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ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA.
The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein.
Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development.
The vertebrate transcriptional activator protein, ZNF143 (also known as STAF for selenocysteine tRNA gene transcription activating factor, or SBF for SPH-binding factor) operates at a multitude of small RNA and protein-coding gene promoters [1-5]. Two separable activation domains within this protein stimulate transcription selectively at either small RNA or mRNA promoters . Initially, attention was focused on the function of ZNF143 for small RNA gene transcription, especially for vertebrate snRNA and selenocysteine tRNA genes [7-9]. Then, several mRNA genes were identified whose proximal promoters contained SPH sites [10-15]. Possibly because of the highly degenerate and relatively long DNA-binding site recognized by ZNF143, it was not recognized for many years that approximately 2000 mammalian protein-coding genes contain SPH (SphI Postoctamer Homology ) elements, or STAF Binding Sites (SBS), in their promoters .
Little is known concerning the phenotypic role(s) of ZNF143 during cellular growth and animal development. A number of cell-cycle-associated gene promoters are regulated by ZNF143 [17-19]. Furthermore, ZNF143 is an important regulator of mammalian embryonic stem cell renewal [20,21]. At the molecular level, recent work has demonstrated that this activator protein interacts with the chromodomain-helicase-DNA binding protein 8 (CHD8), and implicates that human U6 gene transcription is stimulated by ZNF143 through this interaction . Many potential small RNA and protein-coding gene promoters are targeted, but which are most pivotal in vivo?
We used zebrafish embryos as a model system to investigate the role of ZNF143 during vertebrate development. Injection of translation-blocking morpholino oligonucleotides (MOs) resulted in a pleiotropic phenotype including axial defects as well as abnormalities in heart, blood, ear and midbrain hindbrain boundary (MHB). Coinjection of synthetic mRNA encoding zebrafish ZNF143 rescued MO-induced defects, and rescue was dependent on the amino-terminal region of the protein containing activation domains. Expression levels or patterns of the gata1, cmlc2, and pax2a genes were altered following MO knockdown of zebrafish ZNF143. The pax2a gene is likely to be a direct target for ZNF143 because this protein binds the promoter in vitro and specific mutations in SPH sites resulted in reduced transcription in transient transfection experiments.
The zebrafish znf143 cDNA has been identified, and the predicted amino acid sequence contains a high degree of similarity with the human protein (71% overall identity by our measurement) . Furthermore, the zebrafish protein contains highly conserved regions that correspond to the previously identified DNA-binding domain (DBD), mRNA gene activation domain (15 aa repeats) and small RNA gene activation domain of the Xenopus and human proteins  (Figure (Figure1A).1A). To verify the mRNA gene activating potential of zebrafish ZNF143 and demarcate boundaries of this region, we fused fragments encoding zebrafish ZNF143 to the S. cerevisiae GAL4p DNA binding domain (amino acids 1-94), and performed transient transfection assays with such expression plasmids and a firefly luciferase reporter gene transcribed from a minimal promoter driven by GAL4 binding sites. Because transcriptional activating domains of the Xenopus protein were localized to the amino-terminal end previously , we investigated this region only. Amino acids 13-150 of zebrafish ZNF143 contains a potent mRNA gene activation region that functions in both human embryonic kidney (HEK293) cells and zebrafish ZF4 cells (Figure (Figure1B,1B, ,1C).1C). The region including only the four 15 aa repeats (amino acids 47-150) was approximately three-fold less active in both cell types. However, it is possible that this difference could be due to a lower expression level of this fragment (Figure (Figure1B).1B). Importantly, the region of zebrafish ZNF143 between the 15 aa repeats and the zinc finger domain (amino acids 151-228) demonstrated minimal mRNA promoter activation. Within this region has been identified a small RNA gene activating domain in the Xenopus protein .
To begin our studies with zebrafish embryos, we investigated the expression of znf143 RNA using whole-mount in situ hybridization. This RNA was expressed ubiquitously in the early stages that we examined, including 2-4 cell stage embryos and through gastrulation stages (results not shown). znf143 RNA must be inherited maternally, since it was detected prior to onset of zygotic transcription.
