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Chromatin-Associated PKC

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Science Signaling  22 Mar 2011:
Vol. 4, Issue 165, pp. ec85
DOI: 10.1126/scisignal.4165ec85

Roles for protein kinases in the nucleus, indeed directly on chromatin, are emerging as important in mediating gene expression. Sutcliffe et al. show that protein kinase C-θ (PKC-θ) joins this group of chromatin-associated kinases and functions in inducible gene expression in T cells. PKC-θ was present in the nuclei of cultured Jurkat T cells and isolated human CD4+ T cells. Resting memory T cells showed the least PKC-θ, followed by naïve T cells and then effector and activated memory T cells. Chromatin immunoprecipitation (ChIP) analysis of the CD4+ T cell populations showed that PKC-θ was associated with the interleukin 2 (IL-2) promoter primarily in effector and activated memory T cells, which also had the highest amount of IL-2 transcripts. Sequential ChIP analysis suggested that stimulated Jurkat cells showed increased co-occupancy of PKC-θ with the histone kinase MSK-1 and RNA polymerase II (Pol II) (both of which promote gene expression) at inducible genes, such as CD69, TNF-α, and IFN-γ, and washout studies indicated that the recruitment was reversed if the stimulation was removed. Overexpression of wild-type PKC-θ promoted the recruitment of factors that promote inducible gene expression (14-3-3ζ and Pol II) to the promoters of induced genes, and overexpression of a kinase-inactive PKC-θ mutant, which had compromised binding to promoters, reduced their recruitment. Inhibition of the catalytic activity of PKC with Rottlerin did not reduce PKC-θ binding to the CD69 promoter but did reduce the accessibility of the chromatin at the promoter of this gene compared to its accessibility after stimulation of the Jurkat cells in the absence of the inhibitor. (Accessibility was measured in a real-time polymerase chain assay that monitored accessibility of DNA to a nuclease.) RNA interference experiments in which PKC-θ was knocked down in Jurkat cells were consistent with a role for PKC-θ in the recruitment of factors that promote gene expression to genes induced in response to stimulation. Knockdown of PKC-θ also reduced the transcripts of the induced gene. Genome-wide ChIP on chip experiments with activated T cells revealed that PKC-θ associated with 141 genes, 62% of which were also occupied by Pol II. In addition, PKC-θ associated with the regulatory regions of 60 microRNA-encoding genes. Examination of the transcript abundance of the three microRNA-encoding genes with the strongest PKC-θ binding revealed a stimulus-dependent decrease in the expression of these microRNA-encoding genes in Jurkat T cells. In addition to association with genes implicated in immune cell function, the authors noted enrichment in protein-encoding and microRNA-encoding genes associated with the epithelial to mesenchymal transition. Consistent with this possibility, the overexpression of PKC-θ in the MCF-7 breast cancer cell line altered cell morphology to one consistent with increased motility; moreover, these cells exhibited increased motility in an in vitro wound-healing assay. Thus, PKC-θ appears to play key roles in inducible gene expression by promoting the formation of transcription complexes at the promoters of protein-encoding and microRNA-encoding genes.

E. L. Sutcliffe, K. L. Bunting, Y. Q. He, J. Li, C. Phetsouphanh, N. Seddiki, A. Zafar, E. J. Hindmarsh, C. R. Parish, A. D. Kelleher, R. L. McInnes, T. Taya, P. J. Milburn, S. Rao, Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes. Mol. Cell 41, 704–719 (2011). [Online Journal]

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