RESUMO
In a variety of organisms, genes placed near heterochromatin are transcriptionally silenced. In order to understand the molecular mechanisms responsible for this block in transcription, high resolution in vivo chromatin structure analysis was performed on two heat shock genes, hsp26 and hsp70. These genes normally reside in euchromatin where GAGA factor and RNA Pol II are present on the promoter prior to heat shock induction. P-element transformation experiments led to the identification of stocks in which these two genes were inserted within heterochromatin of the chromosome 4 telomeric region. These transgenes exhibit silencing that is partially suppressed by mutations in the gene encoding HP1. Micrococcal nuclease analysis revealed that the heterochromatic transgenes were packaged in a more regular nucleosome array than when located in euchromatin. High resolution DNase I analysis demonstrated that GAGA factor and TFIID were not associated with these promoters in heterochromatin; potassium permanganate experiments showed a loss of Pol II association. Taken together, these data suggest that occlusion of trans-acting factors from their cis- acting regulatory elements leading to a block in promoter potentiation is a mechanism for heterochromatin gene silencing.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila/genética , Proteínas de Choque Térmico/genética , Heterocromatina/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Primers do DNA , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/metabolismo , TransgenesRESUMO
Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.
Assuntos
Núcleo Celular/genética , Cromatina/química , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Telômero/genética , Animais , Núcleo Celular/metabolismo , Cromatina/genética , Cromossomos/genética , Proteínas de Drosophila , Olho/metabolismo , Feminino , Genes de Insetos/genética , Genes de Insetos/fisiologia , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Masculino , Modelos Genéticos , Estrutura Molecular , Mutação , Fenótipo , Sequências Repetitivas de Ácido Nucleico/genética , Supressão Genética , Telômero/química , Transgenes/genética , Translocação Genética/genéticaAssuntos
Núcleo Celular/ultraestrutura , Cromatina/genética , Cromatina/ultraestrutura , DNA/isolamento & purificação , Drosophila/genética , Trifosfato de Adenosina/metabolismo , Animais , Autorradiografia , Fracionamento Celular/métodos , Pegada de DNA/métodos , Desoxirribonuclease I , Drosophila/embriologia , Proteínas de Drosophila , Eletroforese em Gel de Ágar/métodos , Embrião não Mamífero/ultraestrutura , Genes de Insetos , Proteínas de Choque Térmico/genética , Indicadores e Reagentes , Larva , Nucleossomos/química , Nucleossomos/ultraestrutura , Radioisótopos de Fósforo , Reação em Cadeia da Polimerase/métodos , Mapeamento por Restrição/métodos , Glândulas Salivares/ultraestruturaRESUMO
In a variety of organisms, euchromatic genes brought into juxtaposition with pericentric heterochromatin show position-effect variegation (PEV), a silencing of gene expression in a subset of the cells in which the gene is normally expressed. Previously, a P-element mobilization screen identified transgenic Drosophila stocks showing PEV of an hsp70-white+ reporter gene; transgenes in many of these stocks map to the chromocenter of polytene chromosome. A screen at an elevated temperature identified two stocks that under standard culture temperatures show complete repression of the hsp70-white+ transgene. The transgenes in both cases map to the chromocenter of polytene chromosomes. Different types of middle repetitive elements are adjacent to seven pericentric transgenes; unique sequences are adjacent to two of the perimetric transgenes. All of the transgenes show suppression of PEV in response to a mutation in the gene encoding heterochromatin protein 1 (HP1). This suppression correlates with a more accessible chromatin structure. The results indicate that a pericentric transgene showing PEV can be associated with different types of DNA sequences, while maintaining a common association with the chromosomal protein HP1.