Next, we co-injected a pair of morpholino oligonucleotides (MOs) that targeted the translational start site and 5' untranslated region (5'UTR) of znf143 mRNA in order to reduce protein levels during embryonic development. Up to nearly 90% of injected embryos exhibited a range of defects at 48 hours post fertilization (hpf) (Figure (Figure2).2). Microinjection of each individual MO resulted in the same range of phenotypes, but at a much lower efficiency (results not shown), supporting the assertion that the observed phenotypes are specific to ZNF143 knockdown. No mutant phenotypes were found when a control MO was injected at the same concentration (results not shown). For quantitation purposes, we sorted the morphant embryos into six classes that are primarily distinguished by increasing severity of axial and other defects. Class 1 embryos show a distinct kink in the tip of the tail (Figure (Figure2B).2B). Class 2 embryos have the same tail kink and also show disorganization in the somites (Figure (Figure2C).2C). Class 3 embryos have a greater degree of tissue disorganization as well as significant pericardial edema (Figure (Figure2D).2D). Class 4 embryos have the added phenotype of severe brain morphology defects, including absent or poorly formed midbrain-hindbrain boundary (MHB) (Figure (Figure2E).2E). Class 5 embryos display the additional phenotype of a considerably shortened axis (Figure (Figure2F).2F). Class 6 morphants (not shown) are morphological monsters, lacking both head and tail structures. In addition to the described effects, we also noted reduced blood formation, rate of circulatory flow and abnormal ear development in significant proportions of morphant embryos falling in the more severe classes (classes 3-6).
Early developmental stages, through most of epiboly, proceeded normally for the ZNF143 morphant embryos, compared to embryos injected with the control MO. However, we detected a delay of approximately 30 min to reach bud stage. Furthermore, subsequent stages for ZNF143 morphants at 6-somite, 10-somite, 18-somite, and 22-somite were delayed approximately one hour.
In order to verify the specificity of MO-knockdown, we co-injected synthetic mRNA encoding zebrafish ZNF143 with the MOs to determine whether protein synthesized from the exogenously supplied mRNA could rescue the phenotypes. We used mRNAs encoding either full length ZNF143 or a truncated variant lacking the transcription activation domains (Δ2-225) in our rescue assays. Both mRNAs lack the normal 5'UTR sequence and encode a myc tag at the amino-terminus of the protein, such that the morpholinos will not block their expression. We first confirmed that the full-length myc-tagged protein is functional for transcriptional activation in transfected ZF4 cells (Figure (Figure3A,3A, compare lanes 2 and 3 with lanes 4 and 5). In addition, the Δ2-225 protein was defective for transcriptional activation in transfected ZF4 cells (Figure (Figure3A,3A, lanes 6 and 7). We detected nearly equivalent levels of expression of the myc-tagged full-length and Δ2-225 proteins in transfected HEK293 cells (Figure (Figure3B).3B). Thus, our constructs should be expressed at equivalent levels when expressed in embryos, even though for unknown reasons we have been unable to detect these proteins by their myc-tags in injected embryos and in transfected ZF4 cells. Importantly, when co-injected with the MOs, full-length zebrafish ZNF143 rescued the general morphant phenotypes in approximately 70% of the injected embryos, and the remaining defects were primarily the least severe classes (Figure (Figure3C,3C, compare columns 1 and 2). The rescue was dependent on the presence of the amino-terminal region of zebrafish ZNF143 containing both activation domains, since co-injection of mRNA lacking codons 2-225 did not rescue the morphant phenotypes (Figure (Figure3C,3C, column 3). These results show that the amino-terminal myc tag does not inactivate the function(s) of zebrafish ZNF143 in vivo, and the observed MO-induced phenotypes are specific to the loss of ZNF143 function and more specifically to ZNF143 transcriptional regulatory capability.
