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Thus there is an unmet need for methods that can discern the precise nature (e.g., activator, non-competitive inhibitor) and temporal order of cofactor function. For example, paused RNA polymerase II is often present 50 bp downstream of the start of transcription but is not engaged in transcription –. Cofactor binding to DNA is not equivalent to cofactor action. Finally, one can determine the temporal ordering of cofactor binding to DNA but no method exists to elucidate the temporal ordering of biological function. Because virtually all possible responses to steroid hormones have been observed with endogenous genes, no general mode of action exists. Methods to identify new cofactors are not generally available and the kinetic mechanism of most factors and cofactors is unknown. The HREs may not regulate the closest gene, which is the default assignment. Not all factor binding sites are functionally active. Nonetheless, limitations to this approach remain. One approach to defining steroid receptor actions at the molecular level has been to pair ChIP assays with genome-wide sequencing to identify all DNA binding sites of selected cofactors in the cellular genome. Other steps include recruitment of chromatin remodeling factors and cofactors that increase or decrease the rates of transcription –. The initially formed, intracellular receptor-steroid complex binds to specific enhancer-like DNA elements (called hormone response elements, or HREs) to eventually modify the rates of transcription of target genes. Steroid-regulated gene induction is an extensively studied paradigm with numerous well-defined events. Ligand-regulated gene induction is a productive experimental system for examining the mechanisms of gene expression. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Cofactors are intimately involved in steroid-regulated gene expression.