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Regulation of the DNA damage response and gene expression by the Dot1L histone methyltransferase and the 53Bp1 tumour suppressor.

FitzGerald J, Moureau S, Drogaris P, O'Connell E, Abshiru N, Verreault A, Thibault P, Grenon M, Lowndes NF - PLoS ONE (2011)

Bottom Line: These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression.In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed.To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.

View Article: PubMed Central - PubMed

Affiliation: Genome Stability Laboratory, School of Natural Sciences, Centre for Chromosome Biology, National University of Ireland Galway, Galway, Ireland.

ABSTRACT

Background: Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.

Methodology/principal findings: Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.

Conclusions/significance: Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.

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Generation of Dot1L deficient cell lines.A. Schematic of the strategy to disrupt the chicken Dot1L SAM binding domain and confirm the deletion by Southern blotting. Exons are numbered. Bsr: blasticidin resistance gene; Hph: hygromycin resistance gene. EV: EcoRV restriction sites. Grey rectangle: probe for Southern blots. The figure is approximately drawn to scale. B. Southern blot to confirm targeted integration of the Dot1L disruption constructs. Digestion of the WT Dot1L locus with EcoRV generates a band of 13.2 kb when hybridised with the probe shown in A. Following targeted integration of the blasticidin construct, the size of the band increases to 16.4 kb. Targeted integration of the hygromycin construct results in a band of 11.4 kb. C. Reverse transcription PCR of the indicated exons of Dot1L in WT and Dot1Lmd/md cells. D. RT-qPCR to quantify the levels of Dot1L RNA in WT and Dot1Lmd/md cell lines.
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pone-0014714-g001: Generation of Dot1L deficient cell lines.A. Schematic of the strategy to disrupt the chicken Dot1L SAM binding domain and confirm the deletion by Southern blotting. Exons are numbered. Bsr: blasticidin resistance gene; Hph: hygromycin resistance gene. EV: EcoRV restriction sites. Grey rectangle: probe for Southern blots. The figure is approximately drawn to scale. B. Southern blot to confirm targeted integration of the Dot1L disruption constructs. Digestion of the WT Dot1L locus with EcoRV generates a band of 13.2 kb when hybridised with the probe shown in A. Following targeted integration of the blasticidin construct, the size of the band increases to 16.4 kb. Targeted integration of the hygromycin construct results in a band of 11.4 kb. C. Reverse transcription PCR of the indicated exons of Dot1L in WT and Dot1Lmd/md cells. D. RT-qPCR to quantify the levels of Dot1L RNA in WT and Dot1Lmd/md cell lines.

Mentions: Cell lines were created in the DT40 wild-type background, also known as WT18. Dot1L−/− and 53Bp1−/−Dot1L−/− cell lines were also generated in the Cre1-DT40 WT cell line [40], [41] to facilitate future generation of multiple knockout cell lines. Cre1-DT40 WT cells express the Cre1 recombinase that can translocate into the nucleus in response to 4-hydroxytamoxifen. This recombinase [42] will recognise tandem repeats of LoxP sites and allow the removal of DNA located between the two repeats. This property allows multiple use of the same selective cassette surrounded by LoxP sites (Figure 1A). Note that the Cre1-DT40 WT cell line has some limitations including a defective response to nocodazole and an ionizing radiation-induced centrosome amplification phenotype [43]. Importantly, Cre1-DT40 WT cells display the same survival rate [43] and γH2AX/53Bp1 focal accumulation as WT18 cells (Figures S6 and S7). All experiments shown in the manuscript were performed with cell lines in the WT18 background. The only exception was the quantification of γH2AX and 53Bp1 foci formation, which were performed in Cre1-DT40 WT and Cre1-DT40 Dot1L−/− cells in comparison with WT18 cells (Figures 4, S6 and S7). However, similar to the Cre1-DT40 Dot1L−/− cells, both γH2AX and 53Bp1 foci were obtained in the WT18 Dot1L−/− cell line (data not shown).


