Analysis of Chromatin Dynamics during Glucocorticoid Receptor Activation

Abstract

dSteroid hormone receptors initiate a genetic program tightly regulated by the chromatin environment of the responsive regions. Using the glucocorticoid receptor (GR) as a model factor for transcriptional initiation, we classified chromatin structure through formaldehyde-assisted isolation of regulatory elements (FAIRE). We looked at dynamic changes in FAIRE signals during GR activation specifically at regions of receptor interaction. We found a distribution of GR-responsive regions with diverse responses to activation and chromatin modulation. The majority of GR binding regions demonstrate increases in FAIRE signal in response to ligand. However, the majority GR-responsive regions shared a similar FAIRE signal in the basal chromatin state, suggesting a common chromatin structure for GR recruitment. Supporting this notion, global FAIRE sequencing (seq) data indicated an enrichment of signal surrounding the GR binding site prior to activation. Brg-1 knockdown showed response elementspecific effects of ATPase-dependent chromatin remodeling. FAIRE induction was universally decreased by Brg-1 depletion, but to varying degrees in a target specific manner. Taken together, these data suggest classes of nuclear receptor response regions that react to activation through different chromatin regulatory events and identify a chromatin structure that classifies the majority of response elements tested. N uclear receptors are a superfamily of transcription factors that initiate specific genetic programs important in virtually all physiological processes, including development, metabolism, differentiation, and growth (12). Activity of the receptors is induced by ligands specific for each family member. After ligand activation, receptors translocate to the nucleus and dimerize on specific response elements within the genome (22). Upon target binding, nuclear receptors are then able modulate gene output by the recruitment of cofactors and transcriptional machinery (1,42). This stepwise model of transcriptional regulation by nuclear receptors is regulated at various stages throughout the activation process.The organization of chromatin structure provides an impediment to DNA-protein interactions that has been demonstrated to be a critical regulator of nuclear receptor recruitment to its target response element as well as transcriptional activation of target genes. Model transcriptional systems, such as the mouse mammary tumor virus (MMTV) promoter, have been invaluable in gaining an understanding of the critical role chromatin plays in nuclear receptor mediated gene transcription (45). Upon DNA binding by the receptor, cofactors are recruited to the promoter to alter chromatin structure and loosen DNA-histone interactions. The first class of cofactors possess enzyme activity that alters posttranslational modifications on the histone proteins (40). The best-characterized cofactors within this group are the histone acetyltransferases (HATs) and methyltransferases (KMTs), which acetylate or methylate histone tails within the core nu...

Glucocorticoid receptor control of transcription: precision and plasticity via allostery

“…Similar results were obtained using formaldehyde-assisted isolation of regulatory elements and deep sequencing (FAIRE seq; see Supplementary information S1 (table)). In this case, pre-existing FAIRE seq signal indicative of open chromatin increased on glucocorticoid stimulation, suggesting that GR interacts with pre-existing open chromatin and then further alters the chromatin environment 39 . By contrast, negative regulatory DNA sequence (NRS) motifs were identified proximal to a subset of GBSs as anti-correlates to GR genomic occupancy.…”

Dynamics of chromatin accessibility and long-range interactions in response to glucocorticoid pulsing

“…The most specific long-range interactions between promoters and enhancers were found within the boundaries of TADs (Burd et al 2012), and the loci within a TAD are proposed to be sufficiently dynamic to allow reproducible contacts between a gene and its regulatory elements Dekker 2012, 2013). Consistent with this idea, we found that the 100-kb regions around GR-responsive genes are preferentially associated with hormone-responsive, dynamic DHSs.…”

Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus

“…ChIP assays were performed as previously described using ERα antibody (Santa Cruz Biotechnology, Santa Cruz, CA) [15]. Sequencing libraries were prepared from ChIP enriched DNA using the Tru-seq Chip Sample Prep Kit (Illumina, San Diego, CA) according to manufacturer's protocol.…”