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Targets downstream of Cdk8 in Dictyostelium development.

Greene DM, Bloomfield G, Skelton J, Ivens A, Pears CJ - BMC Dev. Biol. (2011)

Bottom Line: Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background.Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry Department, Oxford University, South Parks Road, Oxford OX1 3QU UK. catherine.pears@bioch.ox.ac.uk

ABSTRACT

Background: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production.

Results: Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).

Conclusions: This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

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Late developmental phenotype of cdk8-2 cells. (A) Cells were developed in KK2 buffer at 1 x 107cells/ml with or without cAMP pulsing (50 nm cAMP added to the suspension every 5 minutes). Each strain was shaken for 6hrs at 150 rpm at 22°C before being spread onto filters at a density of 3 x 106 cell/cm2. Photographs were taken after 28hrs. The scale bar in the right hand panel represents 200 μm. (B) Expression of SpiA in cdk8-2 cells. Fruiting bodies formed after cAMP pulsing were harvested after 24hrs or 28 hrs. RNA was extracted from these samples and resolved on a 1% formaldehyde gel, transferred to a nylon membrane and probed with a 32P labelled fragment of the spiA gene. The blot was reprobed with a 32P labelled fragment of the IG7 gene so as to control for loading. (C) Viability of cdk8-2 spores. Fruiting bodies were formed by developing cells in shaken suspension with cAMP pulsing prior to plating. Equal numbers of spores from each strain were treated with heat and detergent and spread onto a bacterial lawn. Colonies resulting from hatching of viable spores were counted after 4-5 days. Each bar represents the mean (±standard deviation) of three independent experiments. Results showing statistically significant difference from Ax2bsR are marked with a * (p < 0.05 by student t-test).
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Figure 2: Late developmental phenotype of cdk8-2 cells. (A) Cells were developed in KK2 buffer at 1 x 107cells/ml with or without cAMP pulsing (50 nm cAMP added to the suspension every 5 minutes). Each strain was shaken for 6hrs at 150 rpm at 22°C before being spread onto filters at a density of 3 x 106 cell/cm2. Photographs were taken after 28hrs. The scale bar in the right hand panel represents 200 μm. (B) Expression of SpiA in cdk8-2 cells. Fruiting bodies formed after cAMP pulsing were harvested after 24hrs or 28 hrs. RNA was extracted from these samples and resolved on a 1% formaldehyde gel, transferred to a nylon membrane and probed with a 32P labelled fragment of the spiA gene. The blot was reprobed with a 32P labelled fragment of the IG7 gene so as to control for loading. (C) Viability of cdk8-2 spores. Fruiting bodies were formed by developing cells in shaken suspension with cAMP pulsing prior to plating. Equal numbers of spores from each strain were treated with heat and detergent and spread onto a bacterial lawn. Colonies resulting from hatching of viable spores were counted after 4-5 days. Each bar represents the mean (±standard deviation) of three independent experiments. Results showing statistically significant difference from Ax2bsR are marked with a * (p < 0.05 by student t-test).

Mentions: In order to examine the newly generated cdk8-2 strain for the presence or absence of the late developmental phenotype, cells were suspended in KK2 buffer and pulsed with 50 nM cAMP every 5mins for 6hrs, before being harvested, washed and plated on KK2 agar. Unlike the cdk8-HL strain, the cdk8-2 cells formed phenotypically normal fruiting bodies, although culmination occurred 3-4hrs later than in the Ax2bsR control strain (Figure 2A). Ax2bsR was created by random insertion of the vector designed for disruption of the cdk8 gene into the genome of Ax2K. Upon culmination, the spiA gene (a gene induced late in spore differentiation and found to be expressed at reduced levels in the original cdk8-HL cells) was expressed at similar levels in both the cdk8-2 and Ax2bsR strains (Figure 2B).


Targets downstream of Cdk8 in Dictyostelium development.

Greene DM, Bloomfield G, Skelton J, Ivens A, Pears CJ - BMC Dev. Biol. (2011)

Late developmental phenotype of cdk8-2 cells. (A) Cells were developed in KK2 buffer at 1 x 107cells/ml with or without cAMP pulsing (50 nm cAMP added to the suspension every 5 minutes). Each strain was shaken for 6hrs at 150 rpm at 22°C before being spread onto filters at a density of 3 x 106 cell/cm2. Photographs were taken after 28hrs. The scale bar in the right hand panel represents 200 μm. (B) Expression of SpiA in cdk8-2 cells. Fruiting bodies formed after cAMP pulsing were harvested after 24hrs or 28 hrs. RNA was extracted from these samples and resolved on a 1% formaldehyde gel, transferred to a nylon membrane and probed with a 32P labelled fragment of the spiA gene. The blot was reprobed with a 32P labelled fragment of the IG7 gene so as to control for loading. (C) Viability of cdk8-2 spores. Fruiting bodies were formed by developing cells in shaken suspension with cAMP pulsing prior to plating. Equal numbers of spores from each strain were treated with heat and detergent and spread onto a bacterial lawn. Colonies resulting from hatching of viable spores were counted after 4-5 days. Each bar represents the mean (±standard deviation) of three independent experiments. Results showing statistically significant difference from Ax2bsR are marked with a * (p < 0.05 by student t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037916&req=5

Figure 2: Late developmental phenotype of cdk8-2 cells. (A) Cells were developed in KK2 buffer at 1 x 107cells/ml with or without cAMP pulsing (50 nm cAMP added to the suspension every 5 minutes). Each strain was shaken for 6hrs at 150 rpm at 22°C before being spread onto filters at a density of 3 x 106 cell/cm2. Photographs were taken after 28hrs. The scale bar in the right hand panel represents 200 μm. (B) Expression of SpiA in cdk8-2 cells. Fruiting bodies formed after cAMP pulsing were harvested after 24hrs or 28 hrs. RNA was extracted from these samples and resolved on a 1% formaldehyde gel, transferred to a nylon membrane and probed with a 32P labelled fragment of the spiA gene. The blot was reprobed with a 32P labelled fragment of the IG7 gene so as to control for loading. (C) Viability of cdk8-2 spores. Fruiting bodies were formed by developing cells in shaken suspension with cAMP pulsing prior to plating. Equal numbers of spores from each strain were treated with heat and detergent and spread onto a bacterial lawn. Colonies resulting from hatching of viable spores were counted after 4-5 days. Each bar represents the mean (±standard deviation) of three independent experiments. Results showing statistically significant difference from Ax2bsR are marked with a * (p < 0.05 by student t-test).
Mentions: In order to examine the newly generated cdk8-2 strain for the presence or absence of the late developmental phenotype, cells were suspended in KK2 buffer and pulsed with 50 nM cAMP every 5mins for 6hrs, before being harvested, washed and plated on KK2 agar. Unlike the cdk8-HL strain, the cdk8-2 cells formed phenotypically normal fruiting bodies, although culmination occurred 3-4hrs later than in the Ax2bsR control strain (Figure 2A). Ax2bsR was created by random insertion of the vector designed for disruption of the cdk8 gene into the genome of Ax2K. Upon culmination, the spiA gene (a gene induced late in spore differentiation and found to be expressed at reduced levels in the original cdk8-HL cells) was expressed at similar levels in both the cdk8-2 and Ax2bsR strains (Figure 2B).

Bottom Line: Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background.Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry Department, Oxford University, South Parks Road, Oxford OX1 3QU UK. catherine.pears@bioch.ox.ac.uk

ABSTRACT

Background: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production.

Results: Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).

Conclusions: This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

Show MeSH
Related in: MedlinePlus