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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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Cell type-specific variation of mitotic DRA transcriptional activity. (A) Total RNA was extracted from asynchronous and mitotic Raji, HeLa CIITA and RJ2.2.5 CIITA cells and active transcription of the DRA gene was analysed with qRT-PCR using two primer sets (DRA exon5–exon5 and DRA exon1–intron1). RNA from untreated and DRB-treated cells was used to measure net active transcription. These primer sets that monitor unspliced (nascent) or spliced (mature/total) transcripts were normalized for differences of their amplification efficiencies using a standard curve of a genomic DNA template. Newly synthesized RNA is presented as percent of mature DRA mRNA. (B) DNA content analysis by PI and flow cytometry of RJ2.2.5 cells expressing CIITA under the control of rtTA-GBD and Dex-Dox treatment (see materials and methods). Cells were blocked by nocodazole prior to CIITA induction by a 6-h treatment with Dex–Dox. (C) RNA was extracted from control and induced, asynchronous and mitotic RJ2.2.5 tetCIITA cells. DRA and CIITA gene expression was analysed by qRT-PCR. (D) Chromatin was extracted from the same samples as in (C), and ChIP assays against RFX5 and CIITA were performed.
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gks1365-F4: Cell type-specific variation of mitotic DRA transcriptional activity. (A) Total RNA was extracted from asynchronous and mitotic Raji, HeLa CIITA and RJ2.2.5 CIITA cells and active transcription of the DRA gene was analysed with qRT-PCR using two primer sets (DRA exon5–exon5 and DRA exon1–intron1). RNA from untreated and DRB-treated cells was used to measure net active transcription. These primer sets that monitor unspliced (nascent) or spliced (mature/total) transcripts were normalized for differences of their amplification efficiencies using a standard curve of a genomic DNA template. Newly synthesized RNA is presented as percent of mature DRA mRNA. (B) DNA content analysis by PI and flow cytometry of RJ2.2.5 cells expressing CIITA under the control of rtTA-GBD and Dex-Dox treatment (see materials and methods). Cells were blocked by nocodazole prior to CIITA induction by a 6-h treatment with Dex–Dox. (C) RNA was extracted from control and induced, asynchronous and mitotic RJ2.2.5 tetCIITA cells. DRA and CIITA gene expression was analysed by qRT-PCR. (D) Chromatin was extracted from the same samples as in (C), and ChIP assays against RFX5 and CIITA were performed.

Mentions: The above results show that the MCE-CIITA-GTM complex in mitosis is maintained in lymphoblastoid but not in non-lymphoblastoid cells. We next addressed the question whether transcription is sustained during mitosis. In most systems studied, association of regulatory factors with DNA does not ensure active gene transcription because either not all essential factors remain bound to chromatin or PTMs such as phopshorylation may inactivate critical factors (18). To this end, we quantified total DRA RNA by qRT-PCR of asynchronous or nocodazole-arrested cells by using exon5–exon5 primer set and newly synthesized RNA by using exon1–intron1 primer set on the same sample in the presence or absence of the transcription elongation inhibitor DRB. To express the latter as a fraction of the mature RNA, we used genomic DNA as a standard to account for differences in amplification efficiencies between the above two primer sets. New RNA synthesis represented 4–5.5% of the total DRA RNA in the asynchronously growing cells shown in Figure 4A. In mitosis newly synthesized DRA RNA was reduced to 33% and 12% of the asynchronous levels of Raji or HeLa CIITA cells, respectively. To correlate active RNA synthesis with the recruitment of RNA PolII in Raji cells, we used ChIP. Results in Supplementary Figure S6 (A and C) show that promoter- or exon-bound (elongating) RNA PolII levels on DRA gene correlate well with the qRT-PCR-measured transcription in the different asynchronous or mitotic cells. Comparison of promoter and exon levels further indicates that there is direct correlation of initiation and elongation events without stalling of PolII in the different set ups. For specificity control, results for CyclinB1 and GAPDH genes are also shown (Supplementary Figure S6B and D).Figure 4.