To determine whether the general morphological defects of ZNF143 morphants could be correlated with specific changes in gene expression, we performed whole mount in situ hybridization to detect developmental gene expression in morphant embryos at several stages. Using goosecoid (gsc) and orthodenticle homolog 2 (otx2) probes to monitor prechordal plate mesoderm and neuroectoderm [24,25], respectively, during gastrulation (shield stage and 75% epiboly for gsc, and 75% epiboly for otx2), we detected no differences between ZNF143 morphants and embryos injected with a control MO (results not shown). In addition, hindbrain development of rhombomeres 3 and 5 appeared normal during early somitogenesis (6- and 10-somite stages) using an early growth response 2a (egr2a; or krox20) probe , although a delay in staining of rhombomere 5 was apparent in the 6-somite embryos (results not shown). This minor variation is consistent with the overall delay detected. Furthermore, we examined the expression of myoD, a marker for somitic muscle . At all stages examined, we detected no discernable difference between wt and ZNF143 morphant embryos (5-6 somite, n = 20; 16 somite, n = 13; 27 hpf, n = 20; data not shown). Next we analyzed the expression of gata1, a marker for hematopoietic progenitors , since blood development is compromised in ZNF143 morphants. At the 16-somite stage, gata1 labels populations of hematopoietic progenitors in the posterior mesoderm flanking the midline (Figure (Figure4A).4A). Consistent with our observation that blood specification is aberrant, ZNF143 morphants exhibited a significant reduction in gata1 expression at this stage (Figure (Figure4B;4B; 50% penetrance, n = 20). At 24 hpf, gata1 is expressed in cells of the intermediate cell mass (ICM; Figure Figure4C),4C), but expression continued to be reduced significantly in ZNF143 morphants (Figure (Figure4D;4D; 37% penetrance, n = 19). Thus, the reduction in blood cells we observed in ZNF143 morphants correlates with a significant reduction in gata1 expression that labels hematopoietic progenitors.
We next analyzed expression of cardiac myosin light chain 2 (cmlc2), a marker for cardiomyocytes, since heart development appeared disrupted after reduction of ZNF143. At 24 hpf, the heart comprises a tube that loops toward the left side of the embryo (Figure (Figure4E).4E). Heart progenitors formed normally in ZNF143 depleted embryos, but morphogenesis of the heart was severely compromised in 55% of MO-injected embryos (Figure (Figure4F,4F, ,4G;4G; n = 38). Phenotypic defects varied in this group from a failure to migrate to the left side of the embryo (Figure (Figure4F),4F), to a failure in tube morphogenesis (Figure (Figure4G).4G). These phenotypic defects in the ZNF143 morphants are not a result of prolonged delay of heart development, because embryos probed for cmlc2 expression at 30 hpf and 37 hpf did not exhibit normal asymmetric looping (26/27 defective). At these later stages, 26% displayed cardia bifida (Figure (Figure4H,4H, ,4I4I).
Because ZNF143 morphants displayed morphological abnormalities in the brain, including defects in the MHB, we analyzed the expression of pax2a, a critical regulator of MHB formation. At the 16-somite stage, pax2a expression marks the optic stalks, mid-hindbrain domain, otic vesicles, pronephros and a small number of spinal cord neurons (Figure (Figure4J).4J). After reduction of ZNF143 function, pax2a expression was reduced in most expression domains of the majority of embryos (68%, n = 19). Expression of pax2a in the MHB was reduced substantially in ZNF143 morphants, and to a lesser degree, expression in the otic vesicles, and pronephros (Figure (Figure4K,4K, ,4L).4L). In contrast, expression in the optic stalks did not appear to be significantly diminished compared to wt. Importantly, this phenotype was rescued by coinjection of wt ZNF143 mRNA (100% rescue, n = 14). At earlier stages (bud, 6-somite, 10-somite) pax2a expression was comparable between ZNF143 morphants and embryos injected with control MO. Furthermore, by 24 hpf, pax2a expression was not significantly different between wt and ZNF143 morphants, with the exception that pax2a expression in a group of hindbrain neurons was reduced or absent in the morphants (Figure (Figure4M,4M, ,4N,4N, arrows; 100% penetrance, n = 18). Thus, normal pax2a expression, particularly at the MHB, depends on ZNF143 function.
