RESUMO
In higher eukaryotes, distance enhancer-promoter interactions are organized by topologically associated domains, tethering elements, and chromatin insulators/boundaries. While insulators/boundaries play a central role in chromosome organization, the mechanisms regulating their functions are largely unknown. In the studies reported here, we have taken advantage of the well-characterized Drosophila bithorax complex (BX-C) to study one potential mechanism for controlling boundary function. The regulatory domains of BX-C are flanked by boundaries, which block crosstalk with their neighboring domains and also support long-distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether readthrough transcription (RT) can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7attP50 and F2attP, in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters, and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that RT can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is therefore possible that RT may be a widely used mechanism to alter boundary function and regulation of gene expression.
Assuntos
Proteínas de Drosophila , RNA Longo não Codificante , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/genética , Genes de Insetos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
Alternative splicing (AS) is a regulatory mechanism of gene expression that greatly expands the coding capacities of genomes by allowing the generation of multiple mRNAs from a single gene. In Drosophila, the mod(mdg4) locus is an extreme example of AS that produces more than 30 different mRNAs via trans-splicing that joins together the common exons and the 3' variable exons generated from alternative promoters. To map the regions required for trans-splicing, we have developed an assay for measuring trans-splicing events and identified a 73-bp region in the last common intron that is critical for trans-splicing of three pre-mRNAs synthesized from different DNA strands. We have also found that conserved sequences in the distal part of the last common intron induce polyadenylation-independent transcription termination and are enriched by paused RNA polymerase II (RNAP II). These results suggest that all mod(mdg4) mRNAs are formed by joining in trans the 5' splice site in the last common exon with the 3' splice site in one of the alternative exons.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insetos , Íntrons/genética , Precursores de RNA/biossíntese , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Trans-Splicing , Fatores de Transcrição/genética , Terminação da Transcrição Genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Imunoprecipitação da Cromatina , Sequência Conservada , Proteínas de Drosophila/metabolismo , Éxons/genética , Genes Reporter , Masculino , Poli A , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA Mensageiro/metabolismoRESUMO
Expression of the reporter gene in transgenic animals depends on the surrounding chromatin environment. Recent genome-wide studies have shown that, in mammals, the entire genome is transcribed. Transcription through a transgene often has a negative effect on the expression of a reporter gene. Here, we compared the ability of well-studied chicken chromatin insulator HS4 and bidirectional transcription terminators from the human genome to support high-level expression of the firefly luciferase gene (Fluc) under control of the previously characterized goat ß-casein gene promoter. The insertion of HS4 or either of the two transcription terminators upstream of the promoter resulted in tenfold enhancement of Fluc expression in the mammary glands of transgenic mice. These results suggest that transcriptional terminators, similar to the HS4 insulator, can be used to improve the reporter gene expression in transgenic animals.
Assuntos
Caseínas/genética , Luciferases de Vaga-Lume/metabolismo , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Regiões Promotoras Genéticas , Terminação da Transcrição Genética , Transgenes/fisiologia , Animais , Galinhas , Feminino , Vetores Genéticos , Cabras , Humanos , Elementos Isolantes , Luciferases de Vaga-Lume/genética , Camundongos , Camundongos Transgênicos , Transgenes/genéticaRESUMO
In higher eukaryotes enhancer-promoter interactions are known to be restricted by the chromatin insulators/boundaries that delimit topologically associated domains (TADs); however, there are instances in which enhancer-promoter interactions span one or more boundary elements/TADs. At present, the mechanisms that enable cross-TAD regulatory interaction are not known. In the studies reported here we have taken advantage of the well characterized Drosophila Bithorax complex (BX-C) to study one potential mechanism for controlling boundary function and TAD organization. The regulatory domains of BX-C are flanked by boundaries which function to block crosstalk with their neighboring domains and also to support long distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether transcriptional readthrough can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7 attP50 and F2 attP , in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that transcriptional readthrough can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is possible that many of them may function in the regulation of TAD organization. Author Summary: Recent studies have shown that much genome in higher eukaryotes is transcribed into non-protein coding lncRNAs. It is though that lncRNAs may preform important regulatory functions, including the formation of protein complexes, organization of functional interactions between enhancers and promoters and the maintenance of open chromatin. Here we examined how transcription from promoters inserted into the Drosophila Bithorax complex can impact the boundaries that are responsible for establishing independent regulatory domains. Surprisingly, we found that even a relatively low level of transcriptional readthrough can impair boundary function. Transcription also affects the activity of enhancers located in BX-C regulatory domains. Taken together, our results raise the possibility that transcriptional readthrough may be a widely used mechanism to alter chromosome structure and regulate gene expression.