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Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements.

Arampatzi P, Gialitakis M, Makatounakis T, Papamatheakis J - Nucleic Acids Res. (2013)

Bottom Line: During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors.In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility.Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, Heraklion 70013, Greece.

ABSTRACT
Transcriptional silencing during mitosis is caused by inactivation of critical transcriptional regulators and/or chromatin condensation. Inheritance of gene expression patterns through cell division involves various bookmarking mechanisms. In this report, we have examined the mitotic and post-mitotic expression of the DRA major histocompatibility class II (MHCII) gene in different cell types. During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors. In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility. Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation.

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PP2A bookmarks the DRA gene for transcription timing upon entry into the G1 phase. (A) RNA was extracted from mitotic HeLa CIITA cells in which siRNA transfection was carried out to knock down PP2Ac gene expression and samples were analysed with qRT-PCR. A scrambled siRNA (siSCR) was used as a negative control. (B) HeLa CIITA cells transfected with siPP2Ac or siSCR blocked in mitotis with nocodazole. Upon release the samples were collected at the indicated time points (0, 2 and 5 h). Flow cytometric analysis of DNA content upon propidium iodide staining is shown on distribution plots. (C) RNA was extracted from the same samples as in (B) and active transcription (DRA and MYC) or total mRNA (CIITA) were measured by qRT-PCR analysis. (D and E) HeLa CIITA cells were mitotically arrested by nocodazole—and then treated with 50 nM okadaic acid for 3 h. DNase I hypersensitivity assay was performed and digestion was measured by qPCR with primers for five different regions on the DRA locus (D), H4 and CD4 promoters (E) in the absence or presence of okadaic acid. Results only from 20 units DNase I treatment are depicted. (F) Schematic representation of the DRA-LCR/XL4 mitotic bookmarking mechanism. Maps are not to scale.
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gks1365-F7: PP2A bookmarks the DRA gene for transcription timing upon entry into the G1 phase. (A) RNA was extracted from mitotic HeLa CIITA cells in which siRNA transfection was carried out to knock down PP2Ac gene expression and samples were analysed with qRT-PCR. A scrambled siRNA (siSCR) was used as a negative control. (B) HeLa CIITA cells transfected with siPP2Ac or siSCR blocked in mitotis with nocodazole. Upon release the samples were collected at the indicated time points (0, 2 and 5 h). Flow cytometric analysis of DNA content upon propidium iodide staining is shown on distribution plots. (C) RNA was extracted from the same samples as in (B) and active transcription (DRA and MYC) or total mRNA (CIITA) were measured by qRT-PCR analysis. (D and E) HeLa CIITA cells were mitotically arrested by nocodazole—and then treated with 50 nM okadaic acid for 3 h. DNase I hypersensitivity assay was performed and digestion was measured by qPCR with primers for five different regions on the DRA locus (D), H4 and CD4 promoters (E) in the absence or presence of okadaic acid. Results only from 20 units DNase I treatment are depicted. (F) Schematic representation of the DRA-LCR/XL4 mitotic bookmarking mechanism. Maps are not to scale.

Mentions: To determine the functional significance of PP2A mediated bookmarking on transcription, we knocked down PP2Ac in HeLa CIITA cells using an siRNA approach. For this, transfected cells were arrested with nocodazole and were subsequently released to proceed to the next G1 phase by removing the inhibitor. An siPP2Ac that specifically reduced PP2Ac mRNA levels (Figure 7A) did not affect the rate of cells entering the G1 phase (Figure 7B) or the kinetics of the CIITA master regulator expression levels, but caused a significant delay in DRA nascent RNA expression (Figure 7C). Conversely, MYC, which is an MLL-bookmarked gene (34), was not affected by PP2A knocked down. Thus, PP2A is preferentially recruited to alternative regulatory regions of the DRA and is required for normal timing of post-mitotic re-initiation of expression. To determine the relation of PP2A recruitment and activity with chromatin condensation, we next examined whether the phosphatase activity affects the chromatin state. For this, we measured the DNase I sensitivity of various regions across the DRA locus using mitotic cells in the absence or presence of the phosphatase inhibitor okadaic acid at levels that selectively inhibits the activity of PP2A (45). Figure 7D shows that both the DRA promoter and the DRA-LCR/XL4 were highly sensitive to digestion in an okadaic-dependent manner similar to the histone H4 promoter, which has been previously shown to lose sensitivity under such conditions (33) (Figure 7E). Two regions of the DRA coding sequence (exons 1 and 5) were also examined and found to be sensitive. Sensitivity was also reduced in the presence of the inhibitor although to a lesser extent. Specificity was shown by the absence of sensitivity in an upstream region of DRA (−2000 bp from the start site) shown in Figure 7D and the silent CD4 gene promoter (Figure 7E). Overall these results assign a novel function to the DRA-LCR/XL4, that of selectively retaining high amounts of factors in mitosis that associate and recruit anti-condensing PP2A activity and facilitate re-initiation of gene expression upon entrance to the next G1 stage.Figure 7.


Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements.

