Gary R. Kunkel

The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation

We have identified a novel zinc finger-containing transcription factor, called Osterix (Osx), that is specifically expressed in all developing bones. In Osx null mice, no bone formation occurs. In endochondral skeletal elements of Osx null mice, mesenchymal cells, together with osteoclasts and blood vessels, invade the mineralized cartilage matrix. However, the mesenchymal cells do not deposit bone matrix. Similarly, cells in the periosteum and in the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. These cells do, however, express Runx2/Cbfa1, another transcription factor required for bone formation. In contrast, Osx is not expressed in Runx2/Cbfa1 null mice. Thus, Osx acts downstream of Runx2/Cbfa1. Because Osx null preosteoblasts express typical chondrocyte marker genes, we propose that Runx2/Cbfa1-expressing preosteoblasts are still bipotential cells.

Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest

Pigment cells of the zebrafish, Danio rerio, offer an exceptionally tractable system for studying the genetic and cellular bases of cell fate decisions. In the zebrafish, neural crest cells generate three types of pigment cells during embryogenesis: yellow xanthophores, iridescent iridophores and black melanophores. In this study, we present evidence for a model whereby melanophores and iridophores descend from a common precursor whose fate is regulated by an interplay between the transcription factors Mitf and Foxd3. Loss of mitfa, a key regulator of melanophore development, resulted in supernumerary ectopic iridophores while loss of foxd3, a mitfa repressor, resulted in fewer iridophores. Double mutants showed a restoration of iridophores, suggesting that one of Foxd3s roles is to suppress mitfa to promote iridophore development. Foxd3 co-localized with pnp4a, a novel marker of early iridophore development, and was necessary for its expression. A considerable overlap was found between iridoblast and melanoblast markers but not xanthoblast markers, which resolved as cells began to differentiate. Cell lineage analyses using the photoconvertible marker, EosFP, revealed that both melanophores and iridophores develop from a mitfa+ precursor. Taken together, our data reveal a Foxd3/mitfa transcriptional switch that governs whether a bi-potent pigment precursor will attain either an iridophore or a melanophore fate.

The transcriptional activator ZNF143 is essential for normal development in zebrafish

BackgroundZNF143 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.ResultsThe 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.ConclusionsNormal 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 Small RNA Gene Activator Protein, SphI Postoctamer Homology-binding Factor/Selenocysteine tRNA Gene Transcription Activating Factor, Stimulates Transcription of the Human Interferon Regulatory Factor-3 Gene

Many small nuclear RNA gene promoters are activated by SphI postoctamer homology (SPH)-binding factor/selenocysteine tRNA gene transcription activating factor (SBF/Staf). Whereas this transcription factor was initially identified by its ability to bind to SPH elements in such promoters, it was more recently shown to have the capacity to activate transcription of a synthetic mRNA gene promoter through a distinct activation domain. Here, we show that the human interferon regulatory factor-3 (IRF-3) gene promoter contains a functional SPH element that is bound by SBF/Staf in vitro and in transfected cells.

Adhesion-dependent Skp2 transcription requires selenocysteine tRNA gene transcription-activating factor (STAF).

Cell adhesion is essential for cell cycle progression in most normal cells. Loss of adhesion dependence is a hallmark of cellular transformation. The F-box protein Skp2 (S-phase kinase-associated protein 2) controls G(1)-S-phase progression and is subject to adhesion-dependent transcriptional regulation, although the mechanisms are poorly understood. We identify two cross-species conserved binding elements for the STAF (selenocysteine tRNA gene transcription-activating factor) in the Skp2 promoter that are essential for Skp2 promoter activity. Endogenous STAF specifically binds these elements in EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) analysis. STAF is sufficient and necessary for Skp2 promoter activity since exogenous STAF activates promoter activity and expression and STAF siRNA (small interfering RNA) inhibits Skp2 promoter activity, mRNA and protein expression and cell proliferation. Furthermore, ectopic Skp2 expression completely reverses the inhibitory effects of STAF silencing on proliferation. Importantly, STAF expression and binding to the Skp2 promoter is adhesion-dependent and associated with adhesion-dependent Skp2 expression in non-transformed cells. Ectopic STAF rescues Skp2 expression in suspension cells. Taken together, these results demonstrate that STAF is essential and sufficient for Skp2 promoter activity and plays a role in the adhesion-dependent expression of Skp2 and ultimately cell proliferation.

