptional effector of the Wnt pathway (Fujimi et al.
2012; Murgan et al. 2015). They also bind Gli proteins, the terminal effectors of the Hedgehog pathway (Koyabu et al. 2001). Opa activates gene expression from con- sensus Ci binding sites in vivo, and ectopic co-expression of Opa and Ci in the fly head primordia produces morphogenetic defects that are intermediate to either one alone (Sen et al. 2010), suggesting that the two proteins are capable of interaction in vivo. In such instances, Zic proteins may influence transcription by altering the output of signal transduction.
3.5.3 Opa Displays Concentration-Dependent Effects
In vivo, Opa can potentiate Run-dependent regulation of slp1 in a concentration- dependent manner (Swantek and Gergen 2004). Clark and Akam present evidence that during segmentation, enhancers of different segmentation genes show sensitivity in
3 Odd-Paired: The Drosophila Zic Gene
their response to the concentration of Opa in the nucleus (Clark and Akam 2016). In our analysis of ventral head development, Opa displayed dominant genetic interactions; it also produced dramatic head defects when overexpressed in third instar eye-antennal discs (Lee et al. 2007). This genetic behavior also suggests that Opa concentration influences its function. Understanding the role of concentration in Opa’s transcriptional behavior may clarify how it functions as a regulator of gene expression.