Since pax2a gene expression was affected after ZNF143 knockdown, we investigated whether this transcription factor might control zebrafish pax2a transcription. Visual inspection of the proximal promoter region sequence indicated three possible SPH motifs (Figure (Figure5A).5A). Radiolabeled probes were prepared that contained either the SPH1 element alone (-289/-131 fragment), or both SPH2 and SPH3 sites (-154/+16 fragment). Both probes bound human ZNF143, showing a single complex that was competed with a specific human U6-1 SPH oligonucleotide, but not with an unrelated sequence containing the octamer (OCT) transcription control element (Figure (Figure5B).5B). Specific oligonucleotide competition for the pax2a DNA fragments was similar to that for the zebrafish U6 small nuclear RNA gene promoter used as a positive control for these experiments (Figure (Figure5B,5B, rightmost panel; ). Since only a single complex dependent on addition of a nonsaturating amount of ZNF143 was detected with the - 154/+16 fragment, we suspected that only one of the two potential SPH elements was bound. DNaseI footprinting was used to determine which SPH element in the - 154/+16 fragment was bound by the protein. Results from footprinting experiments demonstrated binding of the human ZNF143 DNA-binding domain to the SPH2 element, while the SPH3 site was unoccupied at concentrations used for the experiment (Figure (Figure5C.5C. left panel). Therefore, two sites (SPH1 (likely) and SPH2) within the zebrafish pax2a promoter are bound by ZNF143 in vitro.
Next we investigated the transcriptional effect of the SPH sites in the zebrafish pax2a promoter in transient transfection experiments. Luciferase reporter plasmids containing either the wt pax2a promoter (-289/+16), single SPH1 or SPH2 mutants, or the double SPH1/2 mutant were added to zebrafish ZF4 cells. All mutant sites destroyed the highly conserved CCCA sequence of a SPH element (mutant sequences are shown in Methods). The efficacy of the SPH2MUT alteration was demonstrated by the obliteration of the DNaseI footprint in vitro (Figure (Figure5C,5C, lanes 6-9). The zebrafish pax2a proximal promoter (-289/+16) was quite active in transfected ZF4 cells since a typical experiment yielded approximately 7-fold higher normalized firefly luciferase level than from a simple TATA box only promoter (G5 reporter in Figure Figure3A).3A). In transfected ZF4 cells, using a synthetic promoter containing multiple SPH elements, we found only low, basal activity at the same level as from a promoter lacking SPH sites (Figure (Figure3A,3A, compare columns 1 and 2). In human HEK293 cells the SPH5 promoter activity is approximately 20-fold higher than from the G5 promoter (unpublished results). Hence, unlike HEK293 cells, ZF4 cells do not contain sufficient amounts of active ZNF143 to stimulate potentially responsive promoters. Transcription from the wt pax2a promoter was increased almost two-fold by co-transfection with a ZNF143 expression plasmid (Figure (Figure6).6). The SPH2MUT and doubly mutant promoters were not activated, whereas the relative stimulation of transcription from the SPH1MUT promoter was somewhat reduced (Figure (Figure6,6, filled bars). Not surprisingly, transcription from the various SPHMUT pax2a promoters was not reduced significantly in the absence of exogenous ZNF143 (unfilled bars in Figure Figure6).6). Hence the SPH1 and SPH2 elements are functional for transcriptional activity directed by the zebrafish pax2a gene promoter.
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 . 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 , and have noticed putative SPH elements in many zebrafish small RNA gene promoters (unpublished results). Many fugu small RNA gene promoters contain SPH sites . 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 (Figure3),3), 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 (Figure3A,3A, 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 (Figure3A),3A), 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 [23,29,30]. Although the enhancer(s) of the zebrafish pax2a gene promoter have been characterized partially , 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 . 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.
Because knockdown of the transcriptional activator ZNF143 by injection of translational-blocking MOs causes many significant phenotypic effects in zebrafish embryos, we conclude that this protein is essential for normal development. Phenotypic effects are rescued by coinjection of full-length znf143 mRNA, but not by mRNA lacking coding capacity for the N-terminal region containing activation domains. Hence, the MO-mediated knockdown is specific, and ZNF143 function in vivo requires the N-terminal region. Furthermore, the pax2a gene promoter is at least one likely target of ZNF143 during zebrafish development.
