RESUMO
The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Clonagem Molecular , DNA/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/biossíntese , Bases de Dados como Assunto , Polpa Dentária/metabolismo , Marcação de Genes , Glutationa Transferase/metabolismo , Proteínas Hedgehog , Hibridização In Situ , Luciferases/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Transativadores/metabolismo , Transfecção , Proteína GLI1 em Dedos de Zinco , Dedos de ZincoRESUMO
Transcription factor TFIID is a multiprotein complex composed of the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). TFIID plays an essential role in mediating transcriptional activation by gene-specific activators. Numerous transcriptional activators have been characterized from mammalian cells; however, molecular analysis of the components of mammalian TFIID has been incomplete. Here we describe isolation of cDNAs encoding two TAF subunits of the human transcription factor TFIID. The first cDNA is predicted to encode the C-terminal 947 residues of the 130-kDa human TAF subunit, hTAFII130. The second cDNA encodes the C-terminal 801 residues of the 100-kDa subunit, hTAFII100. Recombinant TAFs expressed in human cells by transient transfections are capable of associating with the endogenous TAFs and TBP to form a TFIID complex in vivo. Protein binding experiments demonstrate that hTAFII130, like its Drosophila homolog dTAFII110, interacts with the glutamine-rich activation domains of the human transcription factor Sp1. Furthermore, hTAFII130 shows reduced binding to the Sp1 mutants with impaired ability to activate transcription, suggesting a role for hTAFII130 as a direct coactivator target for Sp1.