| Popis: |
The genomes of eukaryotic organisms exist in a highly-organized state compartmentalized within the nucleus, packaged with histones and other proteins in a complex called chromatin. Early discoveries revealed chromatin conformation can affect gene expression by studying transposition of the white gene in Drosophila. Open euchromatic regions of the chromosome arm contained more active genes, while heterochromatic closed regions adjacent to the centromere correlated with gene repression. Since that time, researchers have consistently used reporter genes with measurable gene products such as the green fluorescent protein as tools to study transcriptional activation and repression to uncover the chromatin environment of the reporter gene. Here, we report the creation of a modular Chromatin in vivo Assay designed to explore the influence that differences in the DNA sequence or the aforementioned accessory proteins have on the formation and stability of a heterochromatin state. This assay allows for the recruitment of proteins that can modify the chromatin state of a reporter gene, thus influencing transcriptional output of the gene. We demonstrate that promoter structure, determined by presence or absence of CpG dinucleotides in the DNA sequence, has little influence on the ability of the chromatin effector protein heterochromatin protein 1 (HP1) to induce short-term heterochromatin in mouse embryo fibroblasts as measured by transcriptional silencing and the presence of key histone posttranslational modifications. However, promoter structure does have an impact on the ability of the cells to maintain long-term transcriptional silencing after repeated rounds of cell division, termed heterochromatin memory. We also used this assay to demonstrate that the newly-characterized chromatin effector protein m-phase phosphoprotein 8 (MPP8) is capable of inducing transcriptional silencing, albeit less efficiently than HP1. This assay has proven to be a useful tool in elucidating the proteins and pathways that are required for gene silencing and heterochromatin formation and we anticipate its continued use in characterizing the gene-silencing and chromatin-modifying capabilities of chromatin accessory proteins. |