A cDNA containing the zebrafish znf143 gene was obtained from Open Biosystems. We named this plasmid pME18S-FL3/zZNF143. The open reading frame (ORF) contained within this insert was lacking a full-length gene and contained a reading frame error in the coding region near the amino-terminus of the encoded protein. Hence, in order to construct a full-length ORF situatated behind a T7 promoter, three DNA fragments were ligated as follows. The "correct" amino-terminal region fragment was prepared by reverse transcriptase polymerase chain reaction (RT-PCR) using total RNA from zebrafish ZF4 cells and ligated into a pGEM-T vector (Promega). This fragment was excised using KpnI and PvuII and purified by agarose gel electrophoresis. The main body of the znf143 ORF was excised from pME18S-FL3/zZNF143 using PvuII and XhoI and purified by agarose gel electrophoresis. The third DNA fragment was the pBlueScript SK vector (Stratagene) opened at KpnI and XhoI sites, and purified by chromatography on Sepharose CL-4B. The three-fragment ligation reaction was used to transform E. coli XL1-Blue (Stratagene) competent cells. The resulting plasmid was named pBS/zZNF143. A single myc tag was inserted at the amino-terminus of the encoded ZNF143 protein using the QuickChange site-directed mutagenesis protocol. The subsequent Δ2-225 deletion within the zebrafish znf143 ORF started with the parent plasmid pBS/myczZNF143 and was constructed using the QuickChange protocol in which the oligonucleotides base-paired across the deletion endpoints and looped out the template DNA to be deleted. Plasmid minipreps were prepared using a Qiagen miniprep kit, and such preparations were suitable for mRNA synthesis in vitro. Zebrafish ZNF143 expression plasmids used for transient transfection experiments were constructed by amplifying inserts from pBS-based plasmids using PfuUltraHF polymerase (Stratagene), restriction with XbaI and SalI, gel-purification, and ligation into like sites in the pCI-neo vector (Promega). GAL4DBD - zebrafish ZNF143 fusion expression plasmids were constructed in the pCI-neo vector (Promega), and contained inserts encoding the amino-terminal 94 aa of S. cerevisiae Gal4p  fused in-frame to zebrafish ZNF143 fragments amplified by PCR. Luciferase reporter plasmids used for transient transfection experiments were based upon the pGL3-basic vector (Promega) and included the adenovirus major late basal promoter (AdMLT). The pGL3/G5AdMLT ("G5") reporter contained five copies of the S. cerevisiae upstream activating sequence galactose (UASG) element ligated upstream of the TATA box, whereas the "SPH5" reporter contained five copies of the human U6-1 gene SPH element in the same position. The zebrafish pax2a gene promoter (-289 to +16) was amplified from genomic DNA by PCR and ligated into the pGEM-T vector (Promega). The location of the transcriptional start site (+1) is according to the 5'-most expressed sequence tag (EST) in the University of California, Santa Cruz zebrafish genome assembly (July 2007 assembly, chromosome 13, position 29,268,562). SPHMUT plasmids containing clustered point mutations within SPH sites of the pax2a promoter were prepared by the QuickChange protocol. The nucleotide changes introduced into SPHMUT sites were (mutant nucleotides in lower case letters): SPHMUT1: 5'-TAAgatcTCTCTCCTCAT-3'; SPH2MUT: 5'-CTagatctATCCCCCCTC-3' (bottom strand); SPH3MUT: 5'-CAagatctAGCTTCTAAC-3' (top strand). DNA fragments containing wt and mutant pax2a promoters were amplified by PCR using pGEM-based templates, restricted with SacI and NheI, and ligated into the same sites of the pGL3-basic vector. Inserts of all plasmids were sequenced completely to verify deletions, in-frame ligations, and ensure that no other mutations were added to the znf143 ORF or pax2a promoter. DNAs used for transient transfection experiments were purified using the Qiagen plasmid maxi kit, and concentrations were determined by absorbance at 260 nm.
Human HEK293 cells were purchased from ATCC (CRL-1573) and cultured in Dulbecco's Modified Eagle Medium (DMEM), containing high glucose (Gibco 11995), penicillin-streptomycin, and 10% bovine growth serum (Hyclone). Experiments with HEK293 cells were performed under BSL-2 guidelines following approval by the Texas A&M University Institutional Biosafety Committee. Zebrafish ZF4 cells  were obtained from ATCC (CRL-2050) and grown in DMEM/F12 medium (Gibco 11320), penicillin-streptomycin, and 10% bovine growth serum. Cells were transfected in 6-well dishes using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol with amounts of DNA described in the figure legends. Cells were co-transfected with pRL-SV40 (Promega), a renilla luciferase reporter, for normalization. 48 h post-transfection, cell extracts were prepared, and firefly and renilla luciferase activities were measured with a Sirius tube luminometer (Berthold) using reagents and protocols from the Dual-Luciferase Reporter Assay System (Promega). The number of independently transfected samples for each condition is shown in parentheses above the bars in the graphs. Statistical significance (p-values) comparing sets of different experiments was calculated using the Student's T test in Microsoft Excel.
