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. 2013 Feb;250(1):251-9.
doi: 10.1007/s00709-012-0402-x. Epub 2012 Apr 15.

Increased transcription in hydroxyurea-treated root meristem cells of Vicia faba

Konrad Winnicki  1 , Justyna Teresa Polit, Janusz Maszewski
Affiliations

Increased transcription in hydroxyurea-treated root meristem cells of Vicia faba

Konrad Winnicki et al. Protoplasma. 2013 Feb.

Abstract

Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, prevents cells from progressing through S phase by depletion of deoxyribonucleoside triphosphates. Concurrently, disruption of DNA replication leads to double-strand DNA breaks. In root meristems of Vicia faba, HU triggers cell cycle arrest (preferentially in G1/S phase) and changes an overall metabolism by global activation of transcription both in the nucleoplasmic and nucleolar regions. High level of transcription is accompanied by an increase in the content of RNA polymerase II large subunit (POLR2A). Changes in transcription activation and POLR2A content correlate with posttranslational modifications of histones that play a role in opening up chromatin for transcription. Increase in the level of H4 Lys5 acetylation indicates that global activation of transcription following HU treatment depends on histone modifications.

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Figures

Fig. 1
Fig. 1
Frequency distribution (percentage) of nuclear DNA contents in the control (a) and in HU-treated cells (b); nuclear DNA Feulgen staining; a.u. arbitrary units
Fig. 2
Fig. 2
Median fluorescence intensity (a.u. arbitrary units) in the nucleoplasmic region evaluated following 5-EU incorporation into root tip cells from seedlings incubated in H2O and HU; successive phases of the cell cycle in the control plants denoted as Control-G1, Control-S, and Control-G2 while in seedlings incubated in 2.5 mM HU (24 h) denoted as HU-G1, HU-S, and HU-G2. Statistical significance (Kruskal–Wallis test): *p < 0.001 control-G1/HU-G1, control-S/HU-S, control-G2/HU-G2; # p < 0.001 control-G1/control-S, control-G1/control-G2, HU-G1/HU-S, HU-G1/HU-G2; ^ p < 0.001 control-S/control-G2
Fig. 3
Fig. 3
Median fluorescence intensity (a.u. arbitrary units) in the nucleoli evaluated following 5-EU incorporation into root tip cells from seedlings incubated in H2O and HU; successive phases of the cell cycle in the control plants denoted as Control-G1, Control-S, and Control-G2 while in seedlings incubated in 2.5 mM HU (24 h) denoted as HU-G1, HU-S, and HU-G2. Statistical significance (Kruskal–Wallis test): *p < 0.001 control-G1/HU-G1, control-S/HU-S, control-G2/HU-G2; # p < 0.001 control-G1/control-G2; ^ p < 0.001 control-S/control-G2
Fig. 4
Fig. 4
Cytochemical detection of transcription following 5-EU incorporation. a Negative control (without 5-EU), b incubation in H2O, c 24 h incubation with 2.5 mM HU. Bar 50 μm
Fig. 5
Fig. 5
Selected cell nuclei showing intense 5-EU incorporation. a Incubation in H2O, b 24-h incubation with 2.5 mM HU; a’, b’ nuclei stained with DAPI. Arrows point to heterochromatic regions. Bar 10 μm
Fig. 6
Fig. 6
Immunocytochemical detection of polymerase RNA II large subunit (POLR2A). a incubation in H20, b 24 h incubation with 2.5 mM HU; a’, b’ nuclei stained with DAPI. Arrows point to heterochromatic regions. Bar 10 μm
Fig. 7
Fig. 7
Immunoblotting analysis of POLR2A in whole-cell extracts (a) and nuclear protein lysates (b). Molecular weight marker (kilodalton; lane A), electrophoresis and Coomassie staining (lane B), extracts from control seedlings (lane C) and HU-treated seedlings (lane D) immunoblotted with anti-POLR2A (CTD) antibodies
Fig. 8
Fig. 8
Median immunofluorescence intensity (a.u. arbitrary units) evaluated using anti-large subunit of RNA polymerase II (POLR2A) antibodies in seedlings incubated in H2O and HU. Successive phases of the cell cycle in the control plants denoted as Control-G1, Control-S, and Control-G2, while in seedlings incubated in 2.5 mM HU (24 h) denoted as HU-G1, HU-S, and HU-G2. Statistical significance (Kruskal–Wallis test): *p < 0.001 control-G1/HU-G1, *p < 0.001 control-S/HU-S; # p < 0.001 control-G1/control-G2; ^ p = 0.04 control-S/control-G2
Fig. 9
Fig. 9
Immunocytochemical detection of histone H4 Lys5 acetylation. a Incubation in H20, b 24-h incubation with 2.5 mM HU; a’, b’ nuclei stained with DAPI. Arrows point to heterochromatic regions. Bar 10 μm
Fig. 10
Fig. 10
Median immunofluorescence intensity (a.u. arbitrary units) evaluated following detection of histone H4 acetylated Lys5 in seedlings incubated in H2O and HU. Successive phases of the cell cycle in the control plants denoted as Control-G1, Control-S, and Control-G2, while in seedlings incubated in 2.5 mM HU (24 h) denoted as HU-G1, HU-S, and HU-G2. Statistical significance (Kruskal–Wallis test): *p < 0.001 control-G1/HU-G1, control-S/HU-S, control-G2/HU-G2; # p < 0.001 control-G1/control-S, control-G1/control-G2, HU-G1/HU-S, HU-G1/HU-G2
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