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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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NFYA loss compromises recruitment of PP2A to chromatin. (A) Western blotting of asynchronous or mitotic HeLa cell extracts following lentiviral infection with sh-scrambled control (shSCR) or shNFYA. Equivalent protein amounts were probed with antibodies against NFYA, NFYB, PP2Ac and β-tubulin. (B) Flow cytometric analysis of propidium iodide-stained DNA from shSCR or shNFYA infected HeLa cells, untreated or mitotically arrested. (C) ChIP from shSCR or shNFYA infected, asynchronous or mitotic HeLa cells, probing the indicated regions (DRA promoter, DRA-LCR/XL4, DRB1 promoter, XL7 and XL9) with antibodies against NFYA, PP2A, H3 and CREB. Mean and error bars are derived as in Figure 2.
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gks1365-F6: NFYA loss compromises recruitment of PP2A to chromatin. (A) Western blotting of asynchronous or mitotic HeLa cell extracts following lentiviral infection with sh-scrambled control (shSCR) or shNFYA. Equivalent protein amounts were probed with antibodies against NFYA, NFYB, PP2Ac and β-tubulin. (B) Flow cytometric analysis of propidium iodide-stained DNA from shSCR or shNFYA infected HeLa cells, untreated or mitotically arrested. (C) ChIP from shSCR or shNFYA infected, asynchronous or mitotic HeLa cells, probing the indicated regions (DRA promoter, DRA-LCR/XL4, DRB1 promoter, XL7 and XL9) with antibodies against NFYA, PP2A, H3 and CREB. Mean and error bars are derived as in Figure 2.

Mentions: To establish the role of NFY in chromatin recruitment of PP2A, we knocked down the NFYA subunit by transient lentiviral infection. Of the seven different shRNAs tested, we chose one for further use, that is identical to the exon 6 targeting sh plasmid reported by Benatti et al. (44). Two days following infection of shSCR or shNFYA virus, cells were mock- or nocodazole-treated to obtain asynchronous and mitotically arrested cells. Efficient knock down was evaluated by Western analysis (Figure 6A). Cell cycle analysis showed that NFYA knock down reduced progression to the G2/M phase of the cell cycle in either untreated or nocodazole-arrested cells (Figure 6B). Chromatin from those cells was prepared and immunoprecipitated. Results in Figure 6C showed that knock down inhibits gene occupancy by NFYA in both asynchronous and mitotic cells of the DRA, DRB1, the upstream LCR/XL4 and two previously identified intergenic XL elements, XL7 and XL9 (8). Parallel immunoprecipitations showed that reduced PP2A occupancy accompanied and correlated with NFYA recruitment. In particular, even in the case of an increased mitotic occupancy of the LCR/XL4 element by PP2A, knocking down NFYA abrogated PP2A recruitment suggesting a yet another function of this distal MHCII gene regulatory region. In addition, NFYA loss also reduced CREB recruitment as expected because of the cooperative nature of the enhanceosome assembly. As a control for chromatin occupancy and quality, histone H3 was used. Results showed that NFY knock down did not significantly altered H3 recruitment. That was also the case for the XL9 element that is devoid of both MHC-related factors and PP2A, but retained high histone levels.Figure 6.


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

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

NFYA loss compromises recruitment of PP2A to chromatin. (A) Western blotting of asynchronous or mitotic HeLa cell extracts following lentiviral infection with sh-scrambled control (shSCR) or shNFYA. Equivalent protein amounts were probed with antibodies against NFYA, NFYB, PP2Ac and β-tubulin. (B) Flow cytometric analysis of propidium iodide-stained DNA from shSCR or shNFYA infected HeLa cells, untreated or mitotically arrested. (C) ChIP from shSCR or shNFYA infected, asynchronous or mitotic HeLa cells, probing the indicated regions (DRA promoter, DRA-LCR/XL4, DRB1 promoter, XL7 and XL9) with antibodies against NFYA, PP2A, H3 and CREB. Mean and error bars are derived as in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks1365-F6: NFYA loss compromises recruitment of PP2A to chromatin. (A) Western blotting of asynchronous or mitotic HeLa cell extracts following lentiviral infection with sh-scrambled control (shSCR) or shNFYA. Equivalent protein amounts were probed with antibodies against NFYA, NFYB, PP2Ac and β-tubulin. (B) Flow cytometric analysis of propidium iodide-stained DNA from shSCR or shNFYA infected HeLa cells, untreated or mitotically arrested. (C) ChIP from shSCR or shNFYA infected, asynchronous or mitotic HeLa cells, probing the indicated regions (DRA promoter, DRA-LCR/XL4, DRB1 promoter, XL7 and XL9) with antibodies against NFYA, PP2A, H3 and CREB. Mean and error bars are derived as in Figure 2.
Mentions: To establish the role of NFY in chromatin recruitment of PP2A, we knocked down the NFYA subunit by transient lentiviral infection. Of the seven different shRNAs tested, we chose one for further use, that is identical to the exon 6 targeting sh plasmid reported by Benatti et al. (44). Two days following infection of shSCR or shNFYA virus, cells were mock- or nocodazole-treated to obtain asynchronous and mitotically arrested cells. Efficient knock down was evaluated by Western analysis (Figure 6A). Cell cycle analysis showed that NFYA knock down reduced progression to the G2/M phase of the cell cycle in either untreated or nocodazole-arrested cells (Figure 6B). Chromatin from those cells was prepared and immunoprecipitated. Results in Figure 6C showed that knock down inhibits gene occupancy by NFYA in both asynchronous and mitotic cells of the DRA, DRB1, the upstream LCR/XL4 and two previously identified intergenic XL elements, XL7 and XL9 (8). Parallel immunoprecipitations showed that reduced PP2A occupancy accompanied and correlated with NFYA recruitment. In particular, even in the case of an increased mitotic occupancy of the LCR/XL4 element by PP2A, knocking down NFYA abrogated PP2A recruitment suggesting a yet another function of this distal MHCII gene regulatory region. In addition, NFYA loss also reduced CREB recruitment as expected because of the cooperative nature of the enhanceosome assembly. As a control for chromatin occupancy and quality, histone H3 was used. Results showed that NFY knock down did not significantly altered H3 recruitment. That was also the case for the XL9 element that is devoid of both MHC-related factors and PP2A, but retained high histone levels.Figure 6.

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