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

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

Cell type-specific variation of mitotic DRA transcriptional activity. (A) Total RNA was extracted from asynchronous and mitotic Raji, HeLa CIITA and RJ2.2.5 CIITA cells and active transcription of the DRA gene was analysed with qRT-PCR using two primer sets (DRA exon5–exon5 and DRA exon1–intron1). RNA from untreated and DRB-treated cells was used to measure net active transcription. These primer sets that monitor unspliced (nascent) or spliced (mature/total) transcripts were normalized for differences of their amplification efficiencies using a standard curve of a genomic DNA template. Newly synthesized RNA is presented as percent of mature DRA mRNA. (B) DNA content analysis by PI and flow cytometry of RJ2.2.5 cells expressing CIITA under the control of rtTA-GBD and Dex-Dox treatment (see materials and methods). Cells were blocked by nocodazole prior to CIITA induction by a 6-h treatment with Dex–Dox. (C) RNA was extracted from control and induced, asynchronous and mitotic RJ2.2.5 tetCIITA cells. DRA and CIITA gene expression was analysed by qRT-PCR. (D) Chromatin was extracted from the same samples as in (C), and ChIP assays against RFX5 and CIITA were performed.
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Related In: Results  -  Collection

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gks1365-F4: Cell type-specific variation of mitotic DRA transcriptional activity. (A) Total RNA was extracted from asynchronous and mitotic Raji, HeLa CIITA and RJ2.2.5 CIITA cells and active transcription of the DRA gene was analysed with qRT-PCR using two primer sets (DRA exon5–exon5 and DRA exon1–intron1). RNA from untreated and DRB-treated cells was used to measure net active transcription. These primer sets that monitor unspliced (nascent) or spliced (mature/total) transcripts were normalized for differences of their amplification efficiencies using a standard curve of a genomic DNA template. Newly synthesized RNA is presented as percent of mature DRA mRNA. (B) DNA content analysis by PI and flow cytometry of RJ2.2.5 cells expressing CIITA under the control of rtTA-GBD and Dex-Dox treatment (see materials and methods). Cells were blocked by nocodazole prior to CIITA induction by a 6-h treatment with Dex–Dox. (C) RNA was extracted from control and induced, asynchronous and mitotic RJ2.2.5 tetCIITA cells. DRA and CIITA gene expression was analysed by qRT-PCR. (D) Chromatin was extracted from the same samples as in (C), and ChIP assays against RFX5 and CIITA were performed.
Mentions: The above results show that the MCE-CIITA-GTM complex in mitosis is maintained in lymphoblastoid but not in non-lymphoblastoid cells. We next addressed the question whether transcription is sustained during mitosis. In most systems studied, association of regulatory factors with DNA does not ensure active gene transcription because either not all essential factors remain bound to chromatin or PTMs such as phopshorylation may inactivate critical factors (18). To this end, we quantified total DRA RNA by qRT-PCR of asynchronous or nocodazole-arrested cells by using exon5–exon5 primer set and newly synthesized RNA by using exon1–intron1 primer set on the same sample in the presence or absence of the transcription elongation inhibitor DRB. To express the latter as a fraction of the mature RNA, we used genomic DNA as a standard to account for differences in amplification efficiencies between the above two primer sets. New RNA synthesis represented 4–5.5% of the total DRA RNA in the asynchronously growing cells shown in Figure 4A. In mitosis newly synthesized DRA RNA was reduced to 33% and 12% of the asynchronous levels of Raji or HeLa CIITA cells, respectively. To correlate active RNA synthesis with the recruitment of RNA PolII in Raji cells, we used ChIP. Results in Supplementary Figure S6 (A and C) show that promoter- or exon-bound (elongating) RNA PolII levels on DRA gene correlate well with the qRT-PCR-measured transcription in the different asynchronous or mitotic cells. Comparison of promoter and exon levels further indicates that there is direct correlation of initiation and elongation events without stalling of PolII in the different set ups. For specificity control, results for CyclinB1 and GAPDH genes are also shown (Supplementary Figure S6B and D).Figure 4.

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