In this study, we show that MO-induced knockdown of ZNF143 results in many phenotypic effects during zebrafish development. General defects in tail formation were obvious, but we also noticed abnormal heart, blood, ear and MHB development. The specificity of the MO-induced phenotypes was confirmed by experiments showing phenotypic rescue by the full-length protein but not a truncated variant lacking transcriptional activation domains. In the morphants, expression of gata1 (blood), cmlc2 (cardiac) and pax2a (MHB) were reduced or altered spatially. Furthermore, we found two binding sites for ZNF143 in the zebrafish pax2a gene proximal promoter, and disruption of these sites reduced transcription in transfected zebrafish cells. Therefore, one likely mRNA gene target for transcriptional control by this protein in vivo is pax2a, although the number is probably much higher.
It is not surprising that a multitude of phenotypes appear after ZNF143 knockdown because many vertebrate small RNA genes contain SPH sites, and in mammals it has been hypothesized that approximately 2000 mRNA gene promoters contain SPH sites [5
]. It seems likely that the gene encoding ZNF143 is essential for zebrafish viability. The number of promoters containing SPH elements in zebrafish is unknown. Previously, we have demonstrated an important positive role for the SPH site in zebrafish U6
snRNA gene promoters [29
], and have noticed putative SPH elements in many zebrafish small RNA gene promoters (unpublished results). Many fugu small RNA gene promoters contain SPH sites [23
]. At present, we do not know which phenotypic effects or the relative severity of those effects caused by ZNF143 knockdown are the result of altered expression of small RNA genes or mRNA genes. Since knockdown was rescued by coinjection of synthetic mRNA encoding full-length ZNF143, but not by RNA lacking coding potential for N-terminal activation domains (Figure ), we hope to be able to distinguish differential effects of mRNA-activation vs. small RNA activation by this protein using rescue with synthetic RNA encoding deletions or mutations in those separable activation functions.
Since knockdown of ZNF143 caused relatively drastic phenotypic effects in zebrafish embryos, we were surprised that cultured zebrafish ZF4 cells contain undetectable levels of endogenous active protein in a transactivation assay (Figure , compare reporter gene expression from G5 promoter vs. SPH5 promoter in columns 1-3). When these same reporter constructs were used in transfected human HEK293 cells the SPH5 promoter was transcribed at approximately 20-fold higher level due to endogenous ZNF143. Perhaps a small amount of ZNF143 in ZF4 cells is sufficient for their growth, and that amount is below a threshold detectable with relatively large amounts of reporter plasmid added in the transfection experiments. Indeed, mRNA encoding this protein in ZF4 cells was detected by RT-PCR (results not shown). Another possibility is that relative overexpression of ZNF143 in transfected cells suppresses another defect in the small RNA activation pathway in ZF4 cells. Nevertheless, the nonsaturating levels of active ZNF143 in ZF4 cells allowed us to use them as an assay for mRNA activation potential of tagged protein and protein lacking activation domains (Figure ), and we will use this assay in the future to examine activity of mutant proteins.
Our experiments demonstrating brain defects and, specifically, reduced pax2a
expression upon ZNF143 knockdown induced us to inspect the pax2a
proximal promoter for SPH sites. Indeed, ZNF143 binds to the pax2a
promoter in vitro
and activates it in an SPH-site dependent manner when cotransfected into ZF4 cells. Although we used existing human ZNF143 protein reagents in our laboratory for the binding studies, rather than the zebrafish protein, we expect that nucleotide specificity of the human and zebrafish proteins should be undistinguishable. The zinc finger DNA-binding domains are 91% identical, with no changes in the amino acid residues known to be most important for DNA recognition by this motif [23
]. Although the enhancer(s) of the zebrafish pax2a
gene promoter have been characterized partially [31
], we are not aware of previous work to investigate elements within the proximal promoter. We expect that other elements in addition to the SPH1 and SPH2 sites constitute this promoter. Luciferase reporter gene activity of the SPH1+2 mutant promoter was approximately 7-fold higher than that found with a simple TATA-containing promoter in our transient transfection assays (unpublished results). This promoter does not contain a readily identifiable TATA box, a characteristic that is true also of most previously identified SPH-containing mammalian mRNA gene promoters [5
]. It will be interesting to dissect the pax2a
promoter further in order to discern relative roles of SPH elements and other elements with respect to both basal transcription and enhancer-driven expression.
Because of the widespread abundance of SPH sites in mammalian mRNA gene promoters, we searched for them in other important developmental regulators in addition to pax2a. Although several SPH sites appeared to be candidates in the zebrafish gata1 proximal promoter, none bound ZNF143 in vitro (unpublished results). On the other hand, we have found a high-affinity SPH element in the zebrafish fibroblast growth factor receptor-1 (fgfr1) and glycogen synthase kinase 3α promoters (unpublished results). Whether any of the aforementioned genes is controlled in a significant manner by ZNF143 will require further study. Furthermore, because of the large potential number of target promoters in mammals, it is highly likely that the transcription of many other developmental regulator genes is controlled by ZNF143.