Arampatzi P, Gialitakis M, Makatounakis T, Papamatheakis J - Nucleic Acids Res. (2013)

PP2A bookmarks the DRA gene for transcription timing upon entry into the G1 phase. (A) RNA was extracted from mitotic HeLa CIITA cells in which siRNA transfection was carried out to knock down PP2Ac gene expression and samples were analysed with qRT-PCR. A scrambled siRNA (siSCR) was used as a negative control. (B) HeLa CIITA cells transfected with siPP2Ac or siSCR blocked in mitotis with nocodazole. Upon release the samples were collected at the indicated time points (0, 2 and 5 h). Flow cytometric analysis of DNA content upon propidium iodide staining is shown on distribution plots. (C) RNA was extracted from the same samples as in (B) and active transcription (DRA and MYC) or total mRNA (CIITA) were measured by qRT-PCR analysis. (D and E) HeLa CIITA cells were mitotically arrested by nocodazole—and then treated with 50 nM okadaic acid for 3 h. DNase I hypersensitivity assay was performed and digestion was measured by qPCR with primers for five different regions on the DRA locus (D), H4 and CD4 promoters (E) in the absence or presence of okadaic acid. Results only from 20 units DNase I treatment are depicted. (F) Schematic representation of the DRA-LCR/XL4 mitotic bookmarking mechanism. Maps are not to scale.
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gks1365-F7: PP2A bookmarks the DRA gene for transcription timing upon entry into the G1 phase. (A) RNA was extracted from mitotic HeLa CIITA cells in which siRNA transfection was carried out to knock down PP2Ac gene expression and samples were analysed with qRT-PCR. A scrambled siRNA (siSCR) was used as a negative control. (B) HeLa CIITA cells transfected with siPP2Ac or siSCR blocked in mitotis with nocodazole. Upon release the samples were collected at the indicated time points (0, 2 and 5 h). Flow cytometric analysis of DNA content upon propidium iodide staining is shown on distribution plots. (C) RNA was extracted from the same samples as in (B) and active transcription (DRA and MYC) or total mRNA (CIITA) were measured by qRT-PCR analysis. (D and E) HeLa CIITA cells were mitotically arrested by nocodazole—and then treated with 50 nM okadaic acid for 3 h. DNase I hypersensitivity assay was performed and digestion was measured by qPCR with primers for five different regions on the DRA locus (D), H4 and CD4 promoters (E) in the absence or presence of okadaic acid. Results only from 20 units DNase I treatment are depicted. (F) Schematic representation of the DRA-LCR/XL4 mitotic bookmarking mechanism. Maps are not to scale.
Mentions: To determine the functional significance of PP2A mediated bookmarking on transcription, we knocked down PP2Ac in HeLa CIITA cells using an siRNA approach. For this, transfected cells were arrested with nocodazole and were subsequently released to proceed to the next G1 phase by removing the inhibitor. An siPP2Ac that specifically reduced PP2Ac mRNA levels (Figure 7A) did not affect the rate of cells entering the G1 phase (Figure 7B) or the kinetics of the CIITA master regulator expression levels, but caused a significant delay in DRA nascent RNA expression (Figure 7C). Conversely, MYC, which is an MLL-bookmarked gene (34), was not affected by PP2A knocked down. Thus, PP2A is preferentially recruited to alternative regulatory regions of the DRA and is required for normal timing of post-mitotic re-initiation of expression. To determine the relation of PP2A recruitment and activity with chromatin condensation, we next examined whether the phosphatase activity affects the chromatin state. For this, we measured the DNase I sensitivity of various regions across the DRA locus using mitotic cells in the absence or presence of the phosphatase inhibitor okadaic acid at levels that selectively inhibits the activity of PP2A (45). Figure 7D shows that both the DRA promoter and the DRA-LCR/XL4 were highly sensitive to digestion in an okadaic-dependent manner similar to the histone H4 promoter, which has been previously shown to lose sensitivity under such conditions (33) (Figure 7E). Two regions of the DRA coding sequence (exons 1 and 5) were also examined and found to be sensitive. Sensitivity was also reduced in the presence of the inhibitor although to a lesser extent. Specificity was shown by the absence of sensitivity in an upstream region of DRA (−2000 bp from the start site) shown in Figure 7D and the silent CD4 gene promoter (Figure 7E). Overall these results assign a novel function to the DRA-LCR/XL4, that of selectively retaining high amounts of factors in mitosis that associate and recruit anti-condensing PP2A activity and facilitate re-initiation of gene expression upon entrance to the next G1 stage.Figure 7.

Bottom Line: During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors.In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility.Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, Heraklion 70013, Greece.

ABSTRACT
Transcriptional silencing during mitosis is caused by inactivation of critical transcriptional regulators and/or chromatin condensation. Inheritance of gene expression patterns through cell division involves various bookmarking mechanisms. In this report, we have examined the mitotic and post-mitotic expression of the DRA major histocompatibility class II (MHCII) gene in different cell types. During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors. In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility. Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation.

Show MeSH
Related in: MedlinePlus