Fig. 1.

Ultradian glucocorticoid profile exposure directs glucocorticoid receptor binding and modulates RNA polymeraseII recruitment. (A) Blood plasma total corticosterone (cort) levels in rats were sampled every 10 min through jugular cannulae via an automated blood sampling system over 24 hrs (14:10). On average, max amplitudes of hourly pulses varied in a circadian fashion. Modified and re-drawn from Seale, J. V. et al.: Gonadectomy reverses the sexually diergic patterns of circadian and stress-induced hypothalamic-pituitary-adrenal axis activity in male and female rats. J Neuroendocrinol 2004;16:516–24, with permisson. © John Wiley and Sons. (B) Hourly corticosterone (cort) bolus intravenous injections via jugular cannulae into adrenalectomised Sprague Dalwey rats induced rapid and repeatable increases in circulating total cort blood plasma, returning to approximately baseline within 60 min. Resultant GR binding to the gene PER1 GRE and hnRNA production in the liver peaked at 15 min and 30 min respectively, before returning to approximately basal levels by 60 min of each bolus. Plasma cort samples were measured using an enzyme immunoassay. Modified and re-drawn from Stavreva, D. A., et al.: Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription. Nat Cell Biol 2009;11:1093–102, with permission. © Springer Nature. (C) Sprague Dawley rats were infused hourly with either a pulsatile pattern of cort (20 min infusion followed by 40 min cessation) or a matched constant pattern (infused over 60 min at 0.33 rate) at a dose of 3.84 μM. Liver samples were taken at 140 min or 180 min; corresponding to the peak and nadir of the third pulse. Vehicle infusions were used as control. (D) GR and Pol2 enrichment was detected upstream (∼14.5 kb) and within intragenic regions respectively for the gene TSKU. Pulsatile cort induced time dependent increases at the peak (140 min) of the third pulse for both GR and Pol2 enrichment; Pol2 enrichment in fact was reduced to Vehicle within the nadir (180 min) (adjusted P-value $ <0.05, $$$ <0.001). Pattern dependent changes were detected at 180 min for GR whereas both Pol2 time points were significantly increased in response to constant infusion (adjusted P-value * <0.05, *** <0.001). Indicating both GR and Pol2 recruitment to the gene TSKU is cort pattern dependent. Liver Chromatin Immunoprecipitation assays with either a GR or Pol2 antibodies were sequenced using the Illumina HiSeq2000 platform and analysed using the Homer suite of tools. Data was analysed to input control and compared to Veh (data un-published).