RESUMO
Position-effect variegation (PEV) results when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition. When heterochromatin packaging spreads across the heterochromatin/euchromatin border, it causes transcriptional silencing in a stochastic pattern. PEV is intensely studied in Drosophila using the white gene. Screens for dominant mutations that suppress or enhance white variegation have identified many conserved epigenetic factors, including the histone H3 lysine 9 methyltransferase SU(VAR)3-9. Heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9, creating a core memory system. Genetic, molecular, and biochemical analysis of PEV in Drosophila has contributed many key findings concerning establishment and maintenance of heterochromatin with concomitant gene silencing.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Efeitos da Posição Cromossômica/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas do Olho/genética , Rearranjo Gênico/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Modelos Genéticos , Animais , Efeitos da Posição Cromossômica/fisiologia , Drosophila/fisiologia , Rearranjo Gênico/fisiologia , Heterocromatina/fisiologia , Mutação/genéticaRESUMO
The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs) and is thought to require de novo association with transcription factories. We identify two types of factory: "poised transcription factories," containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from "active factories" associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA) gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p(+)S2p(-)) factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p(+)S2p(+)) factories and loops out from its CT. However, the levels of gene association with poised or active transcription factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised transcription factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction.
Assuntos
Inativação Gênica/fisiologia , Ativação Transcricional/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Anticorpos/imunologia , Montagem e Desmontagem da Cromatina/fisiologia , Efeitos da Posição Cromossômica/genética , Efeitos da Posição Cromossômica/fisiologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes/genética , Loci Gênicos/genética , Loci Gênicos/fisiologia , Células Hep G2 , Humanos , Metaloendopeptidases/fisiologia , RNA Polimerase II/fisiologia , Ativação Transcricional/genética , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to approximately 20% of the level in wild-type larvae, lethality occurs in the late larval - pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me(2) and H4K20Me(2). Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo.