Characterizing mechanisms of regulatory specificity in the nuclear receptors and general transcriptional cofactors
Keenan, Jessica Lynn
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Gene regulation, at its most basic level, is controlled by transcription factors (TFs) binding to genomic regulatory elements and recruiting regulatory cofactors (CoFs). Therefore, to understand specificity in gene regulation, we must address how TF-DNA binding translates into target gene specification in the genome and how TF-CoF interactions are regulated within the cell. Towards this goal, we describe a comprehensive study of the DNA binding specificity of the type II nuclear receptor (NR) family of TFs, and introduce a novel high-throughput technique for assaying the many TF-CoF complexes functioning in a cell. The type II nuclear receptors function as heterodimeric TFs with the retinoid X receptor (RXR) to regulate diverse biological processes. DNA-binding specificity has been proposed as a primary mechanism for NR gene regulatory specificity. We use protein-binding microarrays (PBMs) to comprehensively analyze the DNA binding of 12 NR:RXRα heterodimers. We find more promiscuous NR-DNA binding than has been reported, challenging the view that NR binding specificity is defined by half-site spacing. We show that NRs bind DNA using two distinct modes, explaining widespread NR binding to half-sites in vivo. Finally, we show that the current models of NR specificity better reflect binding-site activity rather than binding-site affinity. Our rich dataset and revised NR binding models provide a framework for understanding NR regulatory specificity and will facilitate more accurate analyses of genomic datasets. Central to gene regulation is the recruitment of CoFs (e.g., co-activators and co-repressors) to DNA by site-specific TFs. There are currently no high-throughput approaches to identify and characterize the many TF-cofactor complexes simultaneously operating in a cell. To this end, we have developed the CoRec (Cofactor Recruitment) approach to monitor cofactor recruitment by potentially hundreds of TFs from nuclear lysates. We have used CoRec to examine CoF recruitment in resting and LPS-activated human macrophages, as well as resting and T cell receptor-stimulated human T cells. We demonstrate CoF recruitment to both known and novel regulatory elements and compare regulatory strategies between these two cell types. We anticipate CoRec will be a powerful tool to study the assembly and regulation of nuclear TF-cofactor complexes in a cellular context.