Chromatin remodeling induced by histone deacetylase inhibitors (HDACis) in HeLa, NIH 3T3 and HepG2 cells

摘要

BackgroundIn eukaryotic cells, chromatin results from the associationof DNA with nuclear proteins packaged into hierarchies oforganization. The DNA packaging into chromatin is generallyrecognized as a major mechanism by which theaccess of genomic DNA is restricted. The physical barrierto the underlying DNA is precisely regulated, at least inpart, by the post-translational modifications of histones.Two of the most studied modifications are the acetylationand deacetylation of lysine residues in the nucleosomecore histones, under the control of histone acetyltransferasesand histone deacetylases, respectively. HDACissuch as valproic acid (VPA) and trichostatin A (TSA)induce acetylation of histones and non-histone proteins,which is correlated with nucleosome remodeling and transcriptionalactivation [1]. In some cases the DNA methylationstate and presence of chromatin-associated nonhistoneproteins are also affected by HDACis [2,3]. Herewe investigated by image analysis procedures whetherVPA and TSA, in consequence of their epigenetic action,would affect chromatin supraorganization in different celllines.Materials and methodsHeLa and HepG2 tumoral cells and non-tumoral NIH3T3 fibroblasts were cultivated for 24 h, treated with0.05, 0.5 and 1.0 mM VPA or 10, 20 and 100 ng/mL TSA(1-24 h), and subjected to the Feulgen reaction. The texturalchanges in the chromatin structure of these cellswere studied by scanning microspectrophotometry [4].Decrease in HDAC activity and increase in acetylation ofhistones H3 and H4 were also considered. The effect ofthe HDACis on DNA methylation of HeLa cells wasinvestigated by image analysis. HDACi-induced depletionof the heterochromatin protein 1 (HP1) from NIH 3T3cells was investigated by immunocytochemistry.ResultsNuclear areas covered by “condensed” chromatin, whichwere abundant in untreated controls, appeared reduced inVPA- and TSA-treated cells. Scatter diagrams in whichnuclear relative areas covered with condensed chromatin(Sc %) matched the level of textural contrast between condensedand non-condensed chromatin (AAR), revealeddecreased Sc% values with increasing AAR values underall the tested treatments. Work in progress suggests DNAdemethylation in HeLa cells. Heterochromatin unpackagingand depletion of HP1 from heterochromatin weredetected in most NIH 3T3 cell nuclei.ConclusionsChanges in chromatin supraorganization are induced inthe cell types analyzed after inhibition of histone deacetylasesby VPA and TSA, possibly accompanied by changesin gene expression. The chromatin remodeling promotedby these HDACis is also suggested to be affected by DNAdemethylation in HeLa cells and histone H3K9me3 hypomethylationin NIH 3T3 cells.