Androgen Receptor Responsive Enhancers are flanked by Consistently-Positioned H3-Acetylated Nucleosomes

Chromatin is remodeled and nucleosomes repositioned at genomic elements such as active gene promoters¹ and CTCF insulators² to create nucleosome free regions (NFR). In the case of promoters, this may be caused by RNA polymerase II occupancy and the initiation of divergent transcription, implicating the polymerase starting and then pausing transcription in opposite directions¹. Chromatin states at transcriptional enhancers are less well characterized. Active enhancers most likely loop to engage target gene promoters and modulate cell type-specific gene expression³, but nucleosome positioning in relation to transcription factor occupancy has not been studied extensively at enhancers. The relative positioning of transcription factors and nucleosomes at enhancer regions may well be a pivotal factor influencing transcriptional competency. Recent advances in ChIP-seq technology have allowed precise, high-resolution genome-wide nucleosome location assessments and revealed NFRs at both promoters and enhancers⁴. Here, we show data obtained from a ChIP-seq analysis of histone H3K9,14 acetylation (H3K9,14ac) and androgen receptor (AR) occupancy in cultured prostate cancer cells (LNCaP) after a 4-hour treatment of the cells with the natural AR ligand, dihydrotestosterone (DHT) (Figure 1). We carefully inspected two known AR target genes (KLK3 & KLK2) that have well-characterized DHT-responsive enhancers a few kilobases upstream from their respective TSSs⁵. As previously shown^{6, 7}, H3K9, 14ac signals populated the two loci at the respective gene bodies and enhancers, while AR occupancy showed strong signals at the enhancers and weaker signals at the gene promoters (i.e. the TSS). Unexpectedly, in the ChIP-seq data clear ‘troughs’ were observed not only at the promoters, but even more clearly at the two known enhancers, with AR precisely located between the two H3K9,14ac peaks that form the ‘trough’. Based on the similarity of this pattern to promoters, these H3K9,14ac ‘troughs’ are highly suggestive of positioned nucleosomes flanking an NFR in the enhancers, with AR binding at the NFR. We are in the process of establishing the temporal order of AR occupancy versus histone modifications and NFR formation at these and other enhancers. It is tempting to speculate that AR occupancy drives the histone acetylation and the subsequent creation of NFRs at enhancers in a manner similar to RNA polymerase II affecting promoters, although earlier remodeling steps by FoxA1⁸ or other factors should not be excluded. Another scenario is that AR occupies sites that do contain nucleosomes, but selectively promotes histone acetylation of flanking nucleosomes only – this interpretation is consistent with what is known about liganded AR recruiting acetyl transferases such as p300/CBP to genomic sites⁹. In summary, the findings reported here contribute new insight into the structural dynamics of enhancer regions and their interaction with transcription factors and chromatin structure.

Figure 1.

ChIP-seq data from LNCaP cells treated for 4 hours with 10nM DHT. Single-end 36-bp sequence tags were aligned to the NCBI 36 genome using Maq¹⁰, and the density of correctly oriented tags within sliding 500-bp windows was used to create wiggle tracks on the UCSC genome browser. Densities are presented as the average number of tags per base pair.