RESUMO
A long-standing problem in biology is how to dissect traits for which no tractable model exists. Here, we screen for genes like the nude locus (Foxn1)-genes central to mammalian hair and thymus development-using animals that never evolved hair, thymi, or Foxn1. Fruit flies are morphologically disrupted by the FOXN1 transcription factor and rescued by weak reductions in fly gene function, revealing molecules that potently synergize with FOXN1 to effect dramatic, chaotic change. Strong synergy/effectivity in flies is expected to reflect strong selection/functionality (purpose) in mammals; the more disruptive a molecular interaction is in alien contexts (flies), the more beneficial it will be in its natural, formative contexts (mammals). The approach identifies Aff4 as the first nude-like locus, as murine AFF4 and FOXN1 cooperatively induce similar cutaneous/thymic phenotypes, similar gene expression programs, and the same step of transcription, pre-initiation complex formation. These AFF4 functions are unexpected, as AFF4 also serves as a scaffold in common transcriptional-elongation complexes. Most likely, the approach works because an interaction's power to disrupt is the inevitable consequence of its selected-for power to benefit.
Assuntos
Fatores de Transcrição Forkhead , Pele , Animais , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Nus , Fenótipo , Pele/metabolismo , Timo/metabolismoRESUMO
Translocations between the human Mixed Lineage Leukemia (MLL) and AF4 Family (AFF) member genes, are implicated in leukemia. Mutations to AFFs can disrupt lymphopoesis, CNS development and spermatogenesis. However, despite the growing list of pathologies linked to AFF members, their evolutionary relationship and the structure/function of individual members, remain to be elucidated. Here, we first report that database mining and phylogenetic analysis with AFF proteins from multiple species, revealed two monophyletic sister clades, suggesting a common Bilateria ancestor. We then examined the structure/function of the most recently discovered AFF member, MCEF (also known as AF5q31 or AFF4). In silico, the human MCEF gene was found to have 21 exons, and code for a protein with seven nuclear localization sequences (NLS). In HeLa cells, an MCEF-EGFP fusion protein, localized exclusively to the nucleus. Consequently, we made twenty constructs, expressing MCEF deletion mutants fused to EGFP and/or DsRed fluorescent proteins. Three distinct protein sequences, encoded by three separate MCEF exons, were found to mediate nuclear localization, only two of which were predicted in silico. Importantly, we also found that ectopic expression of MCEF, repressed HIV-1 LTR-directed RNA Polymerase II transcription, at the level of Tat-transactivation. We suggest that portions of MCEF could be exploited for chimeric transcription factor repression (CTFR) of HIV-1.
Assuntos
Produtos do Gene tat/antagonistas & inibidores , HIV-1/fisiologia , Proteínas Nucleares/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sequência Consenso , DNA Complementar/genética , Evolução Molecular , Éxons/genética , Genes Reporter , Repetição Terminal Longa de HIV , Células HeLa , Humanos , Invertebrados/genética , Dados de Sequência Molecular , Proteínas Nucleares/classificação , Filogenia , RNA Polimerase II/metabolismo , Splicing de RNA , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da Espécie , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição , Vertebrados/genética , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Human immunodeficiency virus type 1 (HIV-1) replication is coupled to T-cell activation through its dependence on host cell transcription factors. Despite the enormous sequence variability of these factors, several cis elements for host factors are highly conserved within the 5' long terminal repeats (LTRs) of viruses from AIDS patients; among these is the RBEIII upstream element for the Ras response element binding factor 2 (RBF-2). Here we show that RBF-2 is comprised of a USF1/USF2 heterodimer and TFII-I, which bind cooperatively to RBEIII. Recombinant USF1/USF2 binds to the RBEIII core sequence 160-fold less efficiently than it binds to an E box element, but the interaction with RBEIII is stimulated by TFII-I. Chromosomally integrated HIV-1 LTRs bearing an RBEIII mutation have slightly elevated basal transcription in unstimulated Jurkat cells but are unresponsive to cross-linking of the T-cell receptor or stimulation with phorbol myristate acetate (PMA) and ionomycin. Induction is inhibited by dominant interfering USF and TFII-I but not by the dominant negative I-kappaB protein. USF1, USF2, and TFII-I bind to the integrated wild-type LTR in unstimulated cells and become phosphorylated during the induction of transcription upon stimulation with PMA. These results demonstrate that USF1/USF2 and TFII-I interact cooperatively at the upstream RBEIII element and are necessary for the induction of latent HIV-1 in response to T-cell activation signals.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV/fisiologia , HIV-1/genética , Linfócitos T/virologia , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição/metabolismo , Integração Viral , Replicação Viral , Animais , Células COS , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , HIV-1/fisiologia , Humanos , Células Jurkat , Ativação Linfocitária , Fosforilação , Ligação Proteica , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Transcrição Gênica , Fatores Estimuladores Upstream , Ativação ViralRESUMO
Human immunodeficiency virus type 1 (HIV-1) is classified into subtypes on the basis of phylogenetic analysis of sequence differences. Inter- and intra-subtype polymorphism extends throughout the genome, including the long terminal repeat (LTR). In this study, the importance of the upstream stimulating factor (USF)-binding site (E-box) in the core-negative regulatory element (NRE) of the LTR of HIV-1 subtypes A, B, C, D, E and G was investigated. In vivo, USF was found to repress transcription directed from representative HIV-1 LTR sequences of all the subtypes tested in an epithelial cell line, yet activate the same transcription in a T-cell line. Mutation of the core-NRE USF site of the representative subtype B LTR did not affect the cell-specific, subtype-independent, dual role of USF. In vitro binding assays showed that recombinant USF(43) interacts with the core-NRE from subtypes B and C, but not A, D, E or G. Thus, USF affects LTR-directed transcription in a cell-specific manner, independently of both the HIV-1 subtype from which the LTR was derived and the core-NRE USF site sequences.