RESUMO
The three-dimensional (3D) organization of the genome is important for chromatin regulation. This organization is nonrandom and appears to be tightly correlated with or regulated by chromatin state and scaffolding proteins. To understand how specific DNA and chromatin elements contribute to the functional organization of the genome, we developed a new tool-the tagged chromosomal insertion site (TCIS) system-to identify and study minimal DNA sequences that drive nuclear compartmentalization and applied this system to specifically study the role of cis elements in targeting DNA to the nuclear lamina. The TCIS system allows Cre-recombinase-mediated site-directed integration of any DNA fragment into a locus tagged with lacO arrays, thus enabling both functional molecular studies and positional analysis of the altered locus. This system can be used to study the minimal DNA sequences that target the nuclear periphery (or other nuclear compartments), allowing researchers to understand how genome-wide results obtained, for example, by DNA adenine methyltransferase identification, chromosome conformation capture (HiC), or related methods, connect to the actual organization of DNA and chromosomes at the single-cell level. Finally, TCIS allows one to test roles for specific proteins in chromatin reorganization and to determine how changes in nuclear environment affect chromatin state and gene regulation at a single locus.
Assuntos
Cromatina/fisiologia , Mapeamento Cromossômico , Lâmina Nuclear/fisiologia , Animais , Células Cultivadas , Engenharia Genética , Humanos , Integrases/fisiologia , Camundongos , Mutagênese Insercional , Análise de Sequência de DNARESUMO
Many genes localize at the nuclear periphery through physical interaction with the nuclear pore complex (NPC). We have found that the yeast INO1 gene is targeted to the NPC both upon activation and for several generations after repression, a phenomenon called epigenetic transcriptional memory. Targeting of INO1 to the NPC requires distinct cis-acting promoter DNA zip codes under activating conditions and under memory conditions. When at the nuclear periphery, active INO1 clusters with itself and with other genes that share the GRS I zip code. Here, we show that during memory, the two alleles of INO1 cluster in diploids and endogenous INO1 clusters with an ectopic INO1 in haploids. After repression, INO1 does not cluster with GRS I - containing genes. Furthermore, clustering during memory requires Nup100 and two sets of DNA zip codes, those that target INO1 to the periphery when active and those that target it to the periphery after repression. Therefore, the interchromosomal clustering of INO1 that occurs during transcriptional memory is dependent upon, but mechanistically distinct from, the clustering of active INO1. Finally, while localization to the nuclear periphery is not regulated through the cell cycle during memory, clustering of INO1 during memory is regulated through the cell cycle.
RESUMO
Recruitment of genes to the nuclear periphery upon transcriptional activation is a common phenomenon in Saccharomyces cerevisiae. We have recently identified DNA elements called gene recruitment sequences (GRSs) in the promoters of genes that are recruited to the nuclear periphery. These elements are necessary for peripheral targeting of genes. GRSs also function as DNA zip codes: they are sufficient to target an ectopic locus to the nuclear periphery. Targeting promotes full transcription and involves the interaction of promoters with the Nuclear Pore Complex (NPC). GRSs are widespread across the yeast genome, and are enriched in the promoters of genes induced by protein folding stress. Here, we place these observations in the context of the more global topic of genome organization and speculate about how the position of genes impacts their expression.