Colocalization of PML bodies and PML/RARalpha microspeckles with up- and down-regulated loci and changes of chromatin structure in APL leukemia cells
| Authors | |
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| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | Biophysics of the Genome |
| MU Faculty or unit | |
| Citation | |
| Field | Genetics and molecular biology |
| Keywords | Cytometry |
| Description | The reciprocal translocation between PML and RARalpha genes associated with acute promyelocytic leukemia (APL) results in disintegration of PML bodies and expression of the fusion protein PML/RARalpha in transformed APL cells. Transcription intensity of particular genes were compared between Zn-treated (APL) and untreated (healthy) U937 PIR9 cells, stably transfected by the expression vector carrying the PML/RARalpha fusion gene under the control of Zn-inducible promoter; downregulation of many genes clustered in the locus 19p13.3 and upregulation of some of those in Xq28-Xqter were revealed by microchip analysis and RT-PCR after zinc treatment. We have examined the colocalization of these up- (Xq28-qter)-regulated (19p13.3)chromosomal loci with PML bodies in normal and PML/RARalpha microspeckles in transformed APL cells respectively, as well as the changes in chromatin structure of these loci induced in APL cells by PML/RARalpha-altered transcription. Most frequently, colocalization or very close proximity (< PML body diameter) of the one of two homologous loci was observed for loci 19p13.3 (more than 50%) and Xq28-Xqter (25-35%), contrary to the control locus 13q22.3 (containing none clusters of PML/RARalpha-regulated genes) where colocalization was found in only 5-6%. On the other hand, both homologous loci 19p13.3 usually colocalized with PML/RARalpha microspeckles (more than 70% of nuclei) after disintegration of PML bodies in zinc treated PIR9 cells (APL cells). After all-trans retinoic acid (ATRA) treatment of APL cells, reconstituted PML bodies and colocalization frequencies resembling those in normal cells reappeared. Conversely, frequency of colocalizing loci 13q22.3 and Xq28-Xqter remained very similar in normal, APL and ATRA-treated APL cells. The degree of chromatin compaction was evaluated according to the mutual 3D distances between the pairs of DNA probes separated by short molecular distances (1.5-4 Mbp), for the first time in natural DNA sequences. The distances between the probes in the down-regulated region 19p13.3 were significantly shortened (about 15-20%) in APL cells, whereas remained similar for the cen17-17q11.2 (control) and Xq28-Xqter (upregulated)loci. After ATRA treatment the level of chromatin condensation in 19p13.3 locus reverted to original values in normal cells. These results together with the observed high colocalization frequency of PML/RARalpha with the downregulated locus 19p13.3 support the mechanistic model of gene silencing in APL, where binding of PML/RARalpha to the particular loci results in strong attraction of HDACs, consequent histone deacethylation, chromatin condensation and gene silencing. Not conclusive rule of proportion between transcription intensity and chromatin condensation together with unchanged radial distances of loci (nuclear center-to-locus distances) after their activation/silencing however indicate that the higher-order chromatin structure is not necessarily influenced by transcription process. On the other hand, upregulation of PML/RARalpha- activated genes is obviously not caused by direct PML/RARalpha binding to their DNA sequences. |
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