Two radiolabeled pax2a gene proximal promoter probes used for protein-DNA binding assays were prepared by PCR using either 32P-end-labeled CZPAX2A-131 primer (5'-CAACACTTTGTGATTCGCCAACGC-3'), and unlabeled ZPAX2A-289 primer (5'-TGGTACCGCTTCCTTTCCACTTGT-3'), or 32P end-labeled CZPAX2A+16 primer (5'-ATGTGCCTGTTAGAAGCTTTGGGC-3') and unlabeled ZPAX2A-154 primer (5'-GCGTTGGCGAATCACAAAGTGTTG-3'), and pGEM plasmid DNA with wt or SPHMUT pax2a promoter inserts. The zebrafish U6-1 probe was prepared as described . Protein for EMSA experiments was prepared by in vitro transcription/translation of human znf143 as described previously . DNA-protein complex formation, competition with oligonucleotides, and electrophoresis on non-denaturing gels were performed as before . Sequences of the competitor oligonucleotides can be found in  where human U6-1 SPH is called NONOCT(long) and human U6-1 OCT is called OCTCON. DNaseI footprinting assays followed the same protocol as described previously , using purified human ZNF143 zinc finger DNA-binding domain (DBD) (amino acids 236-444) expressed from a pET5a vector in E. coli . The positions of the putative SPH elements were deduced by electrophoresis of T and C dideoxy sequencing reactions on the same gels.
AB strain zebrafish were maintained using standard methods , and with protocols approved by the Texas A&M Animal Care and Use Committee (AUP #2007-90 and 2010-90). MOs and synthetic mRNAs were injected at the one- to two-cell stage. Two MOs were designed to block the translation of zebrafish znf143, dissolved in 1X Danieau buffer, and were coinjected at a concentration of 2 ng/nL, each. The "Start" MO, ordered from Open Biosystems and Gene Tools, LLC, had the sequence, 5'-ATTCACCTGGGCTAACAGCATGATC-3', and the "5'UTR" MO, ordered from Gene Tools, LLC, had the sequence, 5'-CAACAATCCCTTCGTTCGACCACCA-3'. The control MO (5'-CCTCTTACCTCAGTTACAATTTATA-3') was purchased from Gene Tools, LLC. For rescue coinjection experiments, capped and polyadenylated mRNAs were synthesized from pBS/myczZNF143 or the Δ2-225 deletion variant template using the mMESSAGE mMACHINE T7 Ultra kit (Ambion). Synthetic mRNA concentrations were determined using a NanoDrop spectrophotometer. Approximately 3 nL of RNAs were injected at a concentration of 30 pg/nL, an amount that did not cause any toxicity or phenotype when injected without MOs. Phenotypes were classified for quantitation in Figure Figure3C3C at 48 hpf.
Nuclear extracts from HEK293 cells were prepared using the NE-PER kit (Pierce/Thermo Scientific). After electrophoresis on standard SDS denaturing gels, proteins were transferred to nitrocellulose, and probed with rabbit anti-yeast GAL4DBD (Upstate/Millipore), or mouse monoclonal anti-myc (Upstate/Millipore, clone 4A6).
Whole-mount in situ hybridizations with embryos were performed according to standard methods . Probes used were cmlc2 , pax2a , gata1 , myoD , gsc , otx2 , and egr2a (or krox20) . The numbers of independently injected embryos scored for various phenotypes are noted in the text.
KMH designed and carried out experiments shown in Figures Figures2,2, ,3C,3C, and and4.4. ACL participated in the design of the study, the microscopy and the preparation of the manuscript. GRK performed experiments shown in Figures Figures1,1, 2A,B, ,5,5, and and6,6, conceived the project, coordinated the work, and drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by funds from the Department of Biochemistry and Biophysics (TAMU). ACL is a Research Scholar of the American Cancer Society (RSG-06-150-01-DDC). We thank members of the Lekven laboratory (Cathryn Kelton, Anand Narayanan, Kevin Baker, and Amy Whitener) for assistance with fish husbandry, in situ protocols and for supplying probes for myoD, gata1, cmlc2, pax2a, gsc, otx2, and krox20. The Riley laboratory (TAMU) also supplied the probe for pax2a for some experiments.