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P300-mediated gene expression during muscle cell survival

ABSTRACT: Type of experiment: The primary focus of these studies is to define the transcriptional network regulated by ectopic expression of the transcriptional co-activator protein, p300, in order to define its mechanism(s) of action in promoting muscle cell survival. Experimental factors: Our laboratory has generated a murine C2-derived myoblast cell line stably expressing an IGF-II cDNA in antisense orientation (C2AS12 cells). These cells proliferate normally in serum-rich growth medium but progressively die in low-serum differentiation medium. Ectopic expression of p300 (a transcriptional co-coactivator with acetyltransferase activity) prevents the progressive cell death induced by serum withdrawal, however, the mechanism of this action is not understood. Further, over-expression of a mutated form of p300, lacking at protein interaction domain (deltaTAZ2), failed to maintain cell viability although it retains catalytic activity. Wild-type and mutant p300 are delivered using recombinant adenoviruses (Ad-p300 and Ad-p300deltaTAZ2) and their expression is regulated by a second recombinant adenovirus encoding the tetracycline transactivator protein (Ad-tTA), affording regulated expression of p300 forms (Tet-off system) and providing a control for viral load. Using these reagents the overall experiment was as follows: Three parallel series of C2AS12 cells were infected with (1) wt p300 + tTA, (2) p300deltaTAZ2 +tTA, (3) wt p300 +tTA +doxycycline. Infected cells were grown to confluence followed by transfer to low-serum differentiation medium (T0). Cell were isolated at this point and following 24 (T24) hours incubation for RNA isolation. Companion dishes of cells were included for analysis by immunocytochemistry to ensure regulated transgene expression and cell viabilities. Keywords: time course

ORGANISM(S): Mus musculus

PROVIDER: GSE4224 | GEO | 2006/03/01

SECONDARY ACCESSION(S): PRJNA94869

REPOSITORIES: GEO

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Project description:Background: Commercial Atlantic salmon is fed diets with high fat levels to promote fast and cost-eﬀective growth. To avoid negative impact of obesity, food additives that stimulate fat metabolism and immune function are of high interest. TTA, tetradecylthioacetic acid, is a synthetic fatty acid that stimulates mitochondrial β -oxidation most likely by activation of peroxysome proliferator-activated receptors (PPARs). PPARs are important transcription factors regulating multiple functions including fat metabolism and immune responses. Atlantic salmon experiments have shown that TTA supplemented diets signiﬁcantly reduce mortality during natural outbreaks of viral diseases, suggesting a modulatory role of the immune system. Results: To gain new insights into TTA eﬀects on the Atlantic salmon immune system, a factorial, high-throughput microarray experiment was conducted using a 44K oligo nucleotide salmon microarray SIQ2.0 and the Atlantic salmon macrophage-like cell line ASK. The experiment was used to determine the transcriptional eﬀects of TTA, the eﬀects of TTA in poly(I:C) elicited cells and the eﬀects of pretreating the cells with TTA. The expression patterns revealed that a large proportion of genes regulated by TTA were related to lipid metabolism and increased mitochondrial β -oxidation. In addition we found that for a subset of genes TTA antagonized the transcriptional eﬀects of poly(I:C). This, together with the results from qRT-PCR showing an increased transcription of anti-inﬂammatory IL10 by TTA, indicates anti-inﬂammatory eﬀects. Conclusions: We demonstrate that TTA has signiﬁcant eﬀects on macrophage-like salmon cells that are challenged by the artiﬁcial dsRNA poly(I:C). The immune stimulatory eﬀect of TTA in macrophages involves increased lipid metabolism and suppressed inﬂammatory status. Thus, suggesting that TTA directs the macrophage-like cells towards alternative, anti-inﬂammatory, activation. This has positive implications for TTA as a feed additive.

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P300-mediated gene expression during muscle cell survival

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