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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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MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. (A) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA+) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. (B–E) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.
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gks1365-F2: MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. (A) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA+) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. (B–E) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.

Mentions: To examine the mentioned changes in a better defined mitotic environment, we used 1 μΜ nocodazole to arrest Raji and their CIITA-negative derivatives (RJ2.2.5) cells in prometaphase. Mitotic arrest was monitored by flow cytometry using Propidium Iodide (PI) analysis (Figure 2A left). Mitotic cell content was further assessed by double α-H3S10Phos–PI staining (representative results are shown in Supplementary Figure S3). ChIP analysis of MCE components showed that CREB, RFX5 and NFYB remained generally unchanged in mitosis, irrespective of CIITA expression (Figure 2B). Mitotic recruitment of CIITA itself was slightly reduced. To monitor the transcriptional machinery components, antibodies against TBP and RNA PolII were used and results showed that in arrested cells DRA occupancy was reduced ∼50% and 30% the recruitment observed in asynchronous cells, respectively (Figure 2B). As expected, recruitment of these factors was CIITA-dependent, as CIITA-deficient cells were devoid of TBP and RNA PolII whether prior or after the mitotic arrest (Figure 2B). For comparison, a similar analysis performed in RFX5-deficient (RFX5−) cells and its wild type-like derivative generated by exogenous RFX5 expression that rescues MHCII transcription showed that RFX5 was essential for factor occupancy both in asynchronous and mitotic cells (Supplementary Figure S4).Figure 2.


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

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

MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. (A) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA+) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. (B–E) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.
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gks1365-F2: MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. (A) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA+) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. (B–E) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.
Mentions: To examine the mentioned changes in a better defined mitotic environment, we used 1 μΜ nocodazole to arrest Raji and their CIITA-negative derivatives (RJ2.2.5) cells in prometaphase. Mitotic arrest was monitored by flow cytometry using Propidium Iodide (PI) analysis (Figure 2A left). Mitotic cell content was further assessed by double α-H3S10Phos–PI staining (representative results are shown in Supplementary Figure S3). ChIP analysis of MCE components showed that CREB, RFX5 and NFYB remained generally unchanged in mitosis, irrespective of CIITA expression (Figure 2B). Mitotic recruitment of CIITA itself was slightly reduced. To monitor the transcriptional machinery components, antibodies against TBP and RNA PolII were used and results showed that in arrested cells DRA occupancy was reduced ∼50% and 30% the recruitment observed in asynchronous cells, respectively (Figure 2B). As expected, recruitment of these factors was CIITA-dependent, as CIITA-deficient cells were devoid of TBP and RNA PolII whether prior or after the mitotic arrest (Figure 2B). For comparison, a similar analysis performed in RFX5-deficient (RFX5−) cells and its wild type-like derivative generated by exogenous RFX5 expression that rescues MHCII transcription showed that RFX5 was essential for factor occupancy both in asynchronous and mitotic cells (Supplementary Figure S4).Figure 2.

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