HNF4A is required to specify glucocorticoid action in the liver

The glucocorticoid receptor (GR) is a nuclear hormone receptor critical to the regulation of energy metabolism and the inflammatory response. The actions of GR are highly dependent on cell type and environmental context. Here, we demonstrate the necessity for liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver-specificity of GR action. In normal mouse liver, the HNF4 motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites found within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodelled, with both loss and gain of GR recruitment evident. Loss of chromatin accessibility at HNF4A-marked sites leads to loss of GR binding at weak GRE motifs. GR binding and chromatin accessibility are gained at sites characterised by strong GRE motifs, which typically show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is further demonstrated by evidence of an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

This Mendeley Data repository contains supportive data and code for our paper. Methods are described in the text. All experiments conducted on mouse (Mus musculus) liver, all processed data derived from raw sequencing data aligned to the mm10 genome. Differential GR binding analysis incorporated spike-in normalisation (spiked-in Drosophila melanogaster chromatin; reads aligned to dm6 genome).

The "MACS2 GR ChIP-seq peaks, wildtype liver" folder contains the BED files for GR ChIP-seq peaks called in dexamethasone- or vehicle-treated mouse liver (Figures 1 & S1).

The "Output of differential binding and differential expression analyses" folder contains the edgeR, stageR and csaw output, following the methods described in the text, using the code in the "Code for differential binding, differential accessibility and differential expression analyses" folder (Figures 2, 5, S3).

The "HOMER motif discovery" folder contains screenshots of the output from HOMER (http://homer.ucsd.edu/homer/ngs/peakMotifs.html) motif discovery analysis of GR binding sites and sites of differential chromatin accessibility (Figures 1, S1, 2, 4).

The "GIGGLE output" folder contains the output (csv) from GIGGLE analysis of GR binding sites, run through the Cistrome DB Toolkit portal (http://dbtoolkit.cistrome.org/) (Figure 3).

The "Output of csaw differential accessibility analysis" folder contains the output from csaw analysis of ATAC-seq data (Figure 4).

糖皮质激素受体（glucocorticoid receptor, GR）是一类核激素受体，在能量代谢与炎症应答的调控中发挥关键作用。GR的生物学功能高度依赖于细胞类型与环境背景。本研究证实，肝脏谱系决定因子肝细胞核因子4A（hepatocyte nuclear factor 4A, HNF4A）是决定GR作用肝脏特异性的必需因子。在正常小鼠肝脏中，HNF4基序位于开放染色质区域内GR结合位点处，紧邻糖皮质激素反应元件（glucocorticoid response element, GRE）。当缺失HNF4A时，肝脏GR的顺式调控组（cistrome）会发生重塑，GR招募的丢失与获得均有体现：在HNF4A标记的位点处，染色质可及性丧失，导致弱GRE基序处的GR结合丢失；而在以强GRE基序为特征的位点处，GR结合与染色质可及性得以获得，这类位点通常在非肝脏组织中存在GR招募。进一步通过Hnf4a敲除小鼠肝脏经糖皮质激素处理后转录应答发生改变的实验证据，证实了这些受HNF4A调控的GR位点的功能重要性。

本Mendeley Data仓储包含本论文的配套数据与代码，实验方法详见正文。所有实验均以小家鼠（Mus musculus）肝脏为实验材料，所有处理后数据均源自比对至mm10参考基因组的原始测序数据。差异GR结合分析采用了spike-in外源参照标准化策略：以黑腹果蝇（Drosophila melanogaster）染色质作为内参，测序reads比对至dm6参考基因组。

"MACS2 GR ChIP-seq peaks, wildtype liver" 文件夹：包含地塞米松或溶剂处理的小鼠肝脏中通过MACS2调用得到的GR ChIP-seq峰的BED格式文件（对应图1与补充图S1）。

"Output of differential binding and differential expression analyses" 文件夹：包含按照正文所述实验方法，使用"Code for differential binding, differential accessibility and differential expression analyses"文件夹中的代码运行得到的edgeR、stageR及csaw分析结果（对应图2、5、S3）。

"HOMER motif discovery" 文件夹：包含HOMER（http://homer.ucsd.edu/homer/ngs/peakMotifs.html）基序发现分析的结果截图，分析对象为GR结合位点与差异染色质可及性位点（对应图1、S1、2、4）。

"GIGGLE output" 文件夹：包含通过Cistrome DB工具集门户（http://dbtoolkit.cistrome.org/）运行的GR结合位点GIGGLE分析的输出结果（csv格式）（对应图3）。

"Output of csaw differential accessibility analysis" 文件夹：包含csaw对ATAC-seq数据进行差异可及性分析的结果（对应图4）。