379 TREATMENT OF 293T CELLS WITH XENOPUS EGG EXTRACT INDUCES NOT-STRESS-RELATED EPIGENETIC REMODELING OF RIBOSOMAL GENES

Abstract

Undefined mechanisms involved in reprogramming of somatic cells by Xenopus leavis egg extract have, to date, prevented clinical applications for cell replacement therapy. The aim of this study was to evaluate the immediate response of somatic cells to exposure to Xenopus leavis egg extract using ribosomal genes (rDNA) as a sensitive marker for stress and/or chromatin remodeling. Human epithelial 293T cells in standard culture (control), starved in glucose-free medium (stress-control), or treated for 1 h with egg extract were fixed at 6 and 24 h after treatment and analyzed for pre-rRNA synthetic activity by quantitative RT-PCR; level of H3K9me3 by immunofluorescence; and for occupancy of rDNA promoter by markers of normal activity (UBF), stress silencing (SIRT1, SUV39H1), and remodeling (SNF2H) using chromatin immunoprecipitation. Relative levels of pre-rRNA decreased in all treated groups to 20 to 35% of control levels. Chromatin immunoprecipitation did not reveal any significant effect of starvation or extract treatment on UBF and SIRT1 binding to the rDNA promoter, presumably because of the pleiotropic property of these proteins. However, starvation specifically led to enhanced SUV39H1 binding 6 h after treatment, indicating early cell response to stress. No differences were found in SUV39H1 binding between extract-treated cells and control. In contrast, binding of SNF2H at 6 h increased in extract-treated cells but decreased in starved cells. The relative level of H3K9me3 increased first after 24 h of starvation equally in all cells, suggesting later involvement of this histone modification in rDNA silencing. Extract treatment, however, led to a decrease in H3K9me3 level at 6 h, and after 24 h, 2 main cell populations were observed: one (85.4% of cells) that retained decreased H3K9me3 and one (12.4% of cells) with significantly enhanced levels. Moreover, H3K9me3 foci in the last group were associated with the periphery of presumptive nucleoli. Thus, extract treatment apparently does not initiate a stress-induced silencing of the rDNA genes; rather, it activates SNF2H-dependent chromatin remodeling, resulting in a long-term decrease in pre-rRNA synthetic activity.

The research was supported by Carlsberg Foundation 2008-01-0105.