Altered association of transcriptionally active DNA with the nuclear-matrix after heat shock

Abstract

Purpose: Exposure of human cells to heat leads to denaturation and aggregation of proteins. Within the nucleus, it has been suggested that protein aggregation is linked to the selective inhibition by hyperthermia of nucleotide excision repair in transcriptionally active genes. In this study it was investigated in detail whether and how the inhibition of repair of transcriptionally active genes might be related to alterations in their association with the nuclear-matrix. Material and methods: Different protocols for nuclear-matrix isolation (high salt and lithium 3',5'-diiodosalycilate [LIS] extraction of nuclei) were used to compare DNA loop organization and positioning of transcriptionally active genes in both heated and non-heated cells. Results: DNaseI digestion of total genomic DNA in Cu2+- stabilized LIS-extracted nuclei revealed that heat shock perturbed the formation of nuclear-matrix attachment sites. Specific labelling of active genes indicated that the number of nuclear-matrix attachment sites in transcriptionally active DNA was increased due to the heat shock. At the level of individual genes, heat treatment led to stabilization of the 5' matrix attachment site (MAR) in the transcriptionally active adenosine deaminase (ADA) housekeeping gene. Moreover, heat shock resulted in the formation of an additional MAR at the 3' end of the ADA gene. The inactive 754 locus was unassociated, irrespective of a heat shock. Conclusions: The reported changes in chromatin structure might underlie the selective inhibition of repair in transcriptionally active genes and consequently may be mechanistically linked to the sensitization of heated cells to ionizing radiation.