Abstract
The human genome must be packaged and organized in a functional manner for the regulation of DNA replication and transcription. The nuclear scaffold/matrix, consisting of structural and functional nuclear proteins, remains after extraction of nuclei and anchors loops of DNA. In the search for cis-elements functioning as chromatin domain boundaries, we identified 453 nuclear scaffold attachment sites purified by lithium-3,5-iodosalicylate extraction of HeLa nuclei across 30 Mb of the human genome studied by the ENCODE pilot project. The scaffold attachment sites mapped predominately near expressed genes and localized near transcription start sites and the ends of genes but not to boundary elements. In addition, these regions were enriched for RNA polymerase II and transcription factor binding sites and were located in early replicating regions of the genome. We believe these sites correspond to genome-interactions mediated by transcription factors and transcriptional machinery immobilized on a nuclear substructure.
Highlights
The eukaryotic nucleus has a complex architecture in which the genome must be packaged into higher ordered structures in an organized and accessible way that allows for the dynamic processes of DNA transcription, replication, and repair
To monitor the quality of the fractionation, equal amounts of scaffold DNA and total genomic DNA was used as template for quantitative PCR to assess enrichment of the ApoB 39MAR and a negative region within the ApoB gene that had previously been characterized in HeLa cells [11] (Fig. 1C)
We report the identification of 453 biochemically-defined nuclear scaffold attachment regions within 30 Mb of the human genome
Summary
The eukaryotic nucleus has a complex architecture in which the genome must be packaged into higher ordered structures in an organized and accessible way that allows for the dynamic processes of DNA transcription, replication, and repair. While the mechanisms driving large-scale organization are unknown, it is well demonstrated that chromosomes occupy specific spatial territories within the nucleus that are positioned so that active and repressed regions of the genome often occupy different sub-nuclear compartments [1,2] Within these compartments, chromatin is believed to be organized into functional domains whose chromatin structures are marked by differential epigenetic modifications allowing for the proper regulation of gene expression [3]. Loops of DNA emanate from the residual nuclear structure, and enzymatic digestion reveals tightly associated sequences referred to as scaffold or matrix attachment regions (S/MARs) These attachment sites are believed to form the base of chromatin loops in vivo and to have functional consequences on genome organization and regulation. DNA replication and transcription activity is associated with the nuclear scaffold/matrix [16,17,18,19]; origins of replication and expressed genes are attached in scaffold/matrix preparations [20,21]
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