Regulation of the DNA damage response and gene expression by the Dot1L histone methyltransferase and the 53Bp1 tumour suppressor.

FitzGerald J, Moureau S, Drogaris P, O'Connell E, Abshiru N, Verreault A, Thibault P, Grenon M, Lowndes NF - PLoS ONE (2011)

Generation of Dot1L deficient cell lines.A. Schematic of the strategy to disrupt the chicken Dot1L SAM binding domain and confirm the deletion by Southern blotting. Exons are numbered. Bsr: blasticidin resistance gene; Hph: hygromycin resistance gene. EV: EcoRV restriction sites. Grey rectangle: probe for Southern blots. The figure is approximately drawn to scale. B. Southern blot to confirm targeted integration of the Dot1L disruption constructs. Digestion of the WT Dot1L locus with EcoRV generates a band of 13.2 kb when hybridised with the probe shown in A. Following targeted integration of the blasticidin construct, the size of the band increases to 16.4 kb. Targeted integration of the hygromycin construct results in a band of 11.4 kb. C. Reverse transcription PCR of the indicated exons of Dot1L in WT and Dot1Lmd/md cells. D. RT-qPCR to quantify the levels of Dot1L RNA in WT and Dot1Lmd/md cell lines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044716&req=5

pone-0014714-g001: Generation of Dot1L deficient cell lines.A. Schematic of the strategy to disrupt the chicken Dot1L SAM binding domain and confirm the deletion by Southern blotting. Exons are numbered. Bsr: blasticidin resistance gene; Hph: hygromycin resistance gene. EV: EcoRV restriction sites. Grey rectangle: probe for Southern blots. The figure is approximately drawn to scale. B. Southern blot to confirm targeted integration of the Dot1L disruption constructs. Digestion of the WT Dot1L locus with EcoRV generates a band of 13.2 kb when hybridised with the probe shown in A. Following targeted integration of the blasticidin construct, the size of the band increases to 16.4 kb. Targeted integration of the hygromycin construct results in a band of 11.4 kb. C. Reverse transcription PCR of the indicated exons of Dot1L in WT and Dot1Lmd/md cells. D. RT-qPCR to quantify the levels of Dot1L RNA in WT and Dot1Lmd/md cell lines.
Mentions: Cell lines were created in the DT40 wild-type background, also known as WT18. Dot1L−/− and 53Bp1−/−Dot1L−/− cell lines were also generated in the Cre1-DT40 WT cell line [40], [41] to facilitate future generation of multiple knockout cell lines. Cre1-DT40 WT cells express the Cre1 recombinase that can translocate into the nucleus in response to 4-hydroxytamoxifen. This recombinase [42] will recognise tandem repeats of LoxP sites and allow the removal of DNA located between the two repeats. This property allows multiple use of the same selective cassette surrounded by LoxP sites (Figure 1A). Note that the Cre1-DT40 WT cell line has some limitations including a defective response to nocodazole and an ionizing radiation-induced centrosome amplification phenotype [43]. Importantly, Cre1-DT40 WT cells display the same survival rate [43] and γH2AX/53Bp1 focal accumulation as WT18 cells (Figures S6 and S7). All experiments shown in the manuscript were performed with cell lines in the WT18 background. The only exception was the quantification of γH2AX and 53Bp1 foci formation, which were performed in Cre1-DT40 WT and Cre1-DT40 Dot1L−/− cells in comparison with WT18 cells (Figures 4, S6 and S7). However, similar to the Cre1-DT40 Dot1L−/− cells, both γH2AX and 53Bp1 foci were obtained in the WT18 Dot1L−/− cell line (data not shown).

Bottom Line: These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression.In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed.To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.

View Article: PubMed Central - PubMed

Affiliation: Genome Stability Laboratory, School of Natural Sciences, Centre for Chromosome Biology, National University of Ireland Galway, Galway, Ireland.

ABSTRACT

Background: Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.

Methodology/principal findings: Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.

Conclusions/significance: Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.

Show MeSH
Related in: MedlinePlus