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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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DRA locus accessibility in mitotic B lymphoblastoid cells correlates with MCE maintenance. (A) Schematic representation of SspI restriction enzyme site and the flanking primer target sites relative to the DRA gene and its regulatory elements. (B–E). Restriction endonuclease accessibility assay and qPCR analysis to measure the undigested DNA was performed on asynchronous growing and mitotically arrested populations of Raji (B), RJ2.2.5 (C), HeLa CIITA (CIITA+) (D) and HeLa (E) cell lines. Cells were treated with increasing amounts of SspI enzyme (0, 10, 50, 100 and 200 units). SJO is an RFX5-deficient cell line, which is used as a negative control cell line with inaccessible SspI site on the DRA promoter. To normalize for template loading, a primer set spanning a region in the DRA exon 5 was also used that was devoid of the SspI recognition sequence. Map is not to scale.
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gks1365-F3: DRA locus accessibility in mitotic B lymphoblastoid cells correlates with MCE maintenance. (A) Schematic representation of SspI restriction enzyme site and the flanking primer target sites relative to the DRA gene and its regulatory elements. (B–E). Restriction endonuclease accessibility assay and qPCR analysis to measure the undigested DNA was performed on asynchronous growing and mitotically arrested populations of Raji (B), RJ2.2.5 (C), HeLa CIITA (CIITA+) (D) and HeLa (E) cell lines. Cells were treated with increasing amounts of SspI enzyme (0, 10, 50, 100 and 200 units). SJO is an RFX5-deficient cell line, which is used as a negative control cell line with inaccessible SspI site on the DRA promoter. To normalize for template loading, a primer set spanning a region in the DRA exon 5 was also used that was devoid of the SspI recognition sequence. Map is not to scale.

Mentions: To study whether mitotic retention of MCE components correlates with the chromatin state of the DRA gene, we used a restriction endonuclease assay on asynchronous or nocodazole-arrested cells. To this end, we used an SspI site, located 80 bp upstream of the DRA transcription start site (TSS) that was previously shown to be differentially sensitive to digestion correlating with transcription or transcription potential (Figure 3A) (37). Digestion efficiency was quantified by qPCR with primers flanking the SspI site and expressed as percentage of undigested DNA relative to that found in non-digested mock-treated sample. This approach revealed that B lymphoblastoid cell hypersensitivity of this particular site to SspI was retained during mitosis to levels similar to those observed in asynchronous cultures in a CIITA-independent manner (Figure 3B and C) over a range of enzyme concentration. As a control, RFX5-deficient SJO cells were also examined and this particular region was found to be inaccessible to digestion irrespectively of the amount of restriction enzyme used.Figure 3.


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

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

DRA locus accessibility in mitotic B lymphoblastoid cells correlates with MCE maintenance. (A) Schematic representation of SspI restriction enzyme site and the flanking primer target sites relative to the DRA gene and its regulatory elements. (B–E). Restriction endonuclease accessibility assay and qPCR analysis to measure the undigested DNA was performed on asynchronous growing and mitotically arrested populations of Raji (B), RJ2.2.5 (C), HeLa CIITA (CIITA+) (D) and HeLa (E) cell lines. Cells were treated with increasing amounts of SspI enzyme (0, 10, 50, 100 and 200 units). SJO is an RFX5-deficient cell line, which is used as a negative control cell line with inaccessible SspI site on the DRA promoter. To normalize for template loading, a primer set spanning a region in the DRA exon 5 was also used that was devoid of the SspI recognition sequence. Map is not to scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230186&req=5

gks1365-F3: DRA locus accessibility in mitotic B lymphoblastoid cells correlates with MCE maintenance. (A) Schematic representation of SspI restriction enzyme site and the flanking primer target sites relative to the DRA gene and its regulatory elements. (B–E). Restriction endonuclease accessibility assay and qPCR analysis to measure the undigested DNA was performed on asynchronous growing and mitotically arrested populations of Raji (B), RJ2.2.5 (C), HeLa CIITA (CIITA+) (D) and HeLa (E) cell lines. Cells were treated with increasing amounts of SspI enzyme (0, 10, 50, 100 and 200 units). SJO is an RFX5-deficient cell line, which is used as a negative control cell line with inaccessible SspI site on the DRA promoter. To normalize for template loading, a primer set spanning a region in the DRA exon 5 was also used that was devoid of the SspI recognition sequence. Map is not to scale.
Mentions: To study whether mitotic retention of MCE components correlates with the chromatin state of the DRA gene, we used a restriction endonuclease assay on asynchronous or nocodazole-arrested cells. To this end, we used an SspI site, located 80 bp upstream of the DRA transcription start site (TSS) that was previously shown to be differentially sensitive to digestion correlating with transcription or transcription potential (Figure 3A) (37). Digestion efficiency was quantified by qPCR with primers flanking the SspI site and expressed as percentage of undigested DNA relative to that found in non-digested mock-treated sample. This approach revealed that B lymphoblastoid cell hypersensitivity of this particular site to SspI was retained during mitosis to levels similar to those observed in asynchronous cultures in a CIITA-independent manner (Figure 3B and C) over a range of enzyme concentration. As a control, RFX5-deficient SJO cells were also examined and this particular region was found to be inaccessible to digestion irrespectively of the amount of restriction enzyme used.Figure 3.

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