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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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The DRA-LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. (A) DRA-LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA+) and HeLa cell lines shown in Figure 2D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2B. (B) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. (C) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.
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gks1365-F5: The DRA-LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. (A) DRA-LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA+) and HeLa cell lines shown in Figure 2D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2B. (B) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. (C) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.

Mentions: Our results so far establish that cell-type specific mechanisms that involve deficiency of CIITA or MCE assembly controls the transcriptional activity of the DRA gene promoter during mitosis. We next studied the occupation of the distal XY-like regulatory element LCR/XL4 known to mediate important gene regulatory functions (7–10). Results showed (Figure 5A) that all promoter factors studied co-occupied the upstream element but at much lower levels with the notable exception of NFYA and NFYB subunits both in interphase and in mitosis. Promoter-bound factors—especially those that remain bound to gene regulatory region in mitosis—have been implicated in bookmarking via various mechanisms including the anticondensation action of the PP2A phosphatase (24,33). To investigate the latter, we used co-immunoprecipitation of transiently expressed fluorescent protein-MHC factor fusions and myc-tagged PP2A. Results in Figure 5B showed that NFY subunits A, C and B co-immunoprecipitated with PP2A in decreasing order of strength. The interaction with NFYA was further studied and showed that the N-terminal region—between 52–158 amino acids—that include the Q-rich domain were sufficient for this interaction. Of note, NFYA and NFYC and the previously reported PP2A interacting factor TBP (33) contain Q-rich domains that may be involved in the interaction. This interaction was verified by detecting co-immunoprecipitation of the endogenous proteins (Figure 5C).Figure 5.


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

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

The DRA-LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. (A) DRA-LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA+) and HeLa cell lines shown in Figure 2D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2B. (B) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. (C) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230186&req=5

gks1365-F5: The DRA-LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. (A) DRA-LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA+) and HeLa cell lines shown in Figure 2D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2B. (B) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. (C) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.
Mentions: Our results so far establish that cell-type specific mechanisms that involve deficiency of CIITA or MCE assembly controls the transcriptional activity of the DRA gene promoter during mitosis. We next studied the occupation of the distal XY-like regulatory element LCR/XL4 known to mediate important gene regulatory functions (7–10). Results showed (Figure 5A) that all promoter factors studied co-occupied the upstream element but at much lower levels with the notable exception of NFYA and NFYB subunits both in interphase and in mitosis. Promoter-bound factors—especially those that remain bound to gene regulatory region in mitosis—have been implicated in bookmarking via various mechanisms including the anticondensation action of the PP2A phosphatase (24,33). To investigate the latter, we used co-immunoprecipitation of transiently expressed fluorescent protein-MHC factor fusions and myc-tagged PP2A. Results in Figure 5B showed that NFY subunits A, C and B co-immunoprecipitated with PP2A in decreasing order of strength. The interaction with NFYA was further studied and showed that the N-terminal region—between 52–158 amino acids—that include the Q-rich domain were sufficient for this interaction. Of note, NFYA and NFYC and the previously reported PP2A interacting factor TBP (33) contain Q-rich domains that may be involved in the interaction. This interaction was verified by detecting co-immunoprecipitation of the endogenous proteins (Figure 5C).Figure 5.

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