The human chd8 gene is transcribed from two distant upstream promoters.

Chromodomain-Helicase-DNA-binding protein 8 (CHD8) is a chromatin remodeler that is central to regulation of gene expression pathways during brain development. Many loss-of-function mutations in transcribed regions of the chd8 gene have been identified in autism spectrum disorder patients. Nothing is known about transcription of the human chd8 gene. Defects in expression of this gene could represent another mechanism leading to reduced amount of CHD8. We identify two major promoters for the human chd8 gene, both of which are located many thousand base pairs upstream of the coding region. Each proximal promoter directs a similar transcriptional efficiency in transfected cells. At least two elements within 200bp of the 5'flanking regions of these promoters are important to drive highest transcriptional levels in transient transfection experiments. RNA polymerase II occupancy levels at each promoter are roughly equivalent. Lastly, each promoter directs a dispersed set of start sites in a cultured cell line. This work could provide the framework for future studies to investigate the importance of chd8 gene expression for diseases associated with brain and neuronal development.

The transcriptional activator ZNF143 is essential for normal development in zebrafish.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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 Foxd3's 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 ubiquitous transcriptional protein ZNF143 activates a diversity of genes while assisting to organize chromatin structure.

Zinc Finger Protein 143 (ZNF143) is a pervasive C2H2 zinc-finger transcriptional activator protein regulating the efficiency of eukaryotic promoter regions. ZNF143 is able to activate transcription at both protein coding genes and small RNA genes transcribed by either RNA polymerase II or RNA polymerase III. Target genes regulated by ZNF143 are involved in an array of different cellular processes including both cancer and development. Although a key player in regulating eukaryotic genes, the molecular mechanism by with ZNF143 binds and activates genes transcribed by two different polymerases is still relatively unknown. In addition to its role as a transcriptional regulator, recent genomics experiments have implicated ZNF143 as a potential co-factor involved in chromatin looping and establishing higher order structure within the genome. This review focuses primarily on possible activation mechanisms of promoters by ZNF143, with less emphasis on the role of ZNF143 in cancer and development, and its function in establishing higher order chromatin contacts within the genome.

Two paralogous znf143 genes in zebrafish encode transcriptional activator proteins with similar functions but expressed at different levels during early development.

BACKGROUND: ZNF143 is an important transcriptional regulator protein conserved in metazoans and estimated to bind over 2000 promoter regions of both messenger RNA and small nuclear RNA genes. The use of zebrafish is a useful model system to study vertebrate gene expression and development. Here we characterize znf143a, a novel paralog of znf143b, previously known simply as znf143 in zebrafish. This study reveals a comparison of quantitative and spatial expression patterns, transcriptional activity, and a knockdown analysis of both ZNF143 proteins. RESULTS: ZNF143a and ZNF143b have a fairly strong conservation with 65% amino acid sequence identity, and both are potent activators in transient transfection experiments. In situ hybridization analyses of both znf143 mRNAs show that these genes are expressed strongly in regions of the brain at 24 h post fertilization in zebrafish development. A transient knockdown analysis of znf143 expression from either gene using CRISPR interference revealed similar morphological defects in brain development, and caused brain abnormalities in up to 50% of injected embryos. Although present in the same tissues, znf143a is expressed at a higher level in early development which might confer an evolutionary benefit for the maintenance of two paralogs in zebrafish. CONCLUSIONS: znf143a encodes a strong activator protein with high expression in neural tissues during early embryogenesis in zebrafish. Similar to its paralogous gene, znf143b, both znf143 genes are required for normal development in zebrafish.

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.

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/ß-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.

Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies.

Vertebrate U6 small nuclear RNA (snRNA) gene promoters are among the founding members of those recognized by RNA polymerase III in which all control elements for initiation are located in the 5'-flanking region. Previously, one human U6 gene (U6-1) has been studied extensively. We have identified a total of nine full-length U6 loci in the human genome. Unlike human U1 and U2 snRNA genes, most of the full-length U6 loci are dispersed throughout the genome. Of the nine full-length U6 loci, five are potentially active genes (U6-1, U6-2, U6-7, U6-8 and U6-9) since they are bound by TATA-binding protein and enriched in acetylated histone H4 in cultured human 293 cells. These five all contain OCT, SPH, PSE and TATA elements, although the sequences of these elements are variable. Furthermore, these five genes are transcribed to different extents in vitro or after transient transfection of human 293 cells. Of the nine full-length U6 loci, only U6-7 and U6-8 are closely linked and contain highly conserved 5'-flanking regions. However, due to a modest sequence difference in the proximal sequence elements for U6-7 and U6-8, these genes are transcribed at very different levels in transfected cells.

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.

Regulation of aldehyde reductase expression by STAF and CHOP.

Aldehyde reductase is involved in the reductive detoxification of reactive aldehydes that can modify cellular macromolecules. To analyze the mechanism of basal regulation of aldehyde reductase expression, we cloned the murine gene and adjacent regulatory region and compared it to the human gene. The mouse enzyme exhibits substrate specificity similar to that of the human enzyme, but with a 2-fold higher catalytic efficiency. In contrast to the mouse gene, the human aldehyde reductase gene has two alternatively spliced transcripts. A fragment of 57 bp is sufficient for 25% of human promoter activity and consists of two elements. The 3' element binds transcription factors of the Sp1 family. Gel-shift assays and chromatin immunoprecipitation as well as deletion/mutation analysis reveal that selenocysteine tRNA transcription activating factor (STAF) binds to the 5' element and drives constitutive expression of both mouse and human aldehyde reductase. Aldehyde reductase thus becomes the fourth protein-encoding gene regulated by STAF. The human, but not the mouse, promoter also binds C/EBP homologous protein (CHOP), which competes with STAF for the same binding site. Transfection of the human promoter into ethoxyquin-treated mouse 3T3 cells induces a 3.5-fold increase in promoter activity and a CHOP-C/EBP band appears on gel shifts performed with the 5' probe from the human aldehyde reductase promoter. Induction is attenuated in similar transfection studies of the mouse promoter. Mutation of the CHOP-binding site in the human promoter abolishes CHOP binding and significantly reduces ethoxyquin induction, suggesting that CHOP mediates stimulated expression in response to antioxidants in the human. This subtle difference in the human promoter suggests a further evolution of the promoter toward responsiveness to exogenous stress and/or toxins.