Limits...
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.

Show MeSH

Related in: MedlinePlus

Enhanceosome components are dynamically associated with mitotic chromatin. (A) Immunostaining of HeLa cells using antibodies against CREB (upper panel) and RFX5 (lower panel). (B) Subnuclear localization of pECFP-CREB (upper panel) and pEGFP-RFX5 (lower panel) in interphase and mitotic HeLa cells. Nuclear-ID Red (red pseudo-colour) was used for DNA counterstaining. (C and D) FRAP experiments with transiently expressed ECFP-CREB (C), EGFP-RFX5 and Monomeric DsRed-H2A (D) in different stages of the cell cycle. Mitotic cells were chosen by confocal misroscopy. Results show mean values and standard deviation from at least six cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230186&req=5

gks1365-F1: Enhanceosome components are dynamically associated with mitotic chromatin. (A) Immunostaining of HeLa cells using antibodies against CREB (upper panel) and RFX5 (lower panel). (B) Subnuclear localization of pECFP-CREB (upper panel) and pEGFP-RFX5 (lower panel) in interphase and mitotic HeLa cells. Nuclear-ID Red (red pseudo-colour) was used for DNA counterstaining. (C and D) FRAP experiments with transiently expressed ECFP-CREB (C), EGFP-RFX5 and Monomeric DsRed-H2A (D) in different stages of the cell cycle. Mitotic cells were chosen by confocal misroscopy. Results show mean values and standard deviation from at least six cells.

Mentions: Earlier biochemical and subcellular localization studies (15,41) have defined an intricate hierarchy of interactions between the MCE subunits required for enhanceosome formation and subsequent recruitment of CIITA. The MCE is thought to form in a step-wise manner involving the independent assembly of the NFY and RFX subcomplexes first, followed by synergistic binding to chromatin. In spite of abundant biochemical and functional information, little is known about the dynamics of MCE–CIITA and its stability in living cells, especially during the cell cycle. Because during mitosis transcription is generally disrupted, we chose to study whether MHCII factors are retained on chromatin in this stage of the cell cycle. Initially, we performed immunostaining experiments in HeLa cells with antibodies specific for RFX5 and CREB. CREB and to a lesser extend RFX5 proteins were found to be associated with mitotic chromatin (Figure 1A). To further investigate this, fluorescent protein fusions of RFX5 and CREB were expressed and their subcellular localization was studied. Both proteins were observed to be associated with chromatin in various stages of mitosis such as in anaphase as shown in Figure 1B. To demonstrate the specificity of chromatin association, various deletion derivatives were tested. In Supplementary Figure S1 is shown such an example of RFX5 and CREB truncations that harbour the N-terminal regions of these proteins that lack chromatin-binding ability.Figure 1.


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

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

Enhanceosome components are dynamically associated with mitotic chromatin. (A) Immunostaining of HeLa cells using antibodies against CREB (upper panel) and RFX5 (lower panel). (B) Subnuclear localization of pECFP-CREB (upper panel) and pEGFP-RFX5 (lower panel) in interphase and mitotic HeLa cells. Nuclear-ID Red (red pseudo-colour) was used for DNA counterstaining. (C and D) FRAP experiments with transiently expressed ECFP-CREB (C), EGFP-RFX5 and Monomeric DsRed-H2A (D) in different stages of the cell cycle. Mitotic cells were chosen by confocal misroscopy. Results show mean values and standard deviation from at least six cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230186&req=5

gks1365-F1: Enhanceosome components are dynamically associated with mitotic chromatin. (A) Immunostaining of HeLa cells using antibodies against CREB (upper panel) and RFX5 (lower panel). (B) Subnuclear localization of pECFP-CREB (upper panel) and pEGFP-RFX5 (lower panel) in interphase and mitotic HeLa cells. Nuclear-ID Red (red pseudo-colour) was used for DNA counterstaining. (C and D) FRAP experiments with transiently expressed ECFP-CREB (C), EGFP-RFX5 and Monomeric DsRed-H2A (D) in different stages of the cell cycle. Mitotic cells were chosen by confocal misroscopy. Results show mean values and standard deviation from at least six cells.
Mentions: Earlier biochemical and subcellular localization studies (15,41) have defined an intricate hierarchy of interactions between the MCE subunits required for enhanceosome formation and subsequent recruitment of CIITA. The MCE is thought to form in a step-wise manner involving the independent assembly of the NFY and RFX subcomplexes first, followed by synergistic binding to chromatin. In spite of abundant biochemical and functional information, little is known about the dynamics of MCE–CIITA and its stability in living cells, especially during the cell cycle. Because during mitosis transcription is generally disrupted, we chose to study whether MHCII factors are retained on chromatin in this stage of the cell cycle. Initially, we performed immunostaining experiments in HeLa cells with antibodies specific for RFX5 and CREB. CREB and to a lesser extend RFX5 proteins were found to be associated with mitotic chromatin (Figure 1A). To further investigate this, fluorescent protein fusions of RFX5 and CREB were expressed and their subcellular localization was studied. Both proteins were observed to be associated with chromatin in various stages of mitosis such as in anaphase as shown in Figure 1B. To demonstrate the specificity of chromatin association, various deletion derivatives were tested. In Supplementary Figure S1 is shown such an example of RFX5 and CREB truncations that harbour the N-terminal regions of these proteins that lack chromatin-binding ability.Figure 1.

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