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SDG2-mediated H3K4 methylation is required for proper Arabidopsis root growth and development.

Yao X, Feng H, Yu Y, Dong A, Shen WH - PLoS ONE (2013)

Bottom Line: Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre.Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance.We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, School of Life Sciences, Fudan University, Shanghai, PR China.

ABSTRACT
Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several Arabidopsis TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the sdg2 mutants, SDG2 function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the sdg2 mutant root phenotype and demonstrate that SDG2 is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the sdg2 mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

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Loss of SDG2 synergistically enhances the CAF1 loss-of-function mutant fas2-4 in causing genome DNA damage but not telomere shortening.A, Representative comet images of the wild-type Col and the mutants sdg2-3, fas2-4 and sdg2-3 fas2-4. Note the intact nucleus at the left and comet tail formed by fragmented nuclear DNA to the right on each panel. B, DNA damage levels as measured by the percentage of DNA in the comet tails of nuclei for the wild-type and mutants. The mean value of more than 100 nuclei is shown with a SD bar. C, Relative expression levels of DNA repair genes determined by quantitative RT-PCR analysis. RNA was prepared from 14-day-old Col, sdg2-3, fas2-4 or sdg2-3 fas2-4 seedlings. RT-PCR was performed using gene-specific primers and normalized using ACTIN2 as reference. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with Col value setting as 1. Bars indicate SD. D, Telomere length comparison between wild-type and mutants. Genomic DNA was digested with MseI, and DNA gel blot analysis was performed using a DIG-labeled telomere repeat as the probe. Note that telomeres are shortened to similar degree in fas2-4 and sdg2-3 fas2-4 but not in sdg2-3 as compared to Col.
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pone-0056537-g007: Loss of SDG2 synergistically enhances the CAF1 loss-of-function mutant fas2-4 in causing genome DNA damage but not telomere shortening.A, Representative comet images of the wild-type Col and the mutants sdg2-3, fas2-4 and sdg2-3 fas2-4. Note the intact nucleus at the left and comet tail formed by fragmented nuclear DNA to the right on each panel. B, DNA damage levels as measured by the percentage of DNA in the comet tails of nuclei for the wild-type and mutants. The mean value of more than 100 nuclei is shown with a SD bar. C, Relative expression levels of DNA repair genes determined by quantitative RT-PCR analysis. RNA was prepared from 14-day-old Col, sdg2-3, fas2-4 or sdg2-3 fas2-4 seedlings. RT-PCR was performed using gene-specific primers and normalized using ACTIN2 as reference. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with Col value setting as 1. Bars indicate SD. D, Telomere length comparison between wild-type and mutants. Genomic DNA was digested with MseI, and DNA gel blot analysis was performed using a DIG-labeled telomere repeat as the probe. Note that telomeres are shortened to similar degree in fas2-4 and sdg2-3 fas2-4 but not in sdg2-3 as compared to Col.

Mentions: Dynamic chromatin and stringent protection of genome integrity are important features of plant SCN. Indeed, histone chaperone (CAF-1, NRPs or ASF1) mutants exhibiting root growth defects also show increased DNA damage [25], [26], [54], [55]. We performed comet assay to investigate the level of DNA damage in WT and mutants sdg2-3, fas2-4 and sdg2-3 fas2-4 seedlings. Figure 7A shows typical nuclei comets observed in WT and mutants. As shown in Figure 7B, the percentage of DNA in comet tails was slightly increased in sdg2-3 and fas2-4 seedlings compared to that of WT. In the sdg2-3 fas2-4 double mutant, the DNA damage level was drastically enhanced (Figure 7B), indicating a synergistic role of SDG2 and CAF-1 in the protection of genome integrity. Consistent with increased levels of DNA damage, several genes involved in DNA repair (including RAD51, RAD51c, RAD54 and PARP1) were activated in the mutants (Figure 7C). The sdg2-3 fas2-4 double mutant behaved relatively similar to the single mutants in DNA repair gene activation (Figure 7C), indicating that the observed gene activation is not quantitatively correlated with DNA damage levels. Finally, we investigated telomere length in WT, sdg2-3, fas2-4 and sdg2-3 fas2-4. Consistent with previous reports [55], [56], fas2-4 caused telomere shortening (Figure 7D). In contrast, sdg2-3 had no detectable effect on telomere length (Figure 7D). These results indicate that SDG2 differs from CAF-1 in regulating genome integrity and chromatin function.


