Sp2 Is a Maternally Inherited Transcription Factor Required for Embryonic Development

Abstract

The Sp family of transcription factors is required for the expression of cell cycle-and developmentally regulated genes, and the deregulated expression of a handful of family members is associated with human tumorigenesis. Sp2 is a relatively poorly characterized member of the Sp family that, although widely expressed, exhibits little or no DNA binding or transcriptional activity in human and mouse cell lines. To begin to address the role(s) played by Sp2 in early metazoan development we have cloned and characterized Sp2 from zebrafish (Danio rerio). We report that 1) the intron/exon organization and amino acid sequence of zebrafish Sp2 is closely conserved with its mammalian orthologues, 2) zebrafish Sp2 weakly stimulates an Sp-dependent promoter in vitro and associates with the nuclear matrix in a DNA-independent fashion, 3) zebrafish Sp2 is inherited as a maternal transcript, is transcribed in zebrafish embryos and adult tissues, and is required for completion of gastrulation, and 4) zebrafish lines carrying transgenes regulated by the Sp2 promoter recapitulate patterns of endogenous Sp2 expression.Sp/XKLF proteins are sequence-specific DNA-binding proteins that share a highly conserved, carboxyl-terminal DNA binding domain featuring three "zinc fingers" of the Cys 2 -His 2 class (1-3). Four conserved nucleotides within each zinc finger specify the interaction of the DNA binding domain with a nonameric GC-rich sequence that is often located proximal to sites of transcriptional initiation. Sp proteins comprise one subfamily of Sp/XKLF proteins for which nine members (Sp1-Sp9) have been described in mammals. Each family member carries an amino-terminal trans-activation domain that is comprised of three subdomains, termed A, B, and C (4). The A and B subdomains feature alternating regions enriched in glutamine or serine/threonine residues, whereas the C subdomain carries an abundance of charged amino acids (5, 6). Each subdomain is sufficient to drive transcription when tethered individually to a DNA binding domain, albeit to levels that are less significant than elicited when multiple subdomains are examined together. trans-Activation of target genes by Sp proteins is dependent on post-translational modifications as well as their physical interaction with each other, additional sequence-specific DNA-binding proteins, and components of the basal transcription complex.The biochemical and functional properties of Sp1, Sp3, and Sp4 have been characterized most thoroughly, and each has been shown to regulate the expression of a constellation of genes involved in fundamental biological processes, including cell cycle control, differentiation, development, and oncogenesis (1, 7-9). Sp1, Sp2, and Sp3 are expressed in many, if not all, mammalian tissues, whereas the expression of Sp4 -9 is considerably more restricted. Sp-related proteins have been identified in a wide variety of species, including zebrafish and invertebrates, and mechanisms governing Sp-dependent transcription are evolutionarily conserved. For example,...

Transcription of mouse Sp2 yields alternatively spliced and sub-genomic mRNAs in a tissue- and cell-type-specific fashion

“…5B). It is worth noting that a virtually identical distribution of Sp2 expression has been detected in developing zebrafish embryos using a digoxigenin-labeled RNA probe derived from the zebrafish Sp2 3′ untranslated region [29]. …”

“…In keeping with this supposition we have cloned the zebrafish Sp2 orthologue and shown that Sp2 is required for the completion of gastrulation [29]. Expression of full-length Sp2 message is particularly prominent in the embryonic forebrain of the mouse, suggesting that Sp2 may regulate region-specific genes within the developing central nervous system.…”

“…Phan et al [28] reported that Sp2 abundance is increased significantly in human prostate cancer specimens and the extent of Sp2 expression is directly correlated with the pathological grade of the tumor examined. The zebrafish orthologue of Sp2 has been cloned and shown to be structurally and functionally analogous to its mammalian counterparts [29]. Zebrafish embryos inherit Sp2 as a maternal transcript, Sp2 transcription is triggered in blastula stage embryos, and Sp2 is required for the completion of gastrulation.…”

Neural development is dependent on the function of specificity protein 2 in cell cycle progression

“…Although Sp family members are coexpressed broadly, developmental defects exhibited by subtypespecific knockout mice indicate that their functions may only partially overlap (Supp et al, 1996;Marin et al, 1997;Bouwman et al, 2000;Nguyêñ-Trân et al, 2000;Göllner et al, 2001;van Loo et al, 2003). Recent studies indicate Sp2 is required for early embryonic development in mice and zebrafish (Baur et al, 2010;Xie et al, 2010), and Sp2 overexpression in skin stem cell and progenitor populations is oncogenic (Kim et al, 2010). Acute loss of Sp2 negatively regulates the proliferation of immortalized mouse fibroblasts (Baur et al, 2010).…”

Overexpression of Transcription Factor Sp2 Inhibits Epidermal Differentiation and Increases Susceptibility to Wound- and Carcinogen-Induced Tumorigenesis

“…The Sp2 DNA-binding domain is the least conserved (75%) amongst Sp-family members and binds with high affinity (K d = 225 pM) to a consensus DNA-binding site (5'-GGGCGGGAC-3') that is distinct from that of Sp1 (8–10). Yet, in transient-transfection assays Sp2 only weakly trans -activates promoters carrying consensus Sp2-binding sites or well-characterized Sp-dependent promoters that are readily induced by Sp1 and Sp3 (8, 11). Despite its widespread expression, little or no soluble Sp2 DNA-binding activity has been detected in many human and mouse cell lines (8).…”