Integration of protein kinases into transcription complexes: identifying components of immobilised in vitro pre-initiation complexes.

Regulation of gene expression is essential for coordinated cell growth and development. The de-regulation of certain genes is also recognised to contribute to both heritable and acquired disease. Transcription factors influence the assembly and activity of transcription complexes, which they achieve in part by recruiting co-activators to gene promoters to participate in the dynamic cycle of polymerase binding, initiation and escape from the promoter. Co-activator recruitment and accompanying post-translational modifications to components of promoter complexes appear to differ between genes and as a consequence of varying signal input. Thus a full understanding of transcriptional initiation and control will ultimately require the elucidation of these processes. The method described here was designed to detect the presence of proteins and post-translational modifications in complexes formed in vitro on gene-specific promoters. It has been used, among other things, to detect the recruitment of the Mitogen-Activated Protein (MAP) kinases ERK1 and ERK2 to the promoters of mitogen-responsive genes.

Mitogen-induced recruitment of ERK and MSK to SRE promoter complexes by ternary complex factor Elk-1.

Many eukaryotic genes are acutely regulated by extra-cellular signals. The c-fos serum response element (SRE) mediates transcriptional activation in response to mitogens through serum response factor (SRF)-dependent recruitment of Elk-1, a mitogen-activated protein kinase (MAPK)-responsive transcription factor. How subsequent events at SRE promoters stimulate initiation of transcription has yet to be fully resolved. Here we show that extra-cellular signal-regulated kinase (ERK) and mitogen and stress-activated kinase (MSK) are recruited to SRE promoter complexes in vitro and in vivo. Their recruitment in vitro correlates with Elk-1 binding and for ERK the D domain/KIM of Elk-1 is specifically involved. In vivo, recruitment of ERK and MSK is stimulated by mitogens, correlates with histone H3 phosphorylation and is impaired by Elk-1 knockdown. Immunocytochemistry and confocal microscopy reveal that ERK appears to associate to some extent with initiating rather than elongating RNA polymerase II. Taken together, our data add to the body of evidence implying that ERK and related MAPKs may fulfil a generic role at the promoters of acutely regulated genes.

Anomalous behaviour of the STAT3 binding site in the human c-myc P2 promoter.

The Signal Transducer and Activator of Transcription 3 (STAT3) is necessary for ES cell renewal, plays critical roles during vertebrate development, and has oncogenic potential. STAT3 also mediates cytokine responses notably in the induction of acute phase response genes in the liver. Thus STAT3 is a pleiotropic regulator during cell proliferation and a cell-specific mediator of pro-inflammatory responses. How STAT3 fulfils both roles is unclear. To address this question we attempted to characterise pre-initiation complexes (PICs) on STAT3-responsive promoters containing the c-myc P2 promoter element (P2E) or c-fos Serum-Inducible Element (SIE). Although both promoters mediated cytokine responses in HepG2 cells, poor binding of STAT1 and STAT3 in vitro precluded isolation of active promoter complexes on the P2E. The inability of STAT3 to bind the P2E in vitro correlated with failure of the P2E to mediate cytokine-responsive gene expression in several other cell types. Thus the c-myc P2E behaves as a dual-purpose STAT3 element with anomalous characteristics in HepG2 cells.