Abstract
BackgroundThe 250 kDa P2P-R protein (also known as PACT and Rbbp6) was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins. In addition, P2P-R has been associated with multiple biological functions, such as mitosis, mRNA processing, translation and ubiquitination. In the current studies, the online GeneNetwork system was employed to further probe P2P-R biological functions. Molecular studies were then performed to confirm the GeneNetwork evaluations.ResultsGeneNetwork and associated gene ontology links were used to investigate the coexpression of P2P-R with distinct functional sets of genes in an adipocyte genetic reference panel of HXB/BXH recombinant strains of rats and an eye genetic reference panel of BXD recombinant inbred strains of mice. The results establish that biological networks of 75 and 135 transcription-associated gene products that include P2P-R are co-expressed in a genetically-defined manner in rat adipocytes and in the mouse eye, respectively. Of this large set of transcription-associated genes, >10% are associated with hormone-mediated transcription. Since it has been previously reported that P2P-R can bind the SRC-1 transcription co-regulatory factor (steroid receptor co-activator 1, [Ncoa1]), the possible effects of P2P-R on estrogen-induced transcription were evaluated. Estrogen-induced transcription was repressed 50-70% by the transient transfection of P2P-R plasmid constructs into four different cell types. In addition, knockdown of P2P-R expression using an antisense oligonucleotide increased estrogen-mediated transcription. Co-immunoprecipitation assays confirmed that P2P-R interacts with SRC-1 and also demonstrated that P2P-R interacts with estrogen receptor α.ConclusionsThe findings presented in this study provide strong support for the value of systems genetics, especially GeneNetwork, in discovering new functions of genes that can be confirmed by molecular analysis. More specifically, these data provide evidence that the expression of P2P-R co-varies in a genetically-defined manner with large transcription networks and that P2P-R can function as a co-repressor of estrogen-dependent transcription.
Highlights
The 250 kDa P2P-R protein was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins
When the top 1000 gene transcripts that are coexpressed with P2P-R and have correlation coefficients ≥ 0.67 were exported for gene ontology analysis to WebGestalt, 67 gene products were found to be associated with transcription with p = 2.81e-4 (Figure 1)
In support of the above data based on analysis of the fat cell GeneNetwork database derived from HXB/BHX recombinant inbred rats, limited analysis were performed on the GeneNetwork mouse adipose tissue database wherein it was found that 13.5% of the transcripts whose expression co-varies with P2P-R with a correlation coefficient of ≥ 0.5 has transcription-associated functions
Summary
The 250 kDa P2P-R protein ( known as PACT and Rbbp6) was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins. Systems genetics is an evolving new speciality that can be used to define biological networks and to predict molecular interactions. It is based on the analysis of transcripts whose expression co-vary within genetic populations, such as the BXD strains of mice and the HXB strains of rats or other genetic reference panels. In 2005, we published the first report documenting the ability of the systems genetics tool GeneNetwork to predict interactions between molecules that could be confirmed by molecular analysis [3]. The 3’ UTR of the P2P-R mRNA was found to contain one perfect consensus and two near-perfect consensus Pum binding sequences, while pull-down assays combined with reverse transcription and RT-PCR confirmed that Pum does bind P2P-R mRNA to modulate its expression [3]
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