RESUMO
A nonrandom radial nuclear organization of genes has been well documented. This study provides further evidence that radial positioning depends on features of corresponding â¼1 Mbp chromatin domains (CDs), which represent the basic units of higher-order chromatin organization. We performed a quantitative three-dimensional analysis of the radial nuclear organization of three genes located on chromosome 1 in a DG75 Burkitt lymphoma-derived cell line. Quantitative real-time polymerase chain reaction revealed similar transcription levels for the three selected genes, whereas the total expression strength (TES) calculated as the sum of transcription of all genes annotated within a surrounding window of about 1 Mbp DNA differed for each region. Radial nuclear position of the studied CDs correlated with TES, i.e., the domain with the highest TES occupied the most interior position. Positions of CDs with stable TES values were stably maintained even under experimental conditions, resulting in genome-wide changes of the expression levels of many other genes. Our results strongly support the hypothesis that knowledge of the local chromatin environment is essential to predict the radial nuclear position of a gene.
Assuntos
Núcleo Celular/ultraestrutura , Montagem e Desmontagem da Cromatina/genética , Cromossomos Humanos Par 1/genética , Expressão Gênica , Genes/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Imageamento Tridimensional , Hibridização in Situ Fluorescente , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.