SDG2-mediated H3K4 methylation is required for proper Arabidopsis root growth and development.

Yao X, Feng H, Yu Y, Dong A, Shen WH - PLoS ONE (2013)

Loss of SDG2 synergistically enhances the CAF1 loss-of-function mutant fas2-4 in causing genome DNA damage but not telomere shortening.A, Representative comet images of the wild-type Col and the mutants sdg2-3, fas2-4 and sdg2-3 fas2-4. Note the intact nucleus at the left and comet tail formed by fragmented nuclear DNA to the right on each panel. B, DNA damage levels as measured by the percentage of DNA in the comet tails of nuclei for the wild-type and mutants. The mean value of more than 100 nuclei is shown with a SD bar. C, Relative expression levels of DNA repair genes determined by quantitative RT-PCR analysis. RNA was prepared from 14-day-old Col, sdg2-3, fas2-4 or sdg2-3 fas2-4 seedlings. RT-PCR was performed using gene-specific primers and normalized using ACTIN2 as reference. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with Col value setting as 1. Bars indicate SD. D, Telomere length comparison between wild-type and mutants. Genomic DNA was digested with MseI, and DNA gel blot analysis was performed using a DIG-labeled telomere repeat as the probe. Note that telomeres are shortened to similar degree in fas2-4 and sdg2-3 fas2-4 but not in sdg2-3 as compared to Col.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585709&req=5

pone-0056537-g007: Loss of SDG2 synergistically enhances the CAF1 loss-of-function mutant fas2-4 in causing genome DNA damage but not telomere shortening.A, Representative comet images of the wild-type Col and the mutants sdg2-3, fas2-4 and sdg2-3 fas2-4. Note the intact nucleus at the left and comet tail formed by fragmented nuclear DNA to the right on each panel. B, DNA damage levels as measured by the percentage of DNA in the comet tails of nuclei for the wild-type and mutants. The mean value of more than 100 nuclei is shown with a SD bar. C, Relative expression levels of DNA repair genes determined by quantitative RT-PCR analysis. RNA was prepared from 14-day-old Col, sdg2-3, fas2-4 or sdg2-3 fas2-4 seedlings. RT-PCR was performed using gene-specific primers and normalized using ACTIN2 as reference. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with Col value setting as 1. Bars indicate SD. D, Telomere length comparison between wild-type and mutants. Genomic DNA was digested with MseI, and DNA gel blot analysis was performed using a DIG-labeled telomere repeat as the probe. Note that telomeres are shortened to similar degree in fas2-4 and sdg2-3 fas2-4 but not in sdg2-3 as compared to Col.
Mentions: Dynamic chromatin and stringent protection of genome integrity are important features of plant SCN. Indeed, histone chaperone (CAF-1, NRPs or ASF1) mutants exhibiting root growth defects also show increased DNA damage [25], [26], [54], [55]. We performed comet assay to investigate the level of DNA damage in WT and mutants sdg2-3, fas2-4 and sdg2-3 fas2-4 seedlings. Figure 7A shows typical nuclei comets observed in WT and mutants. As shown in Figure 7B, the percentage of DNA in comet tails was slightly increased in sdg2-3 and fas2-4 seedlings compared to that of WT. In the sdg2-3 fas2-4 double mutant, the DNA damage level was drastically enhanced (Figure 7B), indicating a synergistic role of SDG2 and CAF-1 in the protection of genome integrity. Consistent with increased levels of DNA damage, several genes involved in DNA repair (including RAD51, RAD51c, RAD54 and PARP1) were activated in the mutants (Figure 7C). The sdg2-3 fas2-4 double mutant behaved relatively similar to the single mutants in DNA repair gene activation (Figure 7C), indicating that the observed gene activation is not quantitatively correlated with DNA damage levels. Finally, we investigated telomere length in WT, sdg2-3, fas2-4 and sdg2-3 fas2-4. Consistent with previous reports [55], [56], fas2-4 caused telomere shortening (Figure 7D). In contrast, sdg2-3 had no detectable effect on telomere length (Figure 7D). These results indicate that SDG2 differs from CAF-1 in regulating genome integrity and chromatin function.

Bottom Line: Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre.Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance.We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, School of Life Sciences, Fudan University, Shanghai, PR China.

ABSTRACT
Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several Arabidopsis TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the sdg2 mutants, SDG2 function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the sdg2 mutant root phenotype and demonstrate that SDG2 is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the sdg2 mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

Show MeSH
Related in: MedlinePlus