Zebrafish U6 small nuclear RNA gene promoters contain a SPH element in an unusual location.

Promoters for vertebrate small nuclear RNA (snRNA) genes contain a relatively simple array of transcriptional control elements, divided into proximal and distal regions. Most of these genes are transcribed by RNA polymerase II (e.g., U1, U2), whereas the U6 gene is transcribed by RNA polymerase III. Previously identified vertebrate U6 snRNA gene promoters consist of a proximal sequence element (PSE) and TATA element in the proximal region, plus a distal region with octamer (OCT) and SphI postoctamer homology (SPH) elements. We have found that zebrafish U6 snRNA promoters contain the SPH element in a novel proximal position immediately upstream of the TATA element. The zebrafish SPH element is recognized by SPH-binding factor/selenocysteine tRNA gene transcription activating factor/zinc finger protein 143 (SBF/Staf/ZNF143) in vitro. Furthermore, a zebrafish U6 promoter with a defective SPH element is inefficiently transcribed when injected into embryos.

A complex that contains proteins binding to the PSE and TATA sites in a human U6 small nuclear RNA promoter

The proximal promoter of a human U6 small nuclear RNA (snRNA)-encoding gene contains two separate elements, the proximal sequence element (PSE) and the TATA box. We investigated the interaction of the PSE- and TATA-binding proteins (PBP and TBP) with normal and mutant U6 proximal promoters using an electrophoretic mobility shift assay. We detected a complex containing both PBP and TBP bound to the wild-type U6 promoter. Efficient formation of the triple complex was dependent on the presence of the PSE and the TATA box on the template DNA. Mutant U6 promoters containing an increased spacing between the PSE and TATA box of 5 or 10 bp were impaired in the ability to form a complex that includes TBP. We infer from these results that PBP and TBP interact when their binding sites are properly positioned in a U6 gene promoter.

CHD8short, a naturally-occurring truncated form of a chromatin remodeler lacking the helicase domain, is a potent transcriptional coregulator

Chromodomain-Helicase-DNA binding protein 8 (CHD8) is a member of a large family of eukaryotic ATP-dependent chromatin remodeling complexes. Loss of function alleles of human chd8 are correlated with autism spectrum disorder. The CHD subfamily members contain a tandem pair of chromodomains that are adjacent to a centrally located Snf2-like helicase domain. An alternatively spliced variant mRNA of CHD8 was identified years ago in mammals that encode a truncated form of the protein, called Duplin, that lacks the helicase domain and everything else in the carboxyl direction. We are using zebrafish to explore the functions of CHD8, especially the truncated form that we refer to as CHD8short (CHD8S). The mRNA for CHD8S is expressed differentially during embryonic development. Using a PCR assay we detected expression of putative zebrafish chd8s mRNA that is barely detectable during early embryogenesis (shield stage at 6h), but increases markedly soon thereafter at 80-90% epiboly (9h) and bud stages (10h), with a return to low levels in 16-somite (17h) and 24hpf embryos. Except for high expression during the shield stage, steady-state levels of chd8l (long) mRNA are relatively constant during the same period of development. We subcloned both chd8l and chd8s cDNAs into expression vector plasmids for use in transient transfection experiments in zebrafish ZF4 cells. In some experiments the luciferase reporter gene was driven by a synthetic promoter that is responsive to activation by ZNF143 activator protein, a known interacting protein with CHD8 in mammalian cells. Whereas CHD8L was a modest coactivator, CHD8S was a potent coactivator, a surprising result since CHD8S is lacking a critical domain to function as a chromatin remodeler enzyme. CHD8S coactivator function is dependent on a region of the protein within the first 50 amino-terminal amino acids. In transient transfection experiments using a Lef1/β-catenin reporter gene, CHD8S was a modest repressor, but deletion of 50 or more amino-terminal amino acids converted it to a coactivator. When synthetic chd8s mRNA was injected into zebrafish embryos in order to overexpress CHD8S, we observed significant brain disruption phenotypes.