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Regulation of p110delta PI 3-kinase gene expression.

Kok K, Nock GE, Verrall EA, Mitchell MP, Hommes DW, Peppelenbosch MP, Vanhaesebroeck B - PLoS ONE (2009)

Bottom Line: This also applies to the leukocyte-enriched p110delta catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy.We show that p110delta expression is mainly regulated at the transcriptional level.In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cell Signalling, Institute of Cancer, Queen Mary University of London, Charterhouse Square, London, United Kingdom.

ABSTRACT

Background: Despite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110delta catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy.

Principal findings: We show that p110delta expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110delta gene transcription appears to be constitutive and not subject to acute stimulation. 5'RACE experiments revealed that p110delta mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110delta transcripts are higher in leukocytes compared to non-leukocytes, with the p110delta transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110delta gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a.

Conclusion: This study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110delta.

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Equal p110δ mRNA stability in leukocytes and non-leukocytes.The indicated cell lines were treated with Actinomycin D (4 µg/ml), an inhibitor of de novo RNA synthesis, for the indicated time points followed by quantification of either p110δ mRNA (A) or p110δ protein (B). p110δ mRNA was quantified by real time RT-PCR, using normalisation for 18S RNA. p110δ mRNA levels are presented in a semi-log plot.
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pone-0005145-g003: Equal p110δ mRNA stability in leukocytes and non-leukocytes.The indicated cell lines were treated with Actinomycin D (4 µg/ml), an inhibitor of de novo RNA synthesis, for the indicated time points followed by quantification of either p110δ mRNA (A) or p110δ protein (B). p110δ mRNA was quantified by real time RT-PCR, using normalisation for 18S RNA. p110δ mRNA levels are presented in a semi-log plot.

Mentions: To assess whether high expression of the p110δ protein in cells is due to increased mRNA stability, cells were treated with Actinomycin D, an inhibitor of de novo RNA synthesis, followed by measurement of mRNA decay over time. As can be seen from Figure 3A, leukocyte (A20 and EL4) and non-leukocyte (B16-BL6, 3LL and NIH-3T3) cell lines displayed very similar rates of mRNA degradation upon inhibition of mRNA synthesis, indicating that there is no difference in p110δ mRNA stability between cell types expressing high or low levels of p110δ protein. Also p110δ protein levels were not affected by Actinomycin D treatment, both in leukocytes and non-leukocytes (Figure 3B).


Regulation of p110delta PI 3-kinase gene expression.

Kok K, Nock GE, Verrall EA, Mitchell MP, Hommes DW, Peppelenbosch MP, Vanhaesebroeck B - PLoS ONE (2009)

Equal p110δ mRNA stability in leukocytes and non-leukocytes.The indicated cell lines were treated with Actinomycin D (4 µg/ml), an inhibitor of de novo RNA synthesis, for the indicated time points followed by quantification of either p110δ mRNA (A) or p110δ protein (B). p110δ mRNA was quantified by real time RT-PCR, using normalisation for 18S RNA. p110δ mRNA levels are presented in a semi-log plot.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2663053&req=5

pone-0005145-g003: Equal p110δ mRNA stability in leukocytes and non-leukocytes.The indicated cell lines were treated with Actinomycin D (4 µg/ml), an inhibitor of de novo RNA synthesis, for the indicated time points followed by quantification of either p110δ mRNA (A) or p110δ protein (B). p110δ mRNA was quantified by real time RT-PCR, using normalisation for 18S RNA. p110δ mRNA levels are presented in a semi-log plot.
Mentions: To assess whether high expression of the p110δ protein in cells is due to increased mRNA stability, cells were treated with Actinomycin D, an inhibitor of de novo RNA synthesis, followed by measurement of mRNA decay over time. As can be seen from Figure 3A, leukocyte (A20 and EL4) and non-leukocyte (B16-BL6, 3LL and NIH-3T3) cell lines displayed very similar rates of mRNA degradation upon inhibition of mRNA synthesis, indicating that there is no difference in p110δ mRNA stability between cell types expressing high or low levels of p110δ protein. Also p110δ protein levels were not affected by Actinomycin D treatment, both in leukocytes and non-leukocytes (Figure 3B).

Bottom Line: This also applies to the leukocyte-enriched p110delta catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy.We show that p110delta expression is mainly regulated at the transcriptional level.In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cell Signalling, Institute of Cancer, Queen Mary University of London, Charterhouse Square, London, United Kingdom.

ABSTRACT

Background: Despite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110delta catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy.

Principal findings: We show that p110delta expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110delta gene transcription appears to be constitutive and not subject to acute stimulation. 5'RACE experiments revealed that p110delta mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110delta transcripts are higher in leukocytes compared to non-leukocytes, with the p110delta transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110delta gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a.

Conclusion: This study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110delta.

